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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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).

Abstract

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 µM astragaloside A, 40 µM scutellarin, and 75 µM 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 (µM). The component combination (10 µM astragaloside A, 40 µM scutellarin, and 75 µM 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.

Introduction

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 ....

Protocol

1. Preparation of reagent

  1. 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 °C under closed conditions.
  2. 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.......

Representative Results

The screening of the optimal combination of Ast injection and Eri capsule is shown in Figure 1. 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 µM (Figure 1A) and Eri capsule at concentrations greater than 5 µM (Figure 1A), indicating that the .......

Discussion

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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
1300 series class II biosafety cabinetThermo Fisher Scientific Instruments Company1374
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideGuangzhou saiguo biotech Company1334GR001MTT
ACEA NovoExpress 1.4.0ACEA Biosciences, Inc-
AktWuhan Three Eagles Proteintech Group, Inc10176-2-AP
Analytical flow cytometryThermo Fisher Scientific Instruments Company62-2-1810-1027-0
Annexin V-FITC apoptosis detection kitBeyotime Biotechnology CompanyC1062L
Astragalus injectionHeilongjiang Zhenbao Island Pharmaceutical CompanyA03190612144Ast injection
Astragaloside AChongqing Gao Ren Biotechnology Company84687-43-4
BaxWuhan Three Eagles Proteintech Group, Inc60267-1-Ig
BCA protein quantification kitBeyotime Biotechnology CompanyP0012
Bcl-2Wuhan Three Eagles Proteintech Group, Inc26593-1-AP
Carbon dioxide incubatorsThermo Fisher Scientific Instruments Company0816-2014
Caspase-3Wuhan Three Eagles Proteintech Group, Inc19677-1-AP
Chlorogenic acidChongqing Gao Ren Biotechnology Company327-97-9
Cobalt chloride hexahydrateMerck Biotechnology, Inc.7791-13-1CoCl2
Dimethyl sulfoxideGuangzhou saiguo biotech Company2020112701DMSO
Disodium hydrogen phosphate dodecahydrateChengdu Kolon Chemical Company2020090101
DMEM high sugar mediumThermo scientific Hyclone2110050
DMEM sugar free mediumBeijing Solarbio life sciences Company2029548
ECL luminous fluidLianshuo Biological CompanyWBKLS0500
Electronic balanceHaozhuang Hengping Scientific Instrument Co., Ltd., Shanghai, ChinaFA1204
Electrophoresis instrumentBio-Rad Laboratories (Shanghai) Co., Ltd1658026
Erigeron breviscapus capsuleYunnan Biogu Pharmaceutical CompanyZ53021671Eri capsule
Fetal bovine serumFour Seasons Institute of Biological Engineering20210402FBS
Fluorescent microscopeOlympms CorporationIX71-F32PH
Gel imagerBio-Rad Laboratories (Shanghai) Co., Ltd1708265
Goat anti-mouse secondary antibodyWuhan Three Eagles Proteintech Group, IncSA00001-1
Goat anti-rabbit secondary antibodyWuhan Three Eagles Proteintech Group, IncSA00001- 2
IBM SPSS Statistics version 26.0International Business Machines Corporation, USA-
ImageJ 1.8.0National Institutes of Health, USA-imaging software
Immunol fluorescence staining kitBeyotime Biotechnology CompanyP0196
KClChengdu Kolon Chemical Company2021070901
MarkerThermo Fisher Scientific Instruments Company26616
Microplate readerPerkin Elmer Corporate Management (Shanghai) Co.HH35L2018296
Motic Inverted microscopeNanda Scientific Instruments Co.AE2000LED
NaClChengdu Kolon Chemical Company2014081301
Nonfat milkBeyotime Biotechnology CompanyP0216
Nrf2Wuhan Three Eagles Proteintech Group, Inc16396-1-AP
P-AktWuhan Three Eagles Proteintech Group, Inc66444-1-Ig
PC12 cellsChinese Academy of SciencesCBP60430
Penicillin-Streptomycin solutionThermo scientific HycloneSV30010
Phosphatase protease inhibitor mixtureBeyotime Biotechnology CompanyP1045
Potassium dihydrogen phosphateChengdu Kolon Chemical Company2015082901
Reactive oxygen species assay kitBeyotime Biotechnology CompanyS0033S
RI-PA lysis solutionBeyotime Biotechnology CompanyP0013B
ScutellarinChongqing Gao Ren Biotechnology Company27740-01-8
SDS-PAGE sample loading buffer, 5xBeyotime Biotechnology CompanyP0015L
Sterile filter tips (0.22 µm)Merck Biotechnology, Inc.SLGP033RB
Tris-baseGuangzhou saiguo biotech Company1115GR500 TBS
TrypsinThermo scientific HycloneJ190013
Tween-20Chengdu Kolon Chemical Company2021051301
Vortex oscillatorOHAUS International Co., Ltd., Shanghai, ChinaVXMNAL
β-actinWuhan Three Eagles Proteintech Group, Inc66009-1-Ig

References

  1. Mendelevich, E. G. Chronic cerebral ischemia, and dizziness. Zhurnal Nevrologii i Psikhiatrii Imeni SS Korsakova. 122 (3), 22-26 (2022).
  2. Gao, L. Expert consensus on the diagnosis....

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