补阳还五汤及主要成分对大脑中动脉阻塞再灌注大鼠脑组织细胞焦亡的影响

Effects of Buyang Huanwu Tang and its main components on pyroptosis in brain tissue of rats with middle cerebral artery occlusion and reperfusion

背景:细胞焦亡是脑缺血再灌注损伤的重要病理机制,补阳还五汤是中医临床治疗缺血性脑卒中的经典方剂,细胞焦亡可能是补阳还五汤治疗脑缺血再灌注损伤的有效作用靶点。目的:观察补阳还五汤对大脑中动脉阻塞再灌注大鼠脑组织细胞焦亡的影响及作用机制。方法:将48只SD大鼠随机分为假手术组、模型组、黄芪组及补阳还五汤组。除假手术组外,其余各组均进行大脑中动脉阻塞缺血2 h再灌注72 h处理,黄芪组和补阳还五汤组大鼠均于缺血2 h再灌注后开始连续灌胃相应体积的药物至缺血再灌注72 h,早晚各1次。Zea Longa评分观察大鼠神经功能缺损情况,TTC染色观察大鼠脑梗死体积,苏木精-伊红染色观察大鼠脑组织病理损伤变化,免疫荧光观察脑组织Tunel和Cleaved Caspase-1共表达以及接头蛋白ASC表达情况,免疫组化及Western blot检测大鼠脑组织焦亡相关蛋白表达。结果与结论:①与假手术组相比,模型组大鼠神经功能缺损评分显著升高(P<0.01),与模型组相比,补阳还五汤组及黄芪组大鼠神经功能缺损评分显著下降(P<0.01);②与模型组相比,黄芪组和补阳还五汤组脑梗死体积占比下降(P<0.01);③模型组脑组织神经细胞胞核固缩深染或溶解消失,细胞排列紊乱,与模型组比较,补阳还五汤组及黄芪组脑组织病理损伤较轻;④与假手术组相比,模型组大鼠脑组织Tunel与Cleaved Caspase-1双染色阳性细胞数及ASC免疫荧光表达强度显著增加,Cleaved Caspase-1、NLRP3、白细胞介素18和白细胞介素1β焦亡蛋白表达显著升高(P<0.01);与模型组相比,补阳还五汤组及黄芪组Cleaved Caspase-1与Tunel双染色阳性细胞数、ASC免疫荧光表达强度及Cleaved Caspase-1、NLRP3、白细胞介素18、白细胞介素1β焦亡蛋白表达均明显下降(P<0.01)。结果表明,补阳还五汤及其君药黄芪可有效缓解大脑中动脉阻塞再灌注大鼠脑组织损伤,其机制可能与抑制神经细胞焦亡有关。

BACKGROUND:Cellular pyroptosis is an important pathological mechanism of cerebral ischemia/reperfusion injury.Buyang Huanwu Tang is a classic formula for the clinical treatment of ischemic stroke in traditional Chinese medicine,and cellular pyroptosis may be an effective target of Buyang Huanwu Tang in the treatment of cerebral ischemia/reperfusion injury.OBJECTIVE:To observe the effect and mechanism of Buyang Huanwu Tang on pyroptosis in brain tissues of middle cerebral artery occlusion/reperfusion rats.METHODS:Forty-eight Sprague-Dawley rats were randomly divided into sham operation group,model group,Astragalus membranaceus group and Buyang Huanwu Tang group.Except for the sham operation group,all groups were subjected to middle cerebral artery occlusion for ischemia for 2 hours and reperfusion for 72 hours.The rats in the Astragalus membranaceus group and Buyang Huanwu Tang group were continuously gavaged with the corresponding volume of drugs until ischemia and reperfusion for 72 hours after awakening from the modeling,once in the morning and once in the evening.Zea Longa score was used to observe the neurological deficits of rats.TTC staining was performed to observe cerebral infarct size in rats.Hematoxylin-eosin staining was used to observe the pathological changes of the brain tissue.Immunofluorescence was used to observe the co-expression of Tunel and Cleaved-Caspase-1 in the brain tissue and the expression of the junction protein ASC.Immunohistochemistry and western blot were used to detect the expression of pyroptosis-related proteins in rat brain tissues.RESULTS AND CONCLUSION:(1)Compared with the sham operation group,the neurological deficit score of rats was significantly higher in the model group(P<0.01),and compared with the model group,the neurological deficit score of rats was significantly lower in the Buyang Huanwu Tang group and the Astragalus membranaceus group(P<0.01).(2)Compared with the model group,the volume ratio of cerebral infarction was lower in the Astragalus membranaceus group and Buyang Huanwu Tang group(P<0.01).(3)In the model group,the nuclei of neuronal cells in the brain tissue were deeply stained or lysed,and arrangement of the cells was disorganized.Compared with the model group,the pathologic damage of the brain was less severe in the Buyang Huanwu Tang group and the Astragalus membranaceus group.(4)Compared with the sham operation group,the number of Tunel and Cleaved-Caspase-1 double-positive cells and immunofluorescence intensity of ASC in the brain tissue was significantly increased in the model group,and the expression of Cleaved-Caspase-1,NLRP3,interleukin 18,and interleukin 1βwas significantly elevated in the model group(P<0.01).Compared with the model group,the number of Cleaved-Caspase-1 and Tunel double-positive cells,immunofluorescence intensity of ASC,and the expression of Cleaved-Caspase-1,NLRP3,interleukin 18,and interleukin 1βwere all significantly decreased in the Buyang Huanwu Tang group and the Astragalus membranaceus group(P<0.01).The results indicate that Buyang Huanwu Tang and its monarch drug Astragalus membranaceus can effectively alleviate brain tissue injury in rats with middle cerebral artery occlusion and reperfusion,and its mechanism may be related to the inhibition of neuronal cell pyroptosis.