黄芪-当归通过调控巨自噬和分子伴侣介导的自噬缓解大鼠脑缺血/再灌注损伤

Huangqi-Danggui decoction alleviates rat cerebral ischemia-reperfusion injury by regulating macroautophagy and chaperone-mediated autophagy

目的:探究黄芪-当归复方药(HQDG)通过调控巨自噬与和分子伴侣介导的自噬(CMA)对脑缺血/再灌注(I/R)损伤7 d时大鼠脑组织的作用及机制。方法:将雄性SD大鼠随机分成假手术(sham)组、模型(model)组、HQDG组和血塞通(XST)组。液质联用技术检测HQDG的主要化学成分。左侧改良线栓法制备大鼠大脑中动脉阻塞/再灌注模型,激光散斑血流成像仪观察脑血流变化;Zea Longa评分观察神经功能缺损情况;HE染色观察神经细胞损伤程度;透射电子显微镜观察脑组织神经血管单元和自噬小体数量的变化;免疫组织化学法检测LC3、P62、溶酶体相关膜蛋白2A(LAMP-2A)、热休克蛋白70(HSP70)和肌细胞增强因子2D(MEF2D)的表达;Western blot法检测P62和LC3的表达。结果:与sham组相比,model组大鼠神经功能缺损评分显著升高(P<0.01),神经细胞大量坏死,细胞核溶解,细胞排列紊乱,自噬小体数量增多,脑组织中LC3、LAMP-2A、HSP70和MEF2D蛋白表达水平升高,P62蛋白表达水平降低(P<0.05或P<0.01);与model组相比,HQDG和XST组脑组织神经功能缺损评分显著降低(P<0.01),细胞损伤明显减轻,自噬小体进一步增多,脑组织中LAMP-2A、HSP70、MEF2D和P62蛋白表达水平降低,LC3-II/LC3-I比值升高(P<0.05或P<0.01)。结论:HQDG可通过激活巨自噬、下调CMA减轻大鼠脑I/R损伤,从而发挥神经保护作用。

AIM:To investigate the effect of Huangqi-Danggui decoction(HQDG)on the brain tissue of rats with cerebral ischemia/reperfusion(I/R)injury for 7 d by regulating macroautophagy and chaperone-mediated autophagy(CMA),and to explore its mechanism.METHODS:Male SD rats were randomly divided into sham group,model group,HQDG group and Xuesaitong(XST)group.Determination of main chemical components of HQDG by liquid chromatography-mass spectrometry.The model of middle cerebral artery occlusion/reperfusion in rats was established by the left modified thread embolism method,and the changes of cerebral blood flow were observed by laser speckle blood flow imager.Zea Longa score was used to observe the neurological deficit.HE staining was used to observe the degree of nerve cell injury.The changes of neurovascular unit and autophagosomes in brain tissue were observed by transmission electron microscopy.Immunohistochemical method was used to detect the expression of LC3,P62,lysosome-associated membrane protein-2A(LAMP-2A),heat shock protein 70(HSP70)and myocyte enhancer factor 2D(MEF2D)proteins.Western blot was used to detect the expression of autophagy-related proteins P62 and LC3-II/LC3-I.RESULTS:Compared with the sham group,the neurological deficit score in model group was significantly higher(P<0.01).A large number of nerve cells showed necrosis and nuclear dissolution,with the cell arrangement being disordered.The number of autophagosomes increased.The protein expression levels of LC3,LAMP-2A,HSP70 and MEF2D in brain tissue increased,while the expression level of P62 protein decreased(P<0.05 or P<0.01).Compared with the model group,the scores of neurological deficit in brain tissue in HQDG and XST groups were significantly lower(P<0.01).Cell damage was significantly reduced.The number of autophagosomes further increased.The expression levels of LAMP-2A,HSP70,MEF2D and P62 proteins in brain tissue decreased,while the expression levels of LC3-II/LC3-I protein increased(P<0.05 or P<0.01).CONCLUSION:HQDG can alleviate cerebral ischemia/reperfusion injury in rats and exert neuroprotective effects by activating macroautophagy and reducing CMA.