This research was funded by Key R &#38; D projects of the Sichuan Provincial Department of science and technology (2020YFS0325).

1. Preparation of reagent
<ol>
	<li>Prepare the complete medium by adding 90 mL of DMEM high sugar medium, 10 mL of fetal bovine serum (FBS), and 1 mL of penicillin-streptomycin solution in a 125 mL sterile culture flask. Mix the complete medium, and store at 4 &#176;C under closed conditions.</li>
	<li>Prepare CoCl2 solution by adding 0.02 g of CoCl2 powder (accurately weighed) in 10 mL of DMEM sugar-free medium (dissolved completely) to obtain a 8.4 mM stock solution. Filter the solution with a 0.22 &#181;m bacterial filter, seal it, and store it at 4 &#176;C away from light. 
	NOTE: CoCl2 is unstable and should be stored in an airtight container, protected from light; the validity period of the CoCl2 solution is 7 days.</li>
	<li>Prepare Tris-Hcl buffered salt solution (TBST).
	<ol>
		<li>Accurately weigh 8 g of sodium chloride, 0.2 g of potassium chloride, and 3 g of Tris-base. Add them into a 1 L beaker, and then add 1,000 mL of RO water to dissolve the mixture.</li>
		<li>Adjust the pH to 7.4, add 1 mL of Tween 20, and thoroughly mix to obtain the TBST solution. 
		NOTE: Tween 20 acts as a surfactant to reduce the nonspecific binding of antibodies to antigens and works best at 0.1% concentration.</li>
	</ol>
	</li>
	<li>Prepare MTT (3-(4,5-Dimethylthiazol-2)-2,5-diphenyltetrazolium bromide salt) solution by weighing 0.1 g of MTT powder in 20 mL of PBS solution to obtain a 5 mg/mL stock solution. Filter the solution with a 0.22 &#181;m bacterial filter, seal it, and store it at 4 &#176;C away from light, placed on standby. 
	NOTE: MTT powder is unstable and easily absorbs moisture, so it should be stored in a closed and dry place away from light. The MTT solution expires after 7 days. MTT has a carcinogenic effect, so avoid direct contact with the skin.</li>
	<li>Prepare the Eri capsule solution by removing the capsule shell and accurately weighing 0.18 g of the content in 10 mL of DMEM sugar-free medium to prepare a 100 &#181;M stock solution (based on the concentration of scutellarin). Filter the solution with a 0.2 &#181;m bacterial filter, seal it, and store it in an airtight container at 4 &#176;C. 
	NOTE: Scutellarin is the main active ingredient in Erigeron breviscapus. The concentration of scutellarin in the capsules has been determined by HPLC-UV to be 2.56 mg/g, i.e., 5.54 &#181;mol/g8. The concentration of the preparation is calculated based on the scutellarin concentration. This is convenient for evaluating the pharmacological activity of the preparation and the component.</li>
	<li>Prepare the solution of the Ast injection by adding 5 mL of injection and 5 mL of sugar-free DMEM media in a 15 mL sterile centrifuge tube to prepare a 60 &#181;M stock solution (based on the concentration of astragaloside A). Filter the solution through a 0.2 &#181;m bacterial filter, and store it in an airtight container at 4 &#176;C. 
	NOTE: The volume of injection is 10 mL each, and the concentration of astragaloside A in the injection has been determined by HPLC-ELSD to be 0.094 mg/mL (i.e., 120 &#181;M)9. The preparation should be carried out in a biosafety cabinet and operated in a light-proof manner throughout. The volume of the injection solution and sugar-free medium should be measured accurately and mixed well.</li>
	<li>Prepare astragaloside A solution by accurately weighing 7.85 mg of astragaloside A standard and dissolving it completely in 2 mL of dimethyl sulfoxide (DMSO) to prepare a 5,000 &#181;M stock solution. Filter it with a 0.22 &#181;m bacterial filter and store it airtight at 4 &#176;C. 
	NOTE: Astragaloside A powder is insoluble in the sugar-free medium. When preparing the solution, dissolve it with the appropriate amount of DMSO to keep the final experimental concentration of DMSO below 0.1% to ensure no cytotoxicity.</li>
	<li>Prepare the scutellarin solution by weighing 11.56 mg of scutellarin standard accurately and completely dissolving it with 5 mL of sugar-free DMEM medium to prepare a 5,000 &#181;M stock solution. Filter it with a 0.22 &#181;m bacterial filter and store it airtight at 4 &#730;C.</li>
	<li>Prepare the chlorogenic acid solution by weighing 8.86 mg of chlorogenic acid standard accurately and completely dissolving it with 5 mL of sugar-free DMEM medium to prepare a 5,000 &#181;M stock solution. Filter it with a 0.22 &#181;m bacterial filter and store it airtight at 4 &#730;C. 
	&#8203;NOTE: Chlorogenic acid powder is unstable and easily absorbs water. It should be sealed and stored at 4 &#176;C away from light; the exposure time should be minimized during weighing.</li>
</ol>
2. Cell viability assay
<ol>
	<li>Obtain PC12 cells and culture them in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 &#181;g/mL streptomycin. 
	NOTE: In this study, the cells are obtained from the Chinese Academy of Sciences. PC12 cells are adherent cells that have the general characteristics of neuroendocrine cells and are widely used in neurophysiology and neuropharmacology.</li>
	<li>Culture the cells at 37 &#176;C in an incubator supplemented with 5% CO2 and sub-culture them every 2-3 days. Use cells at passages 4-8 for all experiments.</li>
	<li>Cell culture
	<ol>
		<li>Treat PC12 cells in the logarithmic growth phase (70%-80% cell confluency) with 1 mL of trypsin (0.25%) and observe the cells under a microscope. When the cells become round, stop the trypsin digestion by adding 3 mL of the complete medium.</li>
		<li>Transfer the cells to a sterile 15 mL centrifuge tube and centrifuge the cells at 840 x g for 5 min. Discard the supernatant, resuspend with the complete medium, and transfer the cell suspension to a 1.5 mL microcentrifuge tube. Then count the number of cells using a flow cytometer.
		<ol>
			<li>Start the flow cytometry software, select the corresponding dilution multiple of the cell solution in the counting setting, and click on Density Chart after loading the cell sample from the 1.5 mL microcentrifuge tube.</li>
			<li>Circle the cell population away from the X-axis and Y-axis, right-click the data table below the figure, and select X, Y, Count, and Abs Count. The data under Abs Count in the data table gives the cell counting result.</li>
