去泛素化酶USP36通过调控线粒体裂变与融合减轻缺血再灌注损伤-肾纤维化

Deubiquitinase ubiquitin-specific protease 36 mitigates ischemia/reperfusion injury-induced renal fibrosis by regulating mitochondrial fission and fusion

目的探究去泛素化酶USP36在缺血再灌注-肾纤维化过程的作用及机制。方法建立体内外缺血再灌注损伤肾纤维化模型,利用蛋白免疫印迹检测USP36、线粒体融合蛋白2(MFN2)及纤维化相关蛋白表达,利用马松(Masson)染色和天狼猩红(Sirius Red)染色评估肾组织间质纤维化,透射扫描电镜观察肾组织中线粒体形态,Mito-Tracker染色观察线粒体裂变与融合,蛋白免疫共沉淀检测MFN2蛋白泛素化修饰。两组间差异比较采用t检验,多组间差异比较采用方差分析(ANOVA)。结果小鼠和细胞实验中USP36蛋白表达(1.001±0.125、0.994±0.113)均高于模型组(0.269±0.071、0.195±0.054),差异有统计学意义(t=11.42、14.23,P<0.01);动物实验对照组CollagenⅠ和α-SMA蛋白表达(1.012±0.070、1.008±0.067)低于模型组(2.899±0.187、2.265±0.209),差异有统计学意义(t=21.16、17.66,P<0.01);细胞实验对照组CollagenⅠ和α-SMA蛋白表达(1.039±0.081、0.997±0.096)低于模型组(3.067±0.311、2.619±0.116),差异有统计学意义(t=16.36、18.58,P<0.01);Massion染色和Sirious Red染色显示缺血再灌注损伤引起细胞外基质沉积;动物实验IRI+Vector组CollagenⅠ和α-SMA蛋白表达(3.689±0.460、2.257±0.197)高于IRI+OE-USP36组(1.889±0.314、1.525±0.103),差异有统计学意义(t=7.22、7.36,P<0.01);细胞实验H/R+Vector组CollagenⅠ和α-SMA蛋白表达(3.298±0.168、4.309±0.283)高于H/R+OE-USP36组(1.409±0.127、1.530±0.176),差异有统计学意义(t=20.06、18.67,P<0.01);过表达USP36减少缺血再灌注损伤引起的细胞外基质沉积;IRI+Vector组和H/R+Vector组MFN2蛋白(0.276±0.063、0.247±0.105)低于IRI+OE-USP36组和H/R+OE-USP36组MFN2蛋白表达(0.789±0.201、0.746±0.105),差异有统计学意义(t=8.45、9.27,P<0.01)。在细胞缺氧复氧过程,过表达USP36抑制肾小管上皮细胞线粒体裂变,促进线粒体融合;USP36通过去泛素化MFN2,抑制MFN2蛋白-酶体降解,从而促进线粒体融合。结论缺血再灌注损伤-肾纤维化过程去泛素化酶USP36通过去泛素化MFN2蛋白修饰,抑制MFN2蛋白-酶体降解从而稳定MFN2蛋白,促进线粒体融合,最终减轻肾纤维化。

Objective To investigate the role and mechanism of deubiquitinase ubiquitin-specific protease 36(USP36)during the process of ischemia/reperfusion-induced renal fibrosis.Methods A renal fibrosis model of ischemia-reperfusion injury(IRI)was established in vitro and in vivo.Western blotting was used to detect the expression of USP36,Mitofusin-2(MFN2),and fibrosis related proteins.Masson staining and Sirius Red staining were used to evaluate interstitial fibrosis in renal tissue.Mito Tracker staining was used to observe mitochondrial fission and fusion.Co-immunoprecipitation was used to detect ubiquitination modification of MFN2 protein.T-test was used to compare the differences between two groups,and analysis of variance(ANOVA)was used to compare the differences between multiple groups.Results The USP36 expression in the control group in vivo and in vitro(1.001±0.125,0.994±0.113)was higher than that in the model group(0.269±0.071,0.195±0.054),and the difference was statistically significant(t=11.42,14.23,P<0.01).The expression of CollagenⅠandα-SMA in the experimental animal control group(1.012±0.070,1.008±0.067)was lower than that in the model group(2.899±0.187,2.265±0.209),and the difference was statistically significant(t=21.16,17.66,P<0.01).The expression of CollagenⅠandα-SMA in the cell control group(1.039±0.081,0.997±0.096)was lower than that in the model group(3.067±0.311,2.619±0.116),and the difference was statistically significant(t=16.36,18.58,P<0.01).Massion and Sirious Red staining showed extracellular matrix deposition caused by IRI.The protein expression of CollagenⅠandα-SMA in the animal experiment IRI+Vector group(3.689±0.460,2.257±0.197)was higher than that in the IRI+OE-USP36 group(1.889±0.314,1.525±0.103),and the difference was statistically significant(t=7.22,7.36,P<0.01).The protein expression of CollagenⅠandα-SMA in the H/R+Vector group(3.298±0.168,4.309±0.283)were significantly lower than that in the H/R+OE-USP36 group(1.409±0.127,1.530±0.176,t=20.06,18.67,P<0.01).Overexpression of USP36 reduced extracellular matrix deposition induced by IRI.The expression of MFN2 protein in the IRI+Vector group and H/R+Vector group(0.276±0.063,0.247±0.105)was significantly lower than that in the IRI+OE-USP36 group and H/R+OE-USP36 group(0.789±0.201,0.746±0.105,t=8.45,9.27,P<0.01).In the process of hypoxia and reoxygenation,overexpression of USP36 inhibited mitochondrial fission and promoted mitochondrial fusion in renal tubular epithelial cells.USP36 promoted mitochondrial fusion by deubiquitinