加味涤痰汤对慢性间歇缺氧模型大鼠心肌损伤及miR-200a/Nrf2信号通路的影响

Effect of Jiawei Ditan Decoction on myocardial injury and miR-200aNrf2 signal pathway in rats with chronic intermittent hypoxia

目的基于MiRNA-200a/Nrf2信号轴探讨加味涤痰汤对慢性间歇缺氧大鼠心肌内质网应激和凋亡的影响。方法雄性SD大鼠,随机分为常氧+加味涤痰汤组、常氧对照组、间歇缺氧+加味涤痰汤组、间歇缺氧组,每组10只。间歇缺氧+加味涤痰汤组、间歇缺氧组置于低氧舱中制备间歇缺氧模型。常氧+加味涤痰汤组、间歇缺氧组+加味涤痰汤组每天造模前给予加味涤痰汤干预,常氧对照组给予生理盐水灌胃,实验干预12周。HE染色观察左心室肌结构;TUNEL染色检测细胞凋亡;Q-PCR检测miRNA-200a、Caspase-3、Bax、Bcl-2、Keap1、Nrf2的mRNA的表达;Western blotting检测内质网应激相关蛋白Atf6、p-Perk、p-Ire1、Chop、Grp78,凋亡相关蛋白Bax、Caspase-3、Bcl-2,Keap1、Nrf2的表达水平。结果间歇缺氧组与常氧对照组相比大鼠心肌损伤明显,细胞界限不均,心肌纤维断裂,微血管扩张,并且出现淋巴细胞浸润,心肌细胞轻微水肿;TUNEL染色显示间歇缺氧组心肌凋亡增加,差异具有统计学意义(P<0.01);Bax/Bcl-2比率上调,Chop、Grp78、Caspase-3、Keap1的表达水平升高,Nrf2表达降低,Atf6、p-Perk、p-Ire1表达升高,差异具有统计学意义(P<0.05)。间歇缺氧+加味涤痰汤组与间歇缺氧组相比,心肌细胞结构有所改善,凋亡下降,Bax/Bcl-2比率下调,Chop、Grp78、Caspase-3、Keap1的表达水平降低,Nrf2表达升高,Atf6、p-Perk、p-Ire1表达降低,差异具有统计学意义(P<0.05)。常氧对照+加味涤痰汤组与常氧对照组相比无明显差异(P>0.05)。结论加味涤痰汤可能通过调节MiRNA-200a/Nrf2信号轴减轻内质网应激和细胞凋亡,从而对CIH大鼠心肌损伤起到一定的保护作用。

Objective To investigate the effects of Jiawei Ditan Decoction on myocardial endoplasmic reticulum stress and apoptosis in rats with chronic intermittent hypoxia based on MiRNA-200a/Nrf2 signal axis.Methods Male SD rats were randomly divided into normoxia+Jiawei Ditan decoction group,normoxia control group,intermittent hypoxia+Jiawei Ditan Decoction group and intermittent hypoxia group,with 10 rats in each group.Intermittent hypoxia+Jiawei Diitan Decoction group and intermittent hypoxia group were placed in hypoxic chamber to prepare intermittent hypoxia model.The normoxia+Jiawei Ditan decoction group and the intermittent hypoxia+Jiawei Ditan Decoction group were given Jiawei Ditan decoction intervention every day before modeling,and the normoxia control group was given normal saline gavage for 12 weeks.HE staining was used to observe the structure of left ventricular muscle.TUNEL staining was used to detect cell apoptosis.The mRNA expressions of miRNA-200a,Caspase3,Bax,Bcl-2,Keap1 and Nrf2 were detected by Q-PCR.Western blotting was used to detect the expression levels of endoplasmic reticulum stress-related proteins Atf6,p-Perk,p-Ire1,Chop and Grp78,and apoptosis-related proteins Bax,Caspase3,Bcl-2,Keap1 and Nrf2.Results Compared with the normoxia control group,the intermittent hypoxia group had obvious myocardial injury,uneven cell boundary,myocardial fiber breakage,microvascular dilatation,lymphocytic infiltration and slight myocardial cell edema.TUNEL staining showed that myocardial apoptosis was increased in intermittent hypoxia group(P<0.01).The Bax/Bcl-2 ratio was up-regulated,the expression levels of Chop,Grp78,Caspase3 and Keap1 were increased,the expression of Nrf2 was decreased,and the expression levels of Atf6,P-PERK and P-IRE1 were increased,and the differences were statistically significant(P<0.05).Compared with the intermittent hypoxia group,the myocardial cell structure was improved,apoptosis was decreased,Bax/Bcl-2 ratio was down-regulated,the expression levels of Chop,Grp78,Caspase3 and Keap1 were decreased,the expression of Nrf2 was increased,and the expression of Atf6,p-Perk and p-Ire1 was decreased in the intermittent hypoxia+Jiawei Ditan decoction group.There were significant differences between the two groups(P<0.05).There was no significant difference between the normoxia control group and the normoxia control group(P>0.05).Conclusion Jiawei Ditan decoction may reduce ER stress and apoptosis by regulating MiRNA-200a/Nrf2 signaling axis,which may play a protective role in myocardial injury of CIH rats.