温阳复元方对脑缺血再灌注损伤大鼠JNK1/Bcl-2/Beclin 1信号通路相关蛋白及基因表达的影响

Effects of Wenyang Fuyuan Formula on JNK1/Bcl-2/Beclin 1 signaling pathway-related proteins and gene expression in rats with cerebral ischemia-reperfusion injury

目的:观察温阳复元方对脑缺血再灌注(I/R)损伤大鼠的影响,探讨其可能的作用机制。方法:65只大鼠随机分为假手术组,模型组,温阳复元方低、中、高剂量组,每组13只。制备大脑中动脉闭塞再灌注(MCAO/R)2 h模型,假手术组和模型组以0.9%氯化钠溶液灌胃,温阳复元方低、中、高剂量组分别给予温阳复元方1.5、3、6 mL灌胃,连续7 d。观察成模后各组大鼠TTC染色,比较神经功能缺损评分,HE、尼氏染色观察大鼠脑组织病理形态变化,免疫组化、qRT-PCR和Western Blot检测大鼠脑组织中JNK1、Beclin 1、Bcl-2、LC3B蛋白和mRNA的表达情况。结果:与假手术组比较,模型组大鼠的神经功能缺损评分显著上升(P<0.01),脑组织有明显的白色梗死灶,缺血侧脑组织细胞排列杂乱松散,神经元轮廓模糊,其胞质自溶及核碎裂,尼氏小体减少,脑组织JNK1、Beclin 1、Bcl-2、LC3B蛋白和mRNA表达显著升高(P<0.05,P<0.01);与模型组比较,温阳复元方中、高剂量组神经功能缺损评分显著下降(P<0.05),各给药组脑皮质损伤减轻,水肿减少,存活神经元数量增加;温阳复元方高剂量组JNK1、Beclin 1、Bcl-2、LC3B蛋白和mRNA表达显著降低(P<0.05,P<0.01),蛋白LC3BⅡ/LC3BⅠ比值显著降低(P<0.01)。结论:温阳复元方可能通过抑制JNK1/Bcl-2/Beclin 1信号通路的激活,降低脑缺血再灌注损伤后神经细胞的凋亡和过度自噬,从而起到脑保护作用。

Objective:To observe the effect of Wenyang Fuyuan Formula on cerebral ischemia-reperfusion(I/R)injury rats and to explore its possible mechanism of action.Methods:A total of 65 rats were randomly divided into sham-operated group,model group and low,medium and high dose groups of Wenyang Fuyuan Formula,with 13 rats in each group.A model of MCAO/R 2 h was prepared,the sham-operated and model groups were gavaged with saline,and low,medium and high dose groups of Wenyang Fuyuan Formula were given 1.5,3,6 mL Wenyang Fuyuan Formula for 7 d.TTC staining was observed in each group of rats after modeling to compare the scores of neurological deficits,HE and Nissl staining was observed to observe the pathological and morphological changes of rat brain tissues,and immunohistochemistry,qRT-PCR and Western Blot were used to detect the expression of JNK1,Beclin 1,Bcl-2,and LC3B proteins and mRNAs in the brain tissues of rats.middle cerebral artery occlusion.Results:Compared with the sham-operated group,the neurological deficit scores in the model group were significantly increased(P<0.01),and there were obvious white infarct foci in the brain tissue,were elevated(P<0.05,P<0.01).Compared with the model group,the neurological deficit scores were significantly decreased in the middle and high dose groups of Wenyang Fuyuan Formula(P<0.05),the brain cortical damage was reduced,edema was decreased and the number of surviving neurons was increased in the administered group;the expression of JNK1,Beclin 1,Bcl-2,LC3B protein and mRNA were significantly decreased in the high dose group of Wenyang Fuyuan Formula(P<0.05,P<0.01),and the protein LC3BIⅡ/LC3BⅠratio was significantly lower(P<0.01).Conclusion:Wenyang Fuyuan Formula may reduce apoptosis and excessive autophagy of neuronal cells after cerebral ischemiareperfusion injury by inhibiting the activation of JNK1/Bcl-2/Beclin 1 signaling pathway,thus playing a cerebral protective role.