摘要
目的:探讨含三基序蛋白8(TRIM8)对高糖高脂(HGHF)诱导的小鼠心肌细胞(MCMs)凋亡的影响及机制。方法:将MCMs分为正常糖(NG)组(葡萄糖浓度为5.5 mmol/L)、高糖(HG)组(葡萄糖浓度为33 mmol/L)、高脂(HF)组(软脂酸钠浓度为300μmol/L)和HGHF组(葡萄糖浓度为33 mmol/L,软脂酸钠浓度为300μmol/L);用siRNA沉默MCMs中TRIM8的表达后,再将MCMs分为对照(control)组(仅给予转染试剂)、Scra-siRNA/PBS组(转染scrambled siRNA)、TRIM8-siRNA/PBS组(转染TRIM8特异性siRNA)、Scra-siRNA/HGHF组和TRIM8-siRNA/HGHF组;为探索TRIM8在HGHF诱导的MCMs损伤中的可能作用机制,将MCMs分为HGHF/DMSO组、HGHF+TRIM8-siRNA+DMSO(HGHF+Ts/DMSO)组、HGHF/ML385组和HGHF+TRIM8-siRNA+ML385(HGHF+Ts/ML385)组。采用流式细胞术检测心肌细胞凋亡率;流式细胞术及DHE染色法检测心肌细胞活性氧簇(ROS)的水平;qPCR及Western blot检测TRIM8、核因子E2相关因子2(Nrf2)、谷氨酸-半胱氨酸连接酶催化亚基(GCLC)、血红素加氧酶1(HO-1)和NAD(P)H:醌氧化还原酶1(NQO-1)的表达水平。结果:HGHF可增加MCMs中TRIM8的表达,同时减少Nrf2及其下游基因GCLC、HO-1和NQO-1的表达(P<0.05)。进一步研究发现,与Scra-siRNA/HGHF组相比,TRIM8-siRNA/HGHF组ROS含量和MCMs凋亡率降低(P<0.05),相应的,抗氧化分子Nrf2及其下游基因GCLC、HO-1和NQO-1的表达增高。与此相反,在此基础上加用Nrf2抑制剂ML385能够部分逆转下调TRIM8对HGHF诱导MCMs凋亡的抑制作用(P<0.05)。结论:TRIM8能够通过调节Nrf2抗氧化途径加剧HGHF诱导的小鼠心肌细胞凋亡。
AIM:To investigate the effects of tripartite motif-containing protein 8(TRIM8)on the apoptosis of mouse cardiomyocytes(MCMs)induced by high glucose and high free fatty acid(HGHF)and the underlying mechanism.METHODS:The MCMs were divided into normal glucose(NG)group(glucose at 5.5 mmol/L),high glucose(HG)group(glucose at 33 mmol/L),high free fatty acid(HF)group(sodium palmitate at 300μmol/L)and HGHF group(glucose at 33 mmol/L and sodium palmitate at 300μmol/L).The expression of TRIM8 in the MCMs was knocked down by siRNA,and the MCMs was further divided into control group,scrambled siRNA(Scra-siRNA)/PBS group,TRIM8-siRNA/PBS group,Scra-siRNA/HGHF group and TRIM8-siRNA/HGHF group.To further confirm the specific mechanism of TRIM8 in the MCM injury induced by HGHF,the MCMs were subgrouped into HGHF/DMSO group,HGHF+TRIM8-siRNA+DMSO(HGHF+Ts/DMSO)group,HGHF/ML385 group and HGHF+Ts/ML385 group.Accordingly,apoptosis was analyzed by flow cytometry,and the levels of reactive oxygen species(ROS)were measured by flow cytometry and DHE staining.The expression of TRIM8,nuclear factor E2-related factor 2(Nrf2),glutamate-cysteine ligase catalytic subunit(GCLC),heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO-1)at mRNA and protein levels was determined by qPCR and Western blot.RESULTS:HGHF increased the expression of TRIM8,and suppressed the expression of Nrf2,GCLC,HO-1 and NQO-1 in the MCMs(P<0.05).Compared with Scra-siRNA/HGHF group,the intracellular ROS content and apoptotic rate were decreased in TRIM8-siRNA/HGHF group(P<0.05).Correspondingly,the expression of the antioxidant molecule Nrf2 and its downstream genes GCLC,HO-1 and NQO-1 was increased(P<0.05).In contrast,the addition of Nrf2 inhibitor ML385 partially reversed the inhibitory effect of TRIM8 expression knock-down on HGHF-induced apoptosis of MCMs.CONCLUSION:TRIM8 exacerbates the HGHF-induced cardiomyocyte apoptosis by modulating Nrf2 antioxidative pathway.
作者
唐名扬
宋志平
侯娟妮
冯健
冯娟
裴海峰
杨永健
TANG Ming-yang;SONG Zhi-ping;HOU Juan-ni;FENG Jian;FENG Juan;PEI Hai-Feng;YANG Yong-jian(North Sichuan Medical College,Nanchong 637000,China;Departmentof Cardiovascular Medicine,The General Hospital of Western Theater Command,Chengdu 610083,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2020年第1期29-37,共9页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81670419
No.81873477)
四川省杰出青年基金资助项目(No.2017JQ0012)
关键词
糖尿病心肌病
高糖高脂
含三基序蛋白8
氧化应激
细胞凋亡
核因子E2相关因子2
Diabetic cardiomyopathy
High glucose and high free fatty acid
Tripartite motif-containing protein 8
Oxidative stress
Apoptosis
Nuclear factor E2-related factor 2
