化瘀解毒方对缺血性中风瘀毒互结证模型大鼠脑细胞自噬及血脑屏障通透性的影响

Effects of Huayu Jiedu Formula(化瘀解毒方)on Brain Cell Autophagy and Blood Brain Barrier Permeability in Ischemic Stroke Model Rats with Binding of Stasis and Toxin Syndrome

目的探讨化瘀解毒方预防缺血性中风瘀毒互结证的可能作用机制。方法60只雄性SD大鼠随机分为假手术组、模型组及化瘀解毒方低、高剂量组,每组15只。化瘀解毒方低、高剂量组大鼠分别给予9.5、28.5 g/(kg·d)化瘀解毒方灌胃,假手术组及模型组大鼠给予3 ml生理盐水灌胃,均连续7天。除假手术组外,其余各组大鼠均于灌胃第4天开始给予角叉菜胶腹腔注射,连续3天,灌胃第7天给予大鼠1次背部皮下注射干酵母悬液。末次灌胃24 h后采用大脑中动脉闭塞(MCAO)线栓法制备缺血性中风瘀毒互结证病证结合模型。造模24 h后进行神经功能缺损评分;各组大鼠随机选取5只大鼠采用TTC染色法观察脑组织梗死率;伊文思蓝(EB)渗透法检测脑组织血脑屏障(BBB)通透性;Western blot法检测脑组织自噬关键分子酵母Atg6同系物1(Beclin1)、微管相关蛋白1轻链3(LC3)、选择性自噬接头蛋白(p62)和咬合蛋白(Occludin)蛋白表达。结果与假手术组比较,模型组大鼠脑梗死率和神经功能缺损评分增加,脑组织Beclin1蛋白表达和LC3Ⅱ/LC3Ⅰ值增加,p62、Occludin蛋白表达降低,EB含量上升(P<0.05或P<0.01)。与模型组比较,化瘀解毒方低剂量组脑梗死率、神经功能缺损评分、LC3Ⅱ/LC3Ⅰ值降低,Occludin蛋白表达上升,化瘀解毒方高剂量组脑梗死率、神经功能缺损评分、LC3Ⅱ/LC3Ⅰ值、EB含量、Beclin1蛋白表达下降,p62蛋白和Occludin蛋白表达增加(P<0.05或P<0.01)。与化瘀解毒方低剂量组比较,化瘀解毒方高剂量组大鼠脑梗死率、EB含量、Beclin1蛋白表达、LC3Ⅱ/LC3Ⅰ值显著降低(P<0.05或P<0.01)。结论化瘀解毒方可明显预防缺血性中风瘀毒互结证脑损伤,以高剂量较佳,其作用机制可能是抑制脑细胞自噬,从而保护BBB功能。

Objective To explore the possible mechanism of Huayu jiedu Formula(化瘀解毒方,HJF)in preventing ischemic stroke(IS)with binding of stasis and toxin syndrome.Methods Sixty male SD rats were randomly divided into sham operation group,model group,and the low-and high-dose HJF group,with 15 rats in each group.The rats in the low-and high-dose HJF groups were given 9.5 g/(kg·d)and 28.5 g/(kg·d)of HJF by gavage,respectively,while those in the sham operation group and the model group were given 3 ml of normal saline by gavage,all for 7 consecutive days.Except for the sham operation group,rats were given intraperitoneal injection of carrageenan from the 4 th day of gavage for 3 consecutive days,and were then given subcutaneous injection of dry yeast suspension to the back on the 7 th day of gavage.Twenty-four hours after the last gavage,middle cerebral artery occlusion(MCAO)suture method was used to develop the disease-syndrome combined model of IS with binding of stasis and toxin syndrome.Neurological deficit score was assessed 24 hours after modeling.TTC staining method was used to observe the infarction rate of brain tissue in five randomly selected rats in each group.Evans blue(EB)penetration method was used to detect the permeability of blood brain barrier(BBB)in brain tissue.Western blotting was used to detect the protein expressions of autophagy key molecule yeast Atg6 homolog 1(Beclinl),microtubule-associated protein 1 light chain 3(LC3),selective autophagy adaptor protein(p62)and Occludin in brain tissue.Results Compared to the sham operation group,the model group had increased cerebral infarction rate and neurological deficit score,higher Beclin 1 protein expression and LC3Ⅱ/LC3Ⅰvalue,lower p62 and Occludin protein expression,and higher EB content(P<0.05 or P<0.01).Compared to the model group,the low-dose HJF group had decreased cerebral infarction rate,neurological deficit score and LC3Ⅱ/LC3Ⅰvalue as well as increased Occludin protein expression,while the high-dose HJF group had decreased cerebral infarction rate,neurological deficit score,LC3Ⅱ/LC3Ⅰvalue,EB content and Beclinl protein expression,and increased p62 and Occludin protein expression(P<0.05 or P<0.01).Compared to the low-dose HJF group,the high-dose group had lower cerebral infarction rate,EB content,Beclinl protein expression,and LC3Ⅱ/LC3Ⅰvalue(P<0.05 or P<0.01).Conclusion HJF can effectively prevent the brain damage of IS rats with binding of stasis and toxin syndrome,and high dose is associated with better effect.Its mechanism may be related to the inhibition of the brain cells autophagy,thereby protecting the BBB function.