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 ...

The protocol in this study provides a combination strategy of two herbs for treating hypoxia-damaged PC12 cells, and a reference for optimizing the best combination mode of herbs. This technique can provide a simple, reliable, and efficient in vitro experimental method for screening the effective ingredient combination from herbs, against hypoxial cells. This strategy can also be applied to screen the component combinations against hypoxial cells from other herb preparation combinations, and illustrate their protective mechanism.To begin, sub-culture the PC12 cells every two to three days at 37 degrees Celsius in an incubator supplemented with 5%carbon dioxide to get a passage of 4 to 8 for all subsequent experiments. Treat 70 to 80%confluent PC12 cells with one milliliter of 0.25%trypsin, and observe the cells under a microscope for cell detachment. Then stop the trypsin digestion by adding three milliliters of the complete medium.Spin down the transferred cells in a 15-milliliter centrifuge tube at 840 x g for five minutes. After discarding the supernatant, resuspend the cell pellet with the complete medium and transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. To count the cells, start the flow cytometry software and select the corresponding dilution multiple of the cell solution in the counting setting.Click on Density Chart after loading the cell sample. 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 to get the cell counting results under the data table of Abs.Count. After adjusting the cell concentration approximately to 1 times 10 to the 5th cells per milliliter with the complete medium, add 100 microliters of the cell suspension per well to a 96-well plate and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, to the supernatant-discarded plate add 120 microliters of one milligram per milliliter MTT solution in each well, and incubate for four hours at 37 degrees Celsius.After incubation, once the supernatant is discarded, add 150 microliters of DMSO to each well. Keep the plate under shaking in a vortex oscillator for 10 minutes at a speed of 240 times per minute. Then measure the absorbance at 490 nanometers in a microplate reader.To screen the optimal combination of drugs by homogenous design method, culture the cells as demonstrated earlier and treat the injured PC12 cells with six different proportion concentrations of Astragalus injection and breviscapus capsule. To evaluate the protective effect of two optimal combinations, treat the injured PC12 cells with the two types of combination of drugs. Incubate the cells for 24 hours and calculate the cell viability as demonstrated earlier.Seed the PC12 cells in a 12-well plate and incubate at 37 degree Celsius for 24 hours. Following drug treatment, treat the cells with 250 microliters of trypsin per well and centrifuge the cells at 840 x g for five minutes at room temperature. Then resuspend the cell pellet with 500 microliters of the binding buffer.Add 5 microliters of Annexin V-FITC, 10 microliters of propidium iodide in triplicates for each group, and incubate the cells in the dark for 15 minutes at room temperature to check for apoptosis using flow cytometry. In the flow cytometer software, click Automatic Compensation in the Start menu, select FITC, PE, PerCP, and APC in the Select channel, and select Compensation at Height. Then click OK in the Statistical Item:Median.Click Density Map and circle the effective cell population with the cell counting method demonstrated earlier. With FITC fluorescence as the x-axis parameter and other fluorescence as the y-axis parameter, create a density map. Click on Compensation Matrix in the Start menu and Coefficient in the overflow matrix.Seed the PC12 cells to the coverslips placed on a 24-well plate well in triplicates, and incubate at 37 degrees Celsius for 24 hours. Following drug treatment, rinse the coverslips twice with PBS for five minutes each, fix them with 4%paraformaldehyde for 15 minutes, and permeabilize with 0.5%Triton X-100 for 20 minutes at room temperature. After rinsing the cells, block them with 10%goat serum for one hour at room temperature.Add 200 microliters of diluted primary antibody caspase-3 to each coverslip and incubate overnight at 4 degrees Celsius. The following day, wash three times with TBST buffer for three minutes each, and incubate with 200 microliters of secondary antibody for one hour at room temperature in the dark. After incubation, incubate the coverslips with 300 microliters of 0.5 micrograms per milliliter of DAPI for 10 minutes, and wash the cells three times with TBST for five minutes each.Capture the coverslip images under a fluorescence microscope and statistically analyze the fluorescence intensity with the imaging software. Wash the drug-treated PC12 cell coverslip three times with PBS for three minutes each and incubate with 400 microliters of 10 micromolar dichloro-dihydro-fluorescein diacetate at 37 degrees Celsius for 20 minutes. Wash the cells again with serum-free DMEM twice for three minutes each.Add the fluorescence quenching agent and immediately capture the coverslip images under a fluorescence microscope to calculate the relative fluorescence intensity with the imaging software. Wash the drug-treated PC12 cells in a 6-well plate three times with PBS for five minutes each, and incubate on ice for 30 minutes in the prepared lysis buffer of 100 microliters per well. Following lysis, centrifuge the cells at 16, 000 x g for 20 minutes at 4 degrees Celsius to collect the supernatant.After detecting the protein concentration by Bradford assay, run a 12%SDS-PAGE electrophoresis with 25 micrograms of protein in each lane and transfer the gel to the PVDF membrane. Block the membranes with 5%nonfat milk for 1.5 hours at room temperature, and incubate with five milliliters of the corresponding primary antibodies for 24 hours at 4 degrees Celsius. After incubation and washing the membrane with TBST three times for 10 minutes each, incubate with five milliliters of secondary HRP-conjugated antibodies at room temperature for two hours.Finally, to the TBST-washed membrane, add chemiluminescent HRP substrate for protein band detection as per the manufacturer's instruction. Then capture the images using the chemiluminescence imaging system to quantify the gray values of the proteins, with beta-actin as an internal control. The cell viability on the normal PC12 cells was lower than 95%with Astragalus injection at concentrations greater than 12 micromolar, and greater than 5 micromolar for the breviscapus capsule.Astragalus injection could improve the survival rate of the injured PC12 cells in the concentration range of 6 to 12 micromolar, and breviscapus capsule at 2 to 5 micromolar. Whereas the drug combination at a ratio of 6 to 1.8 micromolar exhibited the highest cell viability. The cell viability of the two combinations on the injured PC12 cells was significantly promoted, and the component combination was superior to the preparation combination.The apoptosis rate showed that the percentages of early, late, and total apoptotic cells were significantly higher in the model group than in the normal group. Compared with the model group, the percentages of apoptotic cells at each stage were significantly lower in the treatment group. The fluorescence intensity of caspase-3 was significantly lower in each treatment group compared with the model group, and was relatively lower in the component combination group.Relative expression of the genes Akt, Bcl-2, and Bax revealed that the component combination is superior to the preparation combination in promoting cell survival, which is related to the stronger anti-apoptosis effect. The component combination exhibited a better anti-oxidative damage effect than that of the preparation combination, and significantly higher expression of the Nrf2 protein. This method can be used to screen and evaluate the component combination with other potential pharmacological activities from the herb combination, such as anti-inflammatory, antibacterial, and more.

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