		</ol>
		</li>
		<li>Adjust the cell concentration to 1 x 105 cells/mL, inoculate 100 &#181;L/well in 96-well plates, and culture at 37 &#176;C and 5% CO2 in an incubator for 24 h.</li>
	</ol>
	</li>
	<li>Screening of safe concentrations of two drugs on normal PC12 cells
	<ol>
		<li>Culture the cells as described in steps 2.1-2.2. Discard the supernatant, and treat the normal cells with different final concentrations of Ast injection (4, 8, 12, 16, 20, and 24 &#181;M) and Eri capsule (0.625, 1.25, 2.5, 5, 10, and 20 &#181;M). Treat six replicate wells of each group for 24 h. 
		NOTE: The drugs are added to the media to achieve the final concentration of the drug (mentioned in step 2.4.1), and then an equal volume of media is added to each well. When aspirating the supernatant, the pipette tip should be placed gently against the well wall to avoid contacting the cells at the bottom of the well. When adding different solutions, add them gently and quickly along the edges of the wells, and pay attention when changing the pipette gun head to avoid affecting the experimental results.</li>
		<li>Discard the supernatant, add 120 &#181;L of MTT solution (1 mg/mL) to each well, and incubate the cells for 4 h at 37 &#176;C.</li>
		<li>After the incubation, discard the supernatant again and add 150 &#181;L of DMSO to each well. Shake the plate for 10 min at a speed of 240 times/min (number of horizontal vibrations per minute) using a vortex oscillator.</li>
		<li>Measure the absorbance of each sample at 490 nm by using a microplate reader. Calculate the cell viability (%), which is the absorbance of the treated group/absorbance of normal the group (control) x 100. 
		NOTE: Ensure that the MTT solution is within the validity period; if the color has changed (to dark green), it cannot be used. When testing the absorbance of cells, a blank well (culture medium and MTT, without cells or drugs) should be set to eliminate the interference of other reagents. A volume of 150 &#181;L of DMSO is added to each well and shaken for 10 min to dissolve the blue-purple crystals better. The absorbance should be detected as soon as possible to ensure the accuracy of the results.</li>
	</ol>
	</li>
	<li>Screening of effective concentrations of two drugs on injured PC12 cells
	<ol>
		<li>Culture the cells for 24 h as mentioned in steps 2.1-2.2, discard the supernatant, and then treat the cells with 20 &#181;L/well of CoCl2 (0.4 mM) combined with the sugar-free medium.</li>
		<li>Simultaneously treat the cells with 100 &#181;L/well of Ast injection (final concentrations are 2, 6, 8, 10, and 12 &#181;M) or 100 &#181;L of Eri capsule (final concentrations are 1, 2, 3, 4, and 5 &#181;M) at 37 &#176;C for another 24 h. Add 120 &#181;L/well of complete medium to the control group. 
		NOTE: CoCl2 induces hypoxic conditions in the cells.</li>
		<li>Measure the absorbance of each sample by the MTT method and calculate the cell viability as described previously in steps 2.4.2 and 2.4.3.</li>
	</ol>
	</li>
	<li>Screening of the optimal combination of two drugs based on the homogeneous design method 
	NOTE: After obtaining the effective concentration range of the Astragalus injection and breviscapus capsule, the uniform design method U7 (74) table was used to obtain six different combinations of the two drugs. Ast injection:Eri capsule: 1) 2:2.6 &#181;M, 2) 4:5 &#181;M, 3) 6:1.8 &#181;M, 4) 8:4.2 &#181;M, 5) 10:1 &#181;M, and 6)12:3.4 &#181;M.
	<ol>
		<li>Culture the cells as described above in step 2.3.1, and treat the injured PC12 cells with six proportion concentrations of Ast injection:Eri capsule as mentioned above.</li>
		<li>Treat the cells for 24 h and calculate the cell viability as described previously in steps 2.4.2 and 2.4.3.</li>
	</ol>
	</li>
	<li>Evaluation of the protective effect of two optimal combinations on injured PC12 cells 
	NOTE: After obtaining the optimal preparation combination (Ast injection:Eri capsule of 6:1.8 &#181;M) and the optimal component combination7 (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid), further evaluation is performed to check their protective effects on injured PC12 cells.
	<ol>
		<li>Culture the cells as described above in step 2.3.1, and treat the injured PC12 cells with the two types of combinations of drugs as discussed in the NOTE above.</li>
		<li>Treat the cells for 24 h and calculate the cell viability as described previously in steps 2.4.2 and 2.4.3.</li>
	</ol>
	</li>
</ol>
3. Annexin V-FITC/PI assay for apoptosis rate
<ol>
	<li>Seed the PC12 cells in a 12-well plate at a concentration of 1.3 x 105 cells/well and culture them at 37 &#176;C for 24 h.</li>
	<li>Group the cells: control group, model group, and two combinations of drugs. Add 1.2 mL/well of complete medium to the control group, add 1.2 mL/well of media containing 0.4 mM CoCl2 to the model group, and add 1.2 mL/well of each of the two combinations containing 0.4 mM CoCl2. Incubate the cells at 37 &#176;C for another 24 h.</li>
	<li>Following drug treatment, treat the cells with 250 &#181;L/well of trypsin, transfer the cells in a 15 mL centrifuge tube, and centrifuge at 840 xg for 5 min at room temperature (RT). Discard the supernatant and resuspend the pellet in 500 &#181;L of the binding buffer.
	<ol>
		<li>Incubate the cells in the dark with 5 &#181;L of Annexin V-FITC and 10 &#181;L of propidium iodide (PI) for 15 min at RT, and then check for apoptosis by flow cytometry. Set three replicate wells in each group. 
		NOTE: The trypsin used in this test cannot contain EDTA because EDTA is a metal ion chelating agent that competes with annexin V to bind calcium ions and affects the affinity of annexin for PI. When apoptosis occurs, phosphoserine, originally on the inner side of the cell membrane, turns outward to the cell surface, and Annexin V-FITC selectively binds to phosphoserine outside the membrane to show green fluorescence.</li>
		<li>Click Automatic Compensation in the Start menu, select FITC, PE, PerCP, and APC in the Select channel, and select Compensation at: Height. Click OK in the Statistical Item: Median. With the same cell counting method mentioned in step 2.3.2, collect the cell samples, click the Density Map, and circle the effective cell population.</li>
