加味补阳还五汤调节PINK1/Parkin信号通路介导的线粒体自噬对脑缺血再灌注小鼠的影响OA

Objective:To investigate the effects of modified Buyang Huanwu Tang on cerebral ischemia-reperfusion injury(CI/RI)in mice via the PTEN-induced putative kinase 1/E3 ubiquitin ligase(PINK1/Parkin)signaling pathway-mediated mitophagy,and to explore the underlying mechanism by which modified Buyang Huanwu Tang improves CI/RI.Methods:Seventy-two male C57BL/6J mice were randomly divided into six groups(n=12 per group):Sham-operated group,middle cerebral artery occlusion/reperfusion(MCAO/R)model group,low-,medium-,and high-dose modified Buyang Huanwu Tang groups(8.84,17.68,35.36 g·kg-1·d-1),and an aspirin group(13.00 mg·kg-1·d-1).Neurological deficit scores were assessed using the Zea-Longa method.Cerebral infarct volume ratio was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Histopathological changes and neuronal injury in brain tissues were observed using hematoxylin-eosin(HE)staining and Nissl staining.Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay.Mitochondrial ultrastructure in brain tissue was observed by transmission electron microscopy(TEM).Serum levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression levels of PINK1,Parkin,microtubule-associated protein 1 light chain 3B(LC3B,LC3 Ⅱ/Ⅰ),and p62 in brain tissues were detected by real-time quantitative reverse transcription PCR(Real-time PCR)and Western blot,respectively.Results:Compared with the sham-operated group,the MCAO/R model group showed significantly increased neurological deficit scores and cerebral infarct volume ratios(P＜0.01).Severe cortical injury on the infarct side was observed,characterized by decreased neuronal density,cytoplasmic vacuolation,nuclear pyknosis,a marked reduction in Nissl bodies,dissolution of Nissl bodies in the cytoplasm of some pyramidal neurons,and blurred cellular boundaries.The number of TUNEL-positive cells increased significantly(P＜0.01).Mitochondria exhibited cristae membrane rupture and matrix vacuolation,with rupture of the outer mitochondrial membrane and formation of autophagosomes,the number of which increased significantly.Serum SOD activity decreased significantly(P＜0.01),while MDA content increased significantly(P＜0.01).In infarcted brain tissues of model mice,the relative mRNA expression and protein levels of PINK1,Parkin and LC3B were significantly increased(P＜0.05,P＜0.01),whereas p62 mRNA and protein expression were significantly decreased(P＜0.05,P＜0.01),showing statistical significance.Compared with the model group,all treatment groups showed significantly decreased neurological deficit scores and cerebral infarct volume ratios(P＜0.01).Neuronal density increased significantly,cytoplasmic vacuolation was alleviated,nuclear morphology tended to be more regular and clearer,Nissl body density increased significantly with reduced dissolution and improved contour clarity.The mitochondrial cristae structure was partially restored,with some mitochondria showing autophagosome encapsulation,and the degree of mitochondrial damage was alleviated.Serum SOD activity increased significantly(P＜0.01),while MDA content decreased significantly.The mRNA and protein expression levels of PINK1,Parkin,and LC3 Ⅱ/Ⅰ were significantly increased(P＜0.05,P＜0.01),while p62 mRNA and protein expression in the low-and medium-dose modified Buyang Huanwu Tang groups were significantly decreased(P＜0.05,P＜0.01),showing statistical significance.Conclusion:Modified Buyang Huanwu Tang can upregulate the protein expression levels of PINK1,Parkin,and LC3 Ⅱ/Ⅰ and downregulate p62 protein expression,suggesting that it may improve CI/RI by regulating the expression of proteins related to the PINK1/Parkin signaling pathway.Regulation of the mitophagy pathway may be one of the mechanisms by which modified Buyang Huanwu Tang alleviates CI/RI in mice.