化浊解毒活血通络方对脑缺血再灌注损伤大鼠细胞自噬相关蛋白的抑制作用

Inhibitory effect of Huazhuo Jiedu Huoxue Tongluo Prescription on autophagy-related proteins in rats with cerebral ischemia-reperfusion injury

目的:研究化浊解毒活血通络方对脑缺血再灌注损伤大鼠细胞自噬相关蛋白的抑制作用。方法:50只SPF级SD大鼠随机分为5组:假手术组、模型组、中药组、抑制剂组、中药+抑制剂组。采用线栓法制备大脑中动脉缺血/再灌注模型,假手术组仅分离血管不进栓,各组予相应干预。给药3 d后观察各组大鼠神经功能评分,TTC检测脑组织梗死面积,HE染色观察脑组织结构变化,透射电镜观察自噬小体超微结构,Western blot法检测自噬相关蛋白微管相关蛋白轻链3(LC3)、Beclin1、p62、Atg5的表达情况。结果:与假手术组比较,模型组神经功能缺损评分显著升高(P<0.05),脑梗死面积显著增大(P<0.05),病理改变严重,电镜下自噬小体、自噬溶酶体清晰可见,LC3、Beclin1、Atg5表达显著升高(P<0.05),P62表达显著降低(P<0.05)。与模型组比较,中药组、抑制剂组和中药+抑制剂组神经功能缺损评分均显著降低(P<0.05),脑梗死面积显著缩小(P<0.05),脑组织病理状态有所改善,电镜下细胞核核膜轻度融合,自噬小体、自噬溶酶体可见但数量较少,LC3、Beclin1、Atg5表达显著降低(P<0.05),P62表达显著升高(P<0.05)。结论:化浊解毒活血通络方对脑缺血再灌注损伤有改善作用,可能与抑制细胞自噬,上调LC3、Beclin1、Atg5蛋白的表达,下调P62蛋白水平有关。

Objective: To study the inhibitory effect of Huazhuo Jiedu Huoxue Tongluo Prescription on autophagyrelated proteins in rats with cerebral ischemia-reperfusion injury. Methods: Fifty SPF SD rats were randomly divided into 5groups: Sham group, MCAO/R group, traditional Chinese medicine(TCM) group, autophagy inhibitor(3-MA) group, and traditional Chinese medicine+autophagy inhibitor(TCM+3-MA) group. The middle cerebral artery ischemia/reperfusion model was established by suture method. In the sham operation group, only the blood vessels were separated without thrombus insertion. Each group was intervened accordingly. After 3 days of administration, the neurological function scores of the rats in each group were observed, the brain tissue infarct size was detected by TTC, the structural changes of brain tissue were observed by HE staining, the ultrastructure of autophagosomes was observed by transmission electron microscopy, and the autophagy-related protein microtubule-associated protein light chain 3(LC3), Beclin1, p62, Atg5 expressionwas detected by Western blot method. Results: Compared with the sham group, the MCAO/R group had higher neurological deficit score(P<0.05), larger area of cerebral infarction(P<0.05), and serious pathological changes. The enzyme body was clearly visible,the expressions of LC3, Beclin1, Atg5 were increased(P<0.05), and the expression of P62 was decreased(P<0.05). Compared with the MCAO/R group, the neurological deficit scores of the TCM group, 3-MA group and TCM+3-MA group were decreased(P<0.05), the area of cerebral infarction was reduced(P<0.05), and the pathological state of brain tissue was improved. The nuclear and nuclear membranes were slightly fused, autophagosomes and autolysosomes were visible but few in number,the expressions of LC3, Beclin1 and Atg5 were decreased(P<0.05), and the expression of P62 was increased(P<0.05).Conclusion: Huazhuo Jiedu Huoxue Tongluo Prescription can improve cerebral ischemia-reperfusion injury, which may be related to inhibiting autophagy, up-regulating the expression of LC3, Beclin1 and Atg5 proteins, and down-regulating the level of P62 protein.