两种脾虚证大鼠肝脏线粒体自噬及细胞凋亡的比较研究

A Comparative Study of the Mitophagy and Apoptosis of Liver in Rats with Two Different Pi Deficiency Syndromes

目的比较脾气虚证和脾不统血证两种脾虚证模型大鼠肝脏线粒体自噬及细胞凋亡水平,探讨脾虚证发生发展过程中,肝脏细胞状态变化及可能的机制。方法32只SD大鼠随机分为4组:正常对照组(8只)、低分子肝素钠对照组(8只)、脾气虚组(8只)、脾不统血组(8只)。采用劳倦过度加饮食失节的方法复制脾气虚证大鼠模型,在脾气虚证的基础上采用低分子肝素钠诱导出血的方法构建脾不统血证大鼠模型;使用化学发光法检测大鼠肝组织三磷酸腺苷(ATP)含量;JC-1荧光探针法检测大鼠肝组织线粒体膜电位;Western Blot法检测各组大鼠肝组织线粒体自噬相关蛋白PINK1、Parkin、LC3Ⅱ/Ⅰ的表达水平和细胞凋亡相关蛋白Bcl-2、Bax、Cleaved caspase-3、线粒体外Cyt-C的表达水平;使用荧光定量法检测PINK1、Parkin、Bcl-2、Bax mRNA水平。结果与正常对照组及低分子肝素钠组比较,两种脾虚证大鼠肝组织线粒体膜电位,ATP含量,PINK1、Bcl-2蛋白及mRNA水平显著降低(P<0.05,P<0.01),线粒体外Cyt-C蛋白水平、Bax mRNA水平显著升高(P<0.01)。与正常对照组比较,两种脾虚证大鼠LC3Ⅱ/Ⅰ蛋白比值、Parkin蛋白水平显著降低(P<0.01)。与脾气虚证大鼠比较,脾不统血证大鼠肝组织线粒体膜电位、ATP含量降低(P<0.05,P<0.01),线粒体外Cyt-C蛋白水平、Bcl-2蛋白及mRNA水平下降(P<0.05,P<0.01),Cleaved caspase-3蛋白水平、Bax蛋白及mRNA水平上升(P<0.05)。结论脾虚证大鼠肝组织细胞凋亡可能机制为,线粒体过度损伤导致线粒体自噬紊乱,进一步导致了细胞凋亡,随着脾虚证的加重,肝组织细胞凋亡程度加深。

Objective To compare mitophagy and apoptosis levels in liver tissues of rats with Pi qi deficiency syndrome(PQDS)and Pi-qi failing to control blood syndrome(PQFCBS),and to explore the changes of liver cell state and its possible mechanism in the development of Pi deficiency syndrome(PDS).Methods Totally 32 SD rats were randomly divided into 4 groups,the normal control group,low molecule Heparin Sodium(LMHS)group,PQDS group,PQFCBS,8 in each group.PDS mice model was duplicated by the method of excessive labor and diet disorder.PQFCBS rat model was built by low molecule Heparin Sodium induced hemorrhage on the basis of PQDS.ATP content was detected by chemiluminescence,and mitochondrial membrane potential by JC-1 fluorescent probe method.The expression levels of mitophagy regulatory proteins(PINK1,Parkin,and LC3Ⅱ/Ⅰratio)and apoptosis related proteins(Bcl-2,Bax,Cleaved caspase-3,and cytoplasm Cyt-C)in hepatic tissue of rats were detected by Western Blot.The mRNA levels of PINK1,Parkin,Bcl-2,and Bax were detected by Real-time fluorescence quantitative PCR.Results The ATP content in the liver and liver mitochondrial membrane potential were significantly lower in PQDS and PQFCBS rats than in normal and LMHS rats(P<0.01).Compared with the normal group and LMHS group,the protein and mRNA levels of PINK1 and Bcl-2 in PQDS and PQFCBS significantly decreased(P<0.05,P<0.01),the protein level of Cyt-C in the cytoplasm without mitochondria and the mRNA level of Bax significantly increased(P<0.01).Compared with the normal group,the protein level of Parkin and LC3Ⅱ/Ⅰratio significantly decreased(P<0.01).Compared with PQDS group,liver mitochondrial membrane potential and ATP content decreased(P<0.05,P<0.01),the protein level of Cyt-C in the cytoplasm without mitochondria,protein and mRNA levels of Bcl-2 significantly decreased(P<0.05,P<0.01),protein levels of Cleaved caspase-3,protein and mRNA levels of Bax significantly increased(P<0.05)in the PQFCBS group.Conclusions The possible mechanism of apoptosis of liver cells in PDS rats might be as follows.Excessive damage of mitochondria caused mitophagy regulatory disorder,thus further leading to cell apoptosis.Along with the deterioration of PDS,apoptosis of liver cells became more serious.