过表达Ubc9酶对乳大鼠心肌细胞低氧损伤的保护作用

Protective effect of overexpression of Ubc9 enzyme against hypoxic injury to neonatal rat cardiomyocytes

目的探讨类泛素样修饰蛋白(SUMO)修饰中的泛素缀合酶类E2I(Ubc9)对体外乳大鼠心肌细胞(NRCM)急性低氧损伤的作用及其可能的机制。方法采用分离纯化细胞法培养NRCM,对NRCM分别转染5,10,20,50和100感染指数(moi)的纯化的大鼠Ubc9基因腺病毒(Adv-Ubc9),转染48 h后在荧光显微镜下分别观察其绿色荧光蛋白(GFP)的荧光强度,并用Western印迹法检测转染效率。NRCM行氧葡萄糖剥夺(OGD)分别处理0,2,4,6,12和24 h后,Western印迹法检测Ubc9、活化的胱天蛋白酶3、自噬相关蛋白P62/SQSTM1(P62)和微管相关蛋白轻链3(LC3)的表达。NRCM转染Adv-Ubc9 50 moi 48 h,使得Ubc9过表达,随后OGD处理6 h,建立心肌细胞急性低氧模型,利用原位末端转移酶标记(TUNEL)技术检测细胞凋亡,Western印迹法检测Ubc9、活化的胱天蛋白酶3、P62、LC3和类泛素化修饰蛋白1(SUMO-1)的表达水平,蛋白免疫共沉淀法检测Vps34和Beclin1的SUMO化修饰水平。NRCM转染Ubc9的小干扰RNA(Ubc9-siRNA)48 h,使Ubc9沉默,随后行OGD处理6 h,Western印迹法检测活化的胱天蛋白酶3的表达水平。NRCM给予巴伐洛霉素A1(BFA1)50 nmol·L^(-1)作用2 h,Western印迹法检测LC3表达水平。结果Adv-Ubc9在50与100 moi时转染成功且效率一致,与单纯OGD组相比,过表达Ubc9能明显降低OGD引起的心肌细胞凋亡(P<0.01)、降低活化的胱天蛋白酶3的表达(P<0.01),而沉默Ubc9使心肌细胞OGD处理后的活化的胱天蛋白酶3表达上调(P<0.05),说明过表达Ubc9能抵抗OGD引起的心肌细胞凋亡。与正常对照组相比,OGD处理后,自噬底物P62发生堆积(P<0.01),自噬蛋白LC3Ⅱ明显下调(P<0.05),自噬流出现障碍;当过表达Ubc9后能明显逆转OGD引起的自噬底物P62堆积(P<0.05),改善自噬流障碍,但对自噬蛋白LC3Ⅱ影响不明显。在此基础上给予BFA1阻断自噬体与溶酶体融合过程后,与OGD+BFA1组相比,Adv-Ubc9+OGD+BFA1组的LC3Ⅱ表达明显上调(P<0.01),提示Ubc9改善自噬流障碍的作用可能与同时促进自噬激活和自噬降解过程有关;蛋白免疫共沉淀结果显示,与正常对照组相比,OGD处理后SUMO-1蛋白表达水平基本无变化,而过表达Ubc9后,SUMO-1蛋白表达明显上调(P<0.01),OGD处理后,Vps34和Beclin1的SUMO化修饰水平下调(P<0.01),而过表达Ubc9后,Vps34和Beclin1的SUMO化修饰明显上调(P<0.01),说明过表达Ubc9可能通过提高与自噬激活和自噬降解过程均相关的Vps34和Beclin1的SUMO化修饰,从而同时促进自噬激活和自噬降解过程改善OGD后的自噬流障碍,进而抵抗OGD引起的心肌细胞凋亡。结论高表达Ubc9酶能显著抑制OGD导致的心肌细胞凋亡,该保护作用可能是增强了Vps34和Beclin1的SUMO化修饰水平,从而同时促进自噬激活(形成自噬体)和自噬降解(自噬体与溶酶体融合)过程,进而改善OGD后自噬流障碍。