		<li>With FITC fluorescence as the X-axis parameter, and other fluorescence parameters as the Y-axis parameter, create a density map. Click on Compensation Matrix in the Start menu and Coefficient in the overflow matrix. The density map information is displayed for the analysis of the apoptosis rate.</li>
	</ol>
	</li>
</ol>
4. Immunofluorescence detection of caspase-3 generation
<ol>
	<li>Seed the PC12 cells onto coverslips at a density of 8 x 104 cells/coverslip and place them into 24-well plates at 37 &#176;C for 24 h. Group the cells as mentioned above in step 3.2; set up three replicate coverslips for each group.</li>
	<li>Following drug treatment, rinse the coverslips twice with PBS (37 &#176;C) for 5 min each, fix them with 4% paraformaldehyde for 15 min, and permeabilize with 0.5% Triton X-100 for 20 min at RT.</li>
	<li>Rinse the cells again and block them with 10% goat serum for 1 h at RT.</li>
	<li>Incubate each coverslip with 200 &#181;L of primary antibody caspase-3 (a key executive protein of apoptosis) diluted at a concentration of 1:250 in 1x TBST, overnight at 4 &#176;C. Wash with 1x TBST (three times for 3 min each), and incubate with 200 &#181;L of secondary antibody (1:300 dilution in 1x TBST) for 1 h at RT in the dark. 
	NOTE: The fluorescence is easily quenched; thus, after incubation of the secondary antibody, the slides should be kept away from light. The primary and secondary antibodies should be stored at 4 &#176;C away from light.</li>
	<li>Add 300 &#181;L of DAPI (0.5 &#181;g/mL) to the coverslips (to detect the nuclei) for 10 min and wash the cells three times with 1x TBST for 5 min each.</li>
	<li>Observe the coverslips under the fluorescence microscope and capture images10. Perform statistical analysis of the fluorescence intensity using the imaging software. 
	&#8203;NOTE: The slides and coverslips used should be clean and sterile. When staining, pay attention to the pH, concentration, and temperature of the dyeing solution, and avoid fluorescence quenching. The fluorescent microscope should be turned on at least 15 min in advance to preheat the mercury lamp and turned off for 30 min before being turned on again. When acquiring images, the exposure parameters of the same dye in the same batch of experiments should be consistent to ensure the accuracy of the results.</li>
</ol>
5. Immunofluorescence assay of ROS level
<ol>
	<li>Culture and treat the cells as discussed in step 4.1.</li>
	<li>Following drug treatment, wash each coverslip three times with PBS for 3 min, and incubate with 400 &#181;L of DCFH-DA (10 &#181;M) at 37 &#176;C for 20 min. 
	NOTE: DCFH-DA is a universal indicator of oxidative stress. It has cell membrane permeability and no fluorescence. Once it enters the cell, it is hydrolyzed by esterase to produce 2&#39;,7&#39;-dichlorodihydrofluorescein (DCFH) and then rapidly oxidized to produce a strong fluorescent product.</li>
	<li>Wash the cells again with serum-free DMEM (twice for 3 min each) and immediately observe the coverslip after adding the fluorescence quenching agent under the fluorescence microscope. Calculate the relative fluorescence intensity by the imaging software. 
	&#8203;NOTE: After loading the DCFH-DA probe, be sure to clean the residual probe that does not enter the cell, otherwise the background will become high.</li>
</ol>
6. Western blot detection of protein expression of Nrf2, p-Akt, Akt, Bcl-2, and Bax11,12
<ol>
	<li>Inoculate PC12 cells in 6-well plates at a concentration of 1 x 106 cells/well and culture at 37 &#176;C for 24 h. Group and treat the cells as aforementioned in step 4.1, and set three replicate wells in each group.</li>
	<li>After drug treatment for 24 h, wash the cells three times with PBS for 5 min each and incubate on ice for 30 min in prepared lysis buffer. Following lysis, centrifuge the cells for 20 min at 16,000 x g and 4 &#176;C and collect the supernatants.</li>
	<li>Detect the protein concentration and adjust according to the instructions through the Bradford assay. Dilute the samples and denature at 100 &#176;C for 10 min. 
	NOTE: The lysis buffer is composed of phosphatase inhibitor, protease inhibitor, and RIPA lysate in a volume ratio of 1:1:50. In the control group, 200 &#181;L/well of lysis buffer is required, and 100 &#181;L/well of lysis buffer is required in the other groups. Generally, the protein concentrations of control, model, and two types of combinations groups are 0.45 mg/mL, 0.25 mg/mL, and 0.32-0.39 mg/mL respectively; their protein concentrations after dilution with the loading buffer are 0.36, 0.2, and 0.256-0.312 mg/mL, respectively.</li>
	<li>To detect proteins with different molecular weights, load 25 &#181;g of protein in each lane, run a 12% SDS-PAGE electrophoresis, and transfer the gel to PVDF membranes. 
	NOTE: During the preparation of SDS gel, it must be fully mixed without bubbles or insoluble particles; otherwise, uneven or irregular bands may occur. When transferring the membranes, ensure there are no bubbles between the PVDF membrane and the gel; otherwise, white spots will appear in the strip. In addition, this step must be performed at low temperatures, and the ice bag should be changed every 30 min.</li>
	<li>Block the membranes with 5% nonfat milk for 1.5 h at RT and incubate with 5 mL of the corresponding primary antibodies (Nrf2 1:1,000, Akt 1:2,000, p-Akt 1:2,000, Bcl-2 1:5,000, and Bax 1:1,000) for 24 h at 4 &#176;C. Use &#946;-actin (1:5,000) antibody as an internal control.</li>
	<li>Wash the membranes with TBST (three times for 10 min each), then incubate with 5 mL of secondary HPR-conjugated antibodies (1:5,000) at RT for 2 h. 
	NOTE: The dilution ratio of the antibodies should not be arbitrarily changed, and the membranes should be washed with TBST sufficiently in strict accordance with the requirements to avoid the background color of the band being too black.</li>
	<li>Finally, wash the membrane with TBST again, and treat it with chemiluminescent HRP substrate (as per the manufacturer&#39;s instructions) for protein band detection. Capture the images using the chemiluminescence imaging system and quantify the gray values of the proteins using the imaging software, with &#946;-actin as the comparative internal control.</li>
</ol>

