Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells

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.......

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 advantages^13,14,15. A large number of studies have shown that Ast can improve the perm.......

Materials

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