Effects of Yiqi-Huoxue formula on autophagy and PI3K/Akt/mTOR sig-naling pathway in rats with cerebral ischemia-reperfusion injury
10.3969/j.issn.1000-4718.2024.11.002
- VernacularTitle:益气活血方对脑缺血再灌注损伤模型大鼠脑组织细胞自噬及PI3K/Akt/mTOR信号通路的影响
- Author:
Tiantian ZHANG
1
,
2
;
Jinxi WANG
;
Guo MAO
;
Yan SHANG
;
Li LI
;
Piao HE
;
Ting ZHANG
;
Liang OU
;
Guoheng HU
Author Information
1. 湖南省中医药研究院附属医院,湖南 长沙 410006
2. 湖南省中医药研究院,湖南 长沙 410013
- Keywords:
Yiqi-Huoxue formula;
cerebral ischemia-reperfusion injury;
PI3K/Akt/mTOR signaling path-way;
autophagy
- From:
Chinese Journal of Pathophysiology
2024;40(11):1993-2004
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To explore the possible mechanism of Yiqi-Huoxue formula(YQHXF)in treating cerebral ischemia-reperfusion injury.METHODS:Male SD rats were randomly divided into six groups,namely,the sham,mod-el,nimodipine,and low-,middle-and high-dose YQHXF groups.The middle cerebral artery occlusion/reperfusion(MCAO/R)model was established in all groups except the sham group.After successful modeling,the YQHXF low-,me-dium-,and high-dose groups were given 3.8,7.5,and 15 g?kg-1?d-1 of YQHXF,respectively,by gavage,while the ni-modipine group was given 12 mg?kg-1?d-1 of nimodipine tablets by gavage.The sham and model groups were given 10 mL?kg-1?d-1 of distilled water by gavage.After 14 days of drug intervention,the rats were euthanized and the neurological func-tion was evaluated.The infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride(TTC)staining and brain histopathological changes were determined by hematoxylin-eosin(HE)staining.Transmission electron microscopy was used to investigate changes in autophagosomes,with immunofluorescence used to assess expression of microtubule-associ-ated protein 1 light chain 3(LC3)protein in the cerebral cortex,Western blot was used to measure protein levels of p-PI3K,PI3K,p-Akt,Akt,p-mTOR,mTOR,LC3B,p62,beclin-1,and Atg5,and RT-qPCR was used to determined LC3 and P62 mRNA expression.RESULTS:Compared with the sham group,the neural function scores of rats in the model group rats were significantly increased,and TTC staining revealed large areas of white cerebral infarction.There was severe pathological damage to the cerebral tissue in the ischemic cortical area,and large numbers of autophagosomes were seen inside the cells.Immunofluorescence staining showed significant numbers of LC3B-positive cells(P<0.01).Protein expression of beclin-1,Atg5,and LC3-Ⅱ/LC3-Ⅰ was significantly upregulated(P<0.01),while that of p62 was markedly downregulated(P<0.01).The expression of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR proteins was also significantly reduced(P<0.01).In addition,the mRNA expression of LC3 was significantly upregulated(P<0.01),with downregulation of P62 mRNA levels(P<0.01).Compared with the model group,both the YQHXF medium-and high-dose groups showed upregulated LC3-Ⅱ/LC3-Ⅰ values after 12 h of reperfusion(P<0.01),followed by downregulation of the ratios(P<0.05)after 3,7,and 14 days of reperfusion.Furthermore,after 14 days of reperfusion,compared with the model group,the middle-and high-dose YQHXF groups and the nimodipine group showed reduced neurological function scores(P<0.01),reduced cerebral infarction volumes(P<0.01),improvements in the pathological damage to cortical tis-sue,and reduced autophagosome formation to varying degrees.At the same time,the number of LC3B-positive cells was reduced(P<0.01).Protein expression of beclin-1,Atg5,and LC3-Ⅱ/LC3-Ⅰ was significantly downregulated,while that of p62 was upregulated(P<0.01).The mRNA expression of LC3 and p62 was consistent with the protein levels(P<0.01).In addition,the expression of p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR proteins was upregulated(P<0.01).CONCLUSION:YQHXF can dynamically regulate autophagy in ischemic brain tissue,with inhibition of excess autophagy by activation of the PI3K/Akt/mTOR signaling pathway,thus reducing the infarct volume,alleviating brain dam-age,and promoting the recovery of neurological function.