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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+astragalus+injection+on+apoptosis+after+cerebral+ischemia-reperfusion+in+rats.">Effects of astragalus injection on apoptosis after cerebral ischemia-reperfusion in rats.</a> Hebei Medicine. 22 (7), 1147-1149 (2016).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengzhan+Xixin+injection+derived+from+a+traditional+Chinese+herb+Erigeron+breviscapusameliorates+cerebral+ischemia/reperfusion+injury+in+rats+via+modulation+of+mitophagy+and+mitochondrial+apoptosis.">Dengzhan Xixin injection derived from a traditional Chinese herb Erigeron breviscapusameliorates cerebral ischemia/reperfusion injury in rats via modulation of mitophagy and mitochondrial apoptosis.</a> Journal of Ethnopharmacology. 288, 114988 (2022).</li><li>Liao, Y., Ni, X., Zhan, C., Cai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+research+progress+of+Dengzhanhua+preparation+in+prevention+and+treatment+of+ischemic+stroke+and+transient+ischemic+attack.">Clinical research progress of Dengzhanhua preparation in prevention and treatment of ischemic stroke and transient ischemic attack.</a> Guangdong Medical Journal. 41 (9), 963-967 (2020).</li><li>Li, C., Li, J., Xu, G., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+chronic+ethanol+consumption+on+apoptosis+and+autophagy+following+transient+focal+cerebral+ischemia+in+male+mice.">Influence of chronic ethanol consumption on apoptosis and autophagy following transient focal cerebral ischemia in male mice.</a> Scientific Reports. 10 (1), 6164 (2020).</li><li>El Khashab, I. H., Abdelsalam, R. M., Elbrairy, A. I., Attia, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chrysin+attenuates+global+cerebral+ischemic+reperfusion+injury+via+suppression+of+oxidative+stress,+inflammation+and+apoptosis.">Chrysin attenuates global cerebral ischemic reperfusion injury via suppression of oxidative stress, inflammation and apoptosis.</a> Biomedicine Pharmacotherapy. 112, 108619 (2019).</li><li>Su, X., He, S., Chen, Z., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+breviscapine+aglycone+on+PI+3+K/Akt+conduction+pathway+in+neonatal+rats+with+hypoxic-ischemic+brain+injury.">Effect of breviscapine aglycone on PI 3 K/Akt conduction pathway in neonatal rats with hypoxic-ischemic brain injury.</a> Journal of Armed Police Logistics College (Medical Edition). 27 (2), 93-96 (2018).</li><li>Wang, X., Shi, C., Pan, H., Meng, X., Ji, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-22+exerts+its+neuroprotective+and+angiogenic+functions+via+regulating+PI3K/Akt+signaling+pathway+in+cerebral+ischemia-reperfusion+rats).">MicroRNA-22 exerts its neuroprotective and angiogenic functions via regulating PI3K/Akt signaling pathway in cerebral ischemia-reperfusion rats).</a> Journal of Neural Transmission. 127 (1), 35-44 (2020).</li><li>Luca, M., Luca, A., Calandra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxidative+damage+in+the+pathogenesis+and+progression+of+alzheimer's+disease+and+vascular+dementia.">The role of oxidative damage in the pathogenesis and progression of alzheimer's disease and vascular dementia.</a> Oxidative Medicine and Cellular Longevity. 2015, 504678 (2015).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lupeol+alleviates+cerebral+ischemia-reperfusion+injury+in+correlation+with+modulation+of+PI3K/Akt+pathway.">Lupeol alleviates cerebral ischemia-reperfusion injury in correlation with modulation of PI3K/Akt pathway.</a> Neuropsychiatric Disease and Treatment. 16 (8), 1381-1390 (2020).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+phosphocreatine+on+oxidative+stress+and+mitochondrial+dysfunction+induced+apoptosis+in+vitro+and+in+vivo:+Involvement+of+dual+PI3K/Akt+and+Nrf2/HO-1+pathways.">Neuroprotective effect of phosphocreatine on oxidative stress and mitochondrial dysfunction induced apoptosis in vitro and in vivo: Involvement of dual PI3K/Akt and Nrf2/HO-1 pathways.</a> Free Radical Biology and Medicine. 120, 228-238 (2018).</li><li>Mu&#241;oz-S&#225;nchez, J., Ch&#225;nez-C&#225;rdenas, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cobalt+chloride+as+a+chemical+hypoxia+model.">The use of cobalt chloride as a chemical hypoxia model.</a> Journal of Applied Toxicology. 39 (4), 556-570 (2019).</li><li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salidroside+attenuates+CoCl2-simulated+hypoxia+injury+in+PC12+cells+partly+by+mitochondrial+protection.">Salidroside attenuates CoCl2-simulated hypoxia injury in PC12 cells partly by mitochondrial protection.</a> European Journal of Pharmacology. 912 (10), 174617 (2021).</li><li>Yin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+potency+of+Tofu+bio-processed+using+Actinomucor+elegans+against+hypoxic+injury+induced+by+cobalt+chloride+in+PC12+cells.">Neuroprotective potency of Tofu bio-processed using Actinomucor elegans against hypoxic injury induced by cobalt chloride in PC12 cells.</a> Molecules. 26 (10), 2983 (2021).</li></ol>

