Name: exploring the two herb combination strategy to treat injured pc12 cells
Uploaded: 2022-11-18T14:40:00
Description: Watch this Scientific Journal Video about Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells at JoVE.com

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...

<ol>
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Chinese Pharmacopoeia, Dengzhan Asarum Granules. Volume I Available from: <a target="_blank" href="https://db.ouryao.com/yd2020/view.php?id=f27dcefa69">https://db.ouryao.com/yd2020/view.php?id=f27dcefa69</a> (2020)</li><li>. Chinese Pharmacopoeia, Astragalus granules. Volume I Available from: <a target="_blank" href="https://db.ouryao.com/yd2020/view.php?id=fbcd0a8bf8">https://db.ouryao.com/yd2020/view.php?id=fbcd0a8bf8</a> (2020)</li><li>Liu, X., 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=N-linoleyltyrosine+protects+PC12+cells+against+oxidative+damage+via+autophagy:+Possible+involvement+of+CB1+receptor+regulation.">N-linoleyltyrosine protects PC12 cells against oxidative damage via autophagy: Possible involvement of CB1 receptor regulation.</a> International Journal of Molecular Medicine. 46 (5), 1827-1837 (2020).</li><li>Fan, 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=Gab1+regulates+SDF-1-induced+progression+via+inhibition+of+apoptosis+pathway+induced+by+PI3K/AKT/Bcl-2/BAX+pathway+in+human+chondrosarcoma.">Gab1 regulates SDF-1-induced progression via inhibition of apoptosis pathway induced by PI3K/AKT/Bcl-2/BAX pathway in human chondrosarcoma.</a> Tumor Biology. 37 (1), 1141-1149 (2016).</li><li>Meng, M., Zhang, L., Ai, D., Wu, H., Peng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Asarone+ameliorates+&#946;-Amyloid-induced+neurotoxicity+in+PC12+cells+by+activating+P13K/Akt/Nrf2+signaling+pathway.">&#946;-Asarone ameliorates &#946;-Amyloid-induced neurotoxicity in PC12 cells by activating P13K/Akt/Nrf2 signaling pathway.</a> Frontiers in Pharmacology. 12, 659955 (2021).</li><li>Li, J., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+treatment+of+chronic+cerebral+ischemia+with+traditional+Chinese+and+western+medicine.">Advances in the treatment of chronic cerebral ischemia with traditional Chinese and western medicine.</a> Xinjiang Journal of Traditional Chinese Medicine. 39 (3), 115-118 (2021).</li><li>Yu, M., Zhan, Q., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discussion+on+the+therapeutic+value+of+traditional+Chinese+medicine+on+cognitive+dysfunction+caused+by+chronic+cerebral+ischemia.">Discussion on the therapeutic value of traditional Chinese medicine on cognitive dysfunction caused by chronic cerebral ischemia.</a> Chinese Medicine Modern Distance Education of China. 11 (19), 155-157 (2013).</li><li>Wang, M., Liu, J., Yao, M., Ren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+research+on+pharmacological+and+neuroprotective+effects+of+traditional+Chinese+medicine+after+cerebral+ischemia.">Advances in research on pharmacological and neuroprotective effects of traditional Chinese medicine after cerebral ischemia.</a> China Journal of Chinese Materia Medica. 45 (3), 513-517 (2020).</li><li>Wang, N., Sun, H., Ma, X. <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+different+doses+of+astragalus+injection+on+neurological+rehabilitation+of+stroke+patients.">Effects of different doses of astragalus injection on neurological rehabilitation of stroke patients.</a> Chinese Journal of Clinical Rational Drug Use. 9 (13), 67-69 (2016).</li><li>Sun, Y. 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. 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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 perm...

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 ...

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 ...

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