OBJECTIVE To investigate the effect of the small ubiquitin-like modifier(SUMOylation)ubiquitin-conjugating enzyme E21(Ubc9) in neonatal rat cardiomyocytes(NRCMs) that have suffered acute hypoxia injury and the possible mechanisms involved in its action. METHODS NRCMs were transfected with 5, 10, 20, 50 and 100 multiplicity of infection(moi) adenovirus harboring Ubc9(Adv-Ubc9)for 48 h, respectively, while the fluorescence intensity of green fluorescence protein(GFP) was observed by fluorescence microscope, and the transfection efficiency was detected by Western blotting. An acute myocardial hypoxia model was established in NRCMs by treating oxygen and glucose deprivation(OGD), protein Ubc9, cleaved-caspase 3, P62/SQSTM1(P62) and microtubules associated protein 1light chain 3(LC3). The rate of transfection was detected by Western blotting at different OGD time points. NRCMs were treated with OGD for 6 h after being transfected with Adv-Ubc9 50 moi to achieve the overexpression of Ubc9 for 48 h. Cell apoptosis was detected by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling assay(TUNEL assay). And the changes of protein P62, LC3,cleaved-caspase 3 and SUMO-1 were detected by Western blotting. SUMOylated Vps34 and SUMOylated Beclin1 were detected by co-immunoprecipitation assay. NRCMs were treated with OGD 6 h after transfection with Ubc9-small interfering RNA(Ubc9-siRNA) to silence Ubc9 for 48 h. Also, cleaved-caspase 3 was detected by Western blotting. NRCMs were treated with bafilomycin A1(BFA1) 50 nmol·L^-1for 2 h to inhibit the process of autolysosome fusion. LC3 was detected by Western blotting. RESULTS Adv-Ubc9 was successfully transfected at 50 and 100 moi. And the efficiency was consistent. Overexpression of Ubc9 could significantly reduce OGD-induced cardiomyocyte apoptosis(P<0.01) and decrease cleaved-caspase 3 expression(P<0.01), while silencing Ubc9 increased the apoptosis of cardiomyocytes induced by OGD, and the expression of cleaved-caspase 3 was up-regulated(P<0.05)compared with OGD group, indicating that overexpression of Ubc9 could resist the apoptosis of cardiomyocytes induced by OGD. Mechanically, the autophagy substrate P62 was accumulated(P<0.01),and LC3 Ⅱ was down-regulated(P<0.05) after OGD, indicating the autophagic flow was impaired.When Ubc9 was overexpressed, the accumulation of autophagy substrate P62 was decreased by OGD(P<0.05), but had no obvious effect on LC3Ⅱ. On this basis, when treated with BFA1, Adv-Ubc9 group induced a higher level of LC3Ⅱ(P<0.01) than Adv-GFP group under OGD, suggesting that Ubc9 may improve autophagic flow by promoting autophagy activation and autophagy degradation process. The level of SUMO-1 protein was not changed after OGD, but it was up-regulated after overexpression of Ubc9(P<0.01). SUMOylated Vps34 and SUMOylated Beclin1 were down-regulated(P<0.01) after OGD, and they were up-regulated(P<0.01) after overexpression of Ubc9 by co-immunoprecipitation.This suggested that Ubc9 overexpression may improve the SUMO modification of Vps34 and Beclin1,which was related to autophagy activation and autophagy degradation to improve autophagic flow after OGD. CONCLUSION Overexpression of Ubc9 could significantly inhibit the apoptosis of cardiomyocytes induced by OGD. The protective effect may be to promote autophagy activation and autophagy degradation processes after OGD by enhancing the SUMO modification level of Vps34 and Beclin1,which are the core components of the class Ⅲ phosphatidylinositol 3-kinase complex of autophagy.