The authors declare no conflicts of interest.

There is still a lack of ideal drugs for the treatment of cerebral ischemia in modern clinical practice2. Under the guidance of supplementing qi and activating the blood circulation method, Ast, Eri, and other preparations have been used in combination in the clinical practice of TCM and have achieved good comprehensive advantages13,14,15. A large number of studies have shown that Ast can improve the permeability of the blood-brain barrier, increase cerebral blood flow, improve the ability of nerve cells to tolerate hypoxia and resist oxygen free radical damage, and can significantly improve neurological dysfunction16,17. It is particularly suitable for senile ischemic cerebrovascular disease16,17. Eri can dilate blood vessels, increase blood supply to the brain, remove ROS, and reduce lipid peroxidation18,19. However, the synergistic effect, pharmacodynamic components, and the mechanism of the combination of the two drugs are still unclear, which is a common problem restricting the clinical application of TCM.
The ischemic and hypoxic environment of chronic cerebral ischemia induces oxidative stress damage and, at the same time, activates the brain cell apoptosis signaling pathway to promote neuronal apoptosis20,21. The PI3K/Akt pathway is a classic anti-apoptotic and pro-survival signal transduction pathway22,23, among which Bcl-2 family proteins and the caspase family are the downstream executive proteins of this signal pathway. Phosphorylated Akt can directly or indirectly regulate the Bcl-2 family and inhibit activation of the downstream pathway caspase-3, thereby exerting an anti-apoptotic effect11. In addition, excess ROS generated by hypoxia can induce oxidative stress injury, while phosphorylated Akt can activate Nrf2 to eliminate excess ROS to combat oxidative stress damage12,24. Therefore, activation of the PI3K/Akt/Nrf2 pathway can effectively prevent and control oxidative stress and apoptosis, thereby reducing hypoxic-ischemic brain injury25,26.
In the study, the preparation combination was screened using the injured PC12 cell model, and the effect and mechanism in this model were compared with the component combination screened previously7. In addition, the maximum nontoxic concentration of Ast injection and Eri capsule is 12 &#181;M (calculated by astragaloside A) and 5 &#181;M (calculated by scutellarin), respectively, while the maximum nontoxic concentration of astragaloside A and scutellarin is 20 &#181;M and 50 &#181;M, respectively7. It shows that the component combination is safer than the preparation combination, with more controllable quality and comprehensive advantages of high efficiency and low toxicity.
The current cell models that can be used to simulate cerebral ischemia mainly include physical hypoxia (induced by OGD) and chemical hypoxia (induced by Na2S2O4 or CoCl2)27. Among them, both OGD and Na2S2O4 injuries have a short hypoxia time and are not suitable for simulating chronic ischemia27. CoCl2-induced hypoxia is one of the most commonly used hypoxia mimics compared to OGD and the use of other hypoxia mimics. It can lead to persistent and stable oxidative damage by ROS and produce typical apoptotic changes in different cell lines27. Therefore, in this study, CoCl2 was used for 24 h to simulate the neuronal hypoxia28,29. Its modeling concentration (0.1, 0.2, 0.4, and 0.8 mM) and the validity period (1 and 7 days) were screened. In addition, given the inevitable lack of glucose and oxygen after cerebral ischemia, the glucose-free and hypoxic environment can better simulate cerebral ischemic injury. This study showed that 0.4 mM CoCl2 combined with a glucose-free medium for 24 h could establish a stable and controllable chronic hypoxic cell model. In the follow-up study, the animal model of chronic cerebral ischemia will be used to further validate the therapeutic advantages of the component combination in vivo.
This cell model can increase the apoptosis rate, caspase-3 fluorescence intensity, and intracellular ROS level (Figure 2 and Figure 4), which can better simulate the pathological changes of chronic cerebral ischemia, such as apoptosis, oxidative stress, etc. Based on this model, it was found that the two types of combinations can inhibit apoptosis and oxidative stress damage. The effect of the component combination on promoting cell survival was significantly better than that of the preparation combination (Figure 1); both combinations could upregulate the expression levels of P-Akt/Akt, Bcl-2/Bax, and Nrf2 in different degrees (Figure 3 and Figure 4). In particular, the component combination had a stronger effect on the up-regulation of Akt/Bcl-2/Bax and Nrf2 signaling pathway. These results indicate that the component combination can better resist cell damage than the preparation combination, which is related to stronger anti-apoptosis and anti-oxidative stress.
In conclusion, these results provide a combination strategy of two herbs to treat injured PC12 cells and provides a new idea for evaluating and optimizing the combined application mode of two herbs. Overall, this study provides a reference for selecting the active component combination of TCM.

Chronic cerebral ischemia, caused by cerebral hypoperfusion, is a common disease in middle-aged and elderly people1. As a long-term occult ischemia disease, it can lead to progressive or persistent neurological dysfunction. The main pathological mechanisms include cell apoptosis and oxidative damage, leading to progressive or persistent neurological dysfunction; this is the pathological basis of Alzheimer's disease, vascular dementia, and other diseases, and seriously affects the quality of life of patients. However, there is still a lack of ideal drugs in modern medicine to treat chronic cerebral ischemia2. At the same time, the combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) has been widely used in the clinical practice of traditional Chinese medicine (TCM)4. The combination strategy has remarkably improved in promoting the recovery of nerve function after cerebral ischemia, which is better than that of a single drug; however, there are large differences in the dosage ratio in the combination of the two drugs4. The effective components and mechanism of action are not well defined, which is the key issue restricting its clinical application.
Previous studies have demonstrated the synergistic effect of the preparation combination of Ast injection and Eri injection in treating cerebral ischemia in rats. It can upregulate the expression of p-Akt protein and downregulate the expression of Bcl-2 associated death promoter (BAD) protein5,6, which is one of the B-lymphoblastoma 2 (Bcl-2) family proteins and has the effect of promoting apoptosis. However, the material basis and mechanism of its synergistic effect are not clear. Further, the injury model of PC12 cells was established with CoCl2 combined glucose-free medium, and the optimal component combination in Ast and Eri has been screened and defined7.
