加味涤痰汤调控miR-149对慢性间歇低氧大鼠肝损伤的影响

Effects of Modified Ditan Decoction in Regulating miR-149 on Liver Injury in Rats with Chronic Intermittent Hypoxia

目的探讨加味涤痰汤调控mi R-149对慢性间歇低氧大鼠肝损伤的影响及作用机制。方法将18只雄性SD大鼠随机分为对照组、模型组和加味涤痰汤组,每组6只。模型组和加味涤痰汤组于低氧箱中造模,造模期间加味涤痰汤组予加味涤痰汤灌胃,连续12周。HE染色观察肝组织病理变化,透射电镜观察肝组织超微结构,TUNEL染色观察肝细胞凋亡情况,检测肝组织丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、超氧化物歧化酶(SOD)、丙二醛(MDA)含量,RT-qPCR和Western blot检测肝组织miR-149、转录激活因子(ATF)6、葡萄糖调节蛋白-78(GRP78)、C/EBP同源蛋白(CHOP)mRNA和蛋白表达。分析肝组织ATF6 mRNA与miR-149 mRNA表达相关性。结果与对照组比较,模型组大鼠肝组织少量炎性细胞浸润,肝细胞排列不规则、不同程度水肿,内质网扩张,核糖体脱落,线粒体膜破裂,肝细胞凋亡率升高(P<0.05),肝组织ALT、AST、MDA含量升高(P<0.05),SOD含量降低(P<0.05),miR-149 mRNA表达降低(P<0.05),ATF6、GRP78、CHOP mRNA和蛋白表达升高(P<0.05);与模型组比较,加味涤痰汤组大鼠肝组织未见明显水肿及炎性细胞浸润,肝细胞排列规则,内质网稍扩张,核糖体分布较均匀,线粒体膜相对完整,肝细胞凋亡率降低(P<0.05),肝组织ALT、AST、MDA含量降低(P<0.05),SOD含量升高(P<0.05),miR-149m RNA表达升高(P<0.05),ATF6、GRP78、CHOPmRNA和蛋白表达降低(P<0.05)。ATF6 mRNA与miR-149 mRNA水平呈显著负相关(r=-0.766)。结论加味涤痰汤可能通过调控miR-149/ATF6通路抑制慢性间歇低氧大鼠肝脏氧化应激反应,改善肝损伤。

Objective To investigate the effects and mechanism of modified Ditan Decoction in regulating miR-149 on liver injury in rats with chronic intermittent hypoxia.Methods Totally 18 male SD rats were randomly divided into control group,model group,and modified Ditan Decoction,with 6 rats in each group.The model group and the modified Ditan Decoction group were modeled in a low oxygen chamber.During the modeling period,the modified Ditan Decoction group was given gavage of the modified Ditan Decoction for 12 consecutive weeks.HE staining was used to observe the pathological changes of liver tissue;the ultrastructural changes of liver tissue were observed by transmission electron microscopy,TUNEL staining was used to observe hepatocyte apoptosis,the contents of ALT,AST,SOD and MDA in liver tissue were detected,the mRNA and protein expression of miR-149,ATF6,GRP78 and CHOP in liver tissue were detected by RT-qPCR and Western blot.The correlation between the mRNA transcription levels of miR-149 and ATF6 was analyzed.Results Compared with the control group,a small amount of inflammatory cell infiltration in liver tissue of the model group,with irregular arrangement of liver cells,varying degrees of edema,expansion of endoplasmic reticulum,ribosome shedding,mitochondrial membrane rupture,and apoptosis rate of liver cells increased(P<0.05),the contents of ALT,AST and MDA significantly increased(P<0.05),while the content of SOD significantly decreased(P<0.05),the expression of miR-149 mRNA in liver tissue decreased(P<0.05),while the mRNA and protein expression of ATF6,GRP78 and CHOP increased(P<0.05).Compared with the model group,no obvious edema or inflammatory cell infiltration was observed in liver tissue of rats in the modified Ditan Decoction group,the liver cells were arranged in a regular manner,with slightly expanded endoplasmic reticulum and more uniform distribution of ribosomes,the mitochondrial membrane was relatively intact,and the apoptosis rate of liver cells decreased(P<0.05),the contents of ALT,AST and MDA decreased(P<0.05),while the content of SOD increased(P<0.05),the expression of miR-149 mRNA in liver tissue significantly increased(P<0.05),while the mRNA and protein expression of ATF6,GRP78 and CHOP significantly decreased(P<0.05).The level of miR-149 mRNA was significantly negatively correlated with that of ATF6 mRNA(r=-0.766).Conclusion Modified Ditan Decoction may inhibit liver oxidative stress response in chronic intermittent hypoxia rats and improve liver injury by regulating the miR-149/ATF6 pathway.