In this study, the effective doses of Ast and Eri are screened by using the injured PC12 cell model. The optimal combination of the two drugs is screened using this model combined with the homogeneous design method. The cell model is used to further evaluate the difference in the effects and mechanisms of the preparation combination and the component combination in injured PC12 cells. This strategy aims to explore the protective effect and regulatory mechanism of the two types of combinations on injured PC12 cells through the PI3K/Akt/Nrf2 signal pathway to determine the best combination mode. This study provides a reference for optimizing the best application mode of TCM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1300 series class II biosafety cabinet</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>1374</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>1334GR001</td>
			<td>MTT</td>
		</tr>
		<tr>
			<td>ACEA NovoExpress&#160;1.4.0</td>
			<td>ACEA Biosciences, Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>10176-2-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical flow cytometry</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>62-2-1810-1027-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V-FITC apoptosis detection kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>C1062L</td>
			<td></td>
		</tr>
		<tr>
			<td>Astragalus injection</td>
			<td>Heilongjiang Zhenbao Island Pharmaceutical Company</td>
			<td>A03190612144</td>
			<td>Ast injection</td>
		</tr>
		<tr>
			<td>Astragaloside A</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>84687-43-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bax</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>60267-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Bcl-2</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>26593-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide incubators</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>0816-2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase-3</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>19677-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorogenic acid</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>327-97-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt chloride hexahydrate</td>
			<td>Merck Biotechnology, Inc.</td>
			<td>7791-13-1</td>
			<td>CoCl2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>2020112701</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate dodecahydrate</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2020090101</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high sugar medium</td>
			<td>Thermo scientific Hyclone</td>
			<td>2110050</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM sugar free medium</td>
			<td>Beijing Solarbio life sciences Company</td>
			<td>2029548</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL luminous fluid</td>
			<td>Lianshuo Biological Company</td>
			<td>WBKLS0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Haozhuang Hengping Scientific Instrument Co., Ltd., Shanghai, China</td>
			<td>FA1204</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis instrument</td>
			<td>Bio-Rad Laboratories (Shanghai) Co., Ltd</td>
			<td>1658026</td>
			<td></td>
		</tr>
		<tr>
			<td>Erigeron breviscapus capsule</td>
			<td>Yunnan Biogu Pharmaceutical Company</td>
			<td>Z53021671</td>
			<td>Eri capsule</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Four Seasons Institute of Biological Engineering</td>
			<td>20210402</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympms Corporation</td>
			<td>IX71-F32PH</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imager</td>
			<td>Bio-Rad Laboratories (Shanghai) Co., Ltd</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>SA00001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary antibody</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>SA00001- 2</td>
			<td></td>
		</tr>
		<tr>
			<td>IBM SPSS Statistics version 26.0</td>
			<td>International Business Machines Corporation, USA</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ 1.8.0</td>
			<td>National Institutes of Health, USA</td>
			<td>-</td>
			<td>imaging software</td>
		</tr>
		<tr>
			<td>Immunol&#160;fluorescence&#160;staining kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0196</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2021070901</td>
			<td></td>
		</tr>
		<tr>
			<td>Marker</td>
			<td>Thermo Fisher Scientific Instruments Company</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Perkin Elmer Corporate Management (Shanghai) Co.</td>
			<td>HH35L2018296</td>
			<td></td>
		</tr>
		<tr>
			<td>Motic Inverted microscope</td>
			<td>Nanda Scientific Instruments Co.</td>
			<td>AE2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2014081301</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat milk</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0216</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrf2</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>16396-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>P-Akt</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>66444-1-Ig</td>
			<td></td>
		</tr>
		<tr>
			<td>PC12 cells</td>
			<td>Chinese Academy of Sciences</td>
			<td>CBP60430</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Thermo scientific Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphatase protease inhibitor mixture</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P1045</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2015082901</td>
			<td></td>
		</tr>
		<tr>
			<td>Reactive oxygen species assay kit</td>
			<td>Beyotime Biotechnology Company</td>
			<td>S0033S</td>
			<td></td>
		</tr>
		<tr>
			<td>RI-PA lysis solution</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scutellarin</td>
			<td>Chongqing Gao Ren Biotechnology Company</td>
			<td>27740-01-8</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE sample loading buffer, 5x</td>
			<td>Beyotime Biotechnology Company</td>
			<td>P0015L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter tips (0.22 &#181;m)</td>
			<td>Merck Biotechnology, Inc.</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Guangzhou saiguo biotech Company</td>
			<td>1115GR500&#160;</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo scientific Hyclone</td>
			<td>J190013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Chengdu Kolon Chemical Company</td>
			<td>2021051301</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex oscillator</td>
			<td>OHAUS International Co., Ltd., Shanghai, China</td>
			<td>VXMNAL</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Wuhan Three Eagles Proteintech Group, Inc</td>
			<td>66009-1-Ig</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring the two herb combination strategy to treat injured pc12 cells

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (&#181;M). The component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

The screening of the optimal combination of Ast injection and Eri capsule is shown in Figure 1.&#160;The cell survival rate of Ast injection and Eri capsule on the normal PC12 cells is shown in Figure 1A. The cell viability was lower than 95% with Ast injection at concentrations greater than 12 &#181;M (Figure 1A) and Eri capsule at concentrations greater than 5 &#181;M (Figure 1A), indicating that the maximum nontoxic concentration was 12 &#181;M and 5 &#181;M, respectively. Their cytotoxicity was greater than that of astragaloside A and scutellarin used alone. The viability of Ast injection and Eri capsule on the injured PC12 cells induced by CoCl2 is shown in Figure 1B,C. Compared with the model group, Ast injection could improve the survival rate of the injured PC12 cells in the concentration range of 6-12 &#181;M (p &#60; 0.05 or p &#60; 0.01), and Eri capsules at the concentration of 2-5 &#181;M could improve the survival rate (p &#60; 0.05 or p &#60; 0.01). Ast injection and Eri capsule at the ratios of 10:1, 8:4.2, and 6:1.8 &#181;M can significantly increase the viability of the injury PC12 cells (p &#60; 0.01) (Figure 1D). The ratio of 6:1.8 &#181;M exhibited the highest cell viability, indicating that it is the optimal preparation combination and pharmacological activity compared with the best component combination.
The evaluation of the protective effects of two kinds of combination on the injured PC12 cells is shown in Figure 2. Compared with the model group, the cell viability of the two combinations was significantly promoted (p &#60; 0.001), and the component combination was superior to the preparation combination (Figure 2A). This suggests that the component combination can promote cell survival better than the preparation combination. The apoptosis rate was tested by flow cytometry (Figure 2B,C). Compared with the normal group, the percentages of early, late, and total apoptotic cells were significantly higher in the model group (p &#60; 0.01 or p &#60; 0.001). Compared with the model group, the percentages of apoptotic cells at each stage were significantly lower in the treatment groups (p &#60; 0.05 or p &#60; 0.01, or p &#60; 0.001). The fluorescence intensity of caspase-3 protein expression in each group is shown in Figure 2D,E. Compared with the normal group, the fluorescence intensity of caspase-3 was significantly higher in the model group. Compared with the model group, the fluorescence intensity of caspase-3 was significantly lower in each treatment group (p &#60; 0.001 or p &#60; 0.01) and was relatively lower in the component combination group than the preparation combination group. These results suggest that the cell model can induce apoptosis, and the anti-apoptosis effect of the component combination is better than that of the preparation combination.
Western blot detected the p-Akt, Akt, Bcl-2, and Bax protein expression (Figure 3). Compared with the normal group, the expression levels of p-Akt/Akt and Bcl-2/Bax were significantly lower in the model group (p &#60; 0.01 or p &#60; 0.001). Compared with the model group, the expression of p-Akt/Akt and Bcl-2/Bax were significantly higher in the two combinations (p &#60; 0.05 or p &#60; 0.01, or p &#60; 0.001), specifically higher in the component combination. The results suggest that the component combination is superior to the preparation combination in promoting cell survival, which is related to the stronger anti-apoptosis effect produced by upregulating the Akt/Bcl-2/Bax signal pathway.
The fluorescence of DCFH can be measured by a fluorescence microscope to determine the level of ROS in the cells (Figure 4A,B). As shown in Figure 4A, the fluorescence was almost invisible in the normal cells, and the fluorescence in the model group was significantly enhanced. The fluorescence in both combinations was significantly reduced compared to the model group. As shown in Figure 4B, the relative fluorescence intensity was significantly weaker in each treatment group compared with the model group (p &#60; 0.001). Fluorescence had a tendency to decrease in the component combination group compared with the preparation combination group, indicating that the cell model can induce oxidative damage. The anti-oxidative damage effect of the component combination is better than that of the preparation combination.
Western blot detected the expression of the Nrf2 protein (Figure 4C). Compared with the normal group, the expression of Nrf2 was significantly reduced in the model group (p &#60; 0.05). Compared with the model group, the expression of Nrf2 protein was significantly higher in the treatment groups (p &#60; 0.01 or p &#60; 0.05), specifically higher in the component combination group (Figure 4D). This suggests that the component combination is better than the preparation combination in promoting cell survival, which is related to the stronger anti-oxidative damage produced by upregulating the Nrf2 signal pathway.
In conclusion, the component combination (10 &#181;M astragaloside A, 40 &#181;M scutellarin, and 75 &#181;M chlorogenic acid) could promote the survival of injured cells better than the preparation combination (6 &#181;M Ast injection and 1.8 &#181;M Eri capsule) through stronger regulation of signal pathways related to apoptosis and oxidative damage.
SPSS statistical software 26.0 was used for statistical analysis, and all data are expressed as the means &#177; standard deviation (SD). For comparisons between groups, the data were evaluated by a one-way ANOVA followed by Tukey&#39;s test. p &#60; 0.05 was considered to indicate a statistically significant difference.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64721/64721fig01.jpg" /> 
Figure 1: Effects of drugs on the viability of normal and injured PC12 cells. (A) Effects of various concentrations of Ast injection and various concentrations of Eri capsule on normal PC12 cells. (B) Screening of the effective concentration of Ast injection on injured PC12 cells. (C) Screening of the effective concentration of Eri capsule on injured PC12 cells. (D) Effects of the combinations of two drugs in different proportions on the survival rate of injured PC12 cells. Statistical values are expressed as the mean &#177; SD from six independent experiments. &#38;&#38;p &#60; 0.01 compared with the control group. *p &#60; 0.05 and **p &#60; 0.01 compared with the model group. <a href="https://www.jove.com/files/ftp_upload/64721/64721fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64721/64721fig02.jpg" /> 
Figure 2: Effects of two combinations on the survival and apoptosis of injured PC12 cells. (A) Effect of the component combination and the preparation combination on the survival rate of injured PC12 cells. (B) Graph of apoptosis rate detected by Annexin V-PI. (C) Statistical histogram of apoptosis rate. (D) The relative fluorescence intensity of caspase-3 protein expression (400x). Scale bars: 20 &#181;m. (E) Statistical histogram of fluorescence intensity of caspase-3 protein expression. Statistical values are expressed as the mean &#177; SD from three independent experiments, except for cell viability for six independent experiments. *p &#60; 0.05, **p &#60; 0.01, and ***p &#60; 0.001 compared with the model group. ##p &#60; 0.01 compared with the preparation combination group. <a href="https://www.jove.com/files/ftp_upload/64721/64721fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64721/64721fig03.jpg" /> 
Figure 3: Effects of the two combinations on the expression levels of p-Akt, Akt, Bcl-2, and Bax. (A) Protein expression of p-Akt and Akt determined by Western blot analysis. (B)&#160;Statistical histogram of p-Akt/Akt ratio statistics in each group. (C) Protein expression of Bcl-2 and Bax determined by Western blot analysis. (D) Statistical histogram of Bcl-2/Bax ratio in each group. Statistical values are expressed as the mean &#177; SD from three independent experiments. *p &#60; 0.05, **p &#60; 0.01, and ***p &#60; 0.001 compared with the model group. <a href="https://www.jove.com/files/ftp_upload/64721/64721fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64721/64721fig04.jpg" /> 
Figure 4: Effect of two combinations on the Nrf2 protein expression and ROS levels. (A) Levels of ROS were detected by fluorescence microscopy (400x). Scale bars: 20 &#181;m. (B)&#160;Statistical histogram of ROS fluorescence intensity in each group. (C) Nrf2 protein expression determined by Western blot analysis.&#160;(D) Statistical histogram of Nrf2 protein expression. Statistical values are expressed as the mean &#177; SD from three independent experiments. *p &#60; 0.05, **p &#60; 0.01, and ***p &#60; 0.001 compared with the model group. <a href="https://www.jove.com/files/ftp_upload/64721/64721fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells at JoVE.com

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in ...

exploring-two-herb-combination-strategy-to-treat-injured-pc12

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1.2K Views.  Chengdu Medical College. This protocol provides a combination strategy of two herbs to treat injured PC12 cells. The protocol provides a reference for optimizing the best application mode of traditional Chinese medicine (TCM).

Video: Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

Catheter Ablation in Combination With Left Atrial Appendage Closure for Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and disordered electrical activity in the atria gives rise to palpitations, fatigue, dyspnea, chest pain and dizziness with or without syncope 4, 5. Patients with AF have a five-fold higher risk of stroke 6.
Oral anticoagulation (OAC) with warfarin is commonly used for stroke prevention in patients with AF and has been shown to reduce the risk of stroke by 64% 7. Warfarin therapy has several major disadvantages, however, including bleeding, non-tolerance, interactions with other medications and foods, non-compliance and a narrow therapeutic range 8-11. These issues, together with poor appreciation of the risk-benefit ratio, unawareness of guidelines, or absence of an OAC monitoring outpatient clinic may explain why only 30-60% of patients with AF are prescribed this drug 8.
The problems associated with warfarin, combined with the limited efficacy and/or serious side effects associated with other medications used for AF 12,13, highlight the need for effective non-pharmacological approaches to treatment. One such approach is catheter ablation (CA), a procedure in which a radiofrequency electrical current is applied to regions of the heart to create small ablation lesions that electrically isolate potential AF triggers 4. CA is a well-established treatment for AF symptoms 14, 15, that may also decrease the risk of stroke. Recent data showed a significant decrease in the relative risk of stroke and transient ischemic attack events among patients who underwent ablation compared with those undergoing antiarrhythmic drug therapy 16.
Since the left atrial appendage (LAA) is the source of thrombi in more than 90% of patients with non-valvular atrial fibrillation 17, another approach to stroke prevention is to physically block clots from exiting the LAA. One method for occluding the LAA is via percutaneous placement of the WATCHMAN LAA closure device. The WATCHMAN device resembles a small parachute. It consists of a nitinol frame covered by fabric polyethyl terephthalate that prevents emboli, but not blood, from exiting during the healing process. Fixation anchors around the perimeter secure the device in the LAA (Figure 1). To date, the WATCHMAN is the only implanted percutaneous device for which a randomized clinical trial has been reported. In this study, implantation of the WATCHMAN was found to be at least as effective as warfarin in preventing stroke (all-causes) and death (all-causes) 18. This device received the Conformit&eacute; Europ&eacute;enne (CE) mark for use in the European Union for warfarin eligible patients and in those who have a contraindication to anticoagulation therapy 19.
Given the proven effectiveness of CA to alleviate AF symptoms and the promising data with regard to reduction of thromboembolic events with both CA and WATCHMAN implantation, combining the two procedures is hoped to further reduce the incidence of stroke in high-risk patients while simultaneously relieving symptoms. The combined procedure may eventually enable patients to undergo implantation of the WATCHMAN device without subsequent warfarin treatment, since the CA procedure itself reduces thromboembolic events. This would present an avenue of treatment previously unavailable to patients ineligible for warfarin treatment because of recurrent bleeding 20 or other warfarin-associated problems.
The combined procedure is performed under general anesthesia with biplane fluoroscopy and TEE guidance. Catheter ablation is followed by implantation of the WATCHMAN LAA closure device. Data from a non-randomized trial with 10 patients demonstrates that this procedure can be safely performed in patients with a CHADS2 score of greater than 1 21. Further studies to examine the effectiveness of the combined procedure in reducing symptoms from AF and associated stroke are therefore warranted.

Catheter ablation is combined with placement of the WATCHMAN Left Atrial Appendage Closure Device to prevent ischemic stroke in a patient with non-valvular atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting millions of individuals worldwide 1-3. The rapid, irregular, and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 dimensional&#160;(3D) diagnostic imaging in the interventional suite and can enhance minimally-invasive therapy beyond the limitations of 2D angiography alone. The role of CBCT has been recognized in transarterial chemo-embolization (TACE) treatment of hepatocellular carcinoma (HCC). The recent introduction of a CBCT technique: dual-phase CBCT (DP-CBCT) improves intra-arterial HCC treatment with drug-eluting beads (DEB-TACE). DP-CBCT can be used to localize liver tumors with the diagnostic accuracy of multi-phasic multidetector computed tomography (M-MDCT) and contrast enhanced magnetic resonance imaging (CE-MRI) (See the tumor), to guide intra-arterially guidewire and microcatheter to the desired location for selective therapy (Reach the tumor), and to evaluate treatment success during the procedure (Treat the tumor). The purpose of this manuscript is to illustrate how DP-CBCT is used in DEB-TACE to see, reach, and treat HCC.

Dual-phase cone-beam computed tomography (DP-CBCT) is a useful intraprocedural imaging technique for transarterial chemo-embolization treatment with drug-eluting beads of hepatocellular carcinoma. DP-CBCT has been used to perform three major steps in oncologic interventional radiology: tumor localization (see), navigation and intraprocedural catheter guidance (reach), and intraprocedural evaluation of treatment success (treat).

The advent of cone-beam computed tomography (CBCT) in the angiography suite has been revolutionary in interventional radiology. CBCT offers 3 ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of epididymal OA since 2000. There are two challenges in classic microscopic vasoepididymostomy. First, anastomosis of the vas deferens and epididymis is performed with double-needle sutures. However, there is a lack of good-quality and cost-effective double-needle sutures in China, which leads to increased difficulty and poor success rates of anastomosis. Second, the separation of the vas deferens does not retain vasculature, although the vas deferens vasculature plays an important role in the blood supply to the vas deferens, epididymis, and testis. This affects the blood supply to the anastomotic area and epididymis.
Therefore, this team has made innovative improvements to address these problems. Good-quality, cost-effective, single-needle sutures, which are easy to purchase in China and other countries, were used in microsurgical longitudinal intussusception vasoepididymostomy. This can optimize the operation procedure and shorten the operation time while ensuring the success rate of the anastomosis. The surgical method of preserving the vas deferens vessels was innovatively proposed because the etiology of epididymal OA is mostly inflammatory in China. The protection of the blood supply to the vas deferens and epididymis is maximized using microsurgical forceps to separate and protect the vasculature. Patency reached 81.7% in the postoperative follow-up, indicating a better surgical treatment effect.

The present protocol describes a vessel-sparing, longitudinal intussusception vasoepididymostomy using readily available single-needle sutures in China, a safe and effective procedure, which may improve patients' patency and natural pregnancy rates.

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT).
This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied ...

Ultrasound Evaluation of Achilles Tendon Morphology

Measurement of Healthy and Injured Triceps Surae Morphology

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally classified as an overuse injury associated with fusiform tendon thickening, neovascularization, and interstitial tendon degeneration. Current literature suggests these structural changes are associated with&#160;symptoms and lower physical activity levels, as well as&#160;symptoms and lower extremity function in the long term. Surgically and non-surgically managed Achilles tendon ruptures result in increased tendon cross-sectional area (CSA) and a lengthened Achilles tendon. Both structural outcomes have clinical implications, as larger CSA positively predicts function, whereas increased tendon lengthening predicts reduced function after Achilles tendon rupture. Given the relationship between structural changes associated with Achilles tendon injuries for both injury severity and injury recovery, it is critical to be able to quantify Achilles tendon structure reliably and accurately. Silbernagel&#39;s group has established a valid and reliable method for efficiently evaluating triceps surae muscle and tendon structure. In this protocol, B-mode musculoskeletal ultrasound imaging is used to measure triceps surae structure, including Achilles tendon thickness and CSA, soleus thickness, and the presence of additional findings (calcifications and bursitis). B-mode extended field-of-view is used to measure Achilles tendon length and gastrocnemius anatomical CSA. Finally, power Doppler is used to identify intratendinous neovascularization. Quantification of triceps surae structure allows for comparison between limbs as well as longitudinal changes in response to exercise and treatment for healthy individuals and those with Achilles tendon injuries. This protocol has been used in many research studies to date and proves valuable in understanding the relationship between tendon structure and injury development, severity, and recovery. As ultrasound devices are becoming more affordable and portable, this protocol proves promising as a clinical tool, given its quick and efficient methods.

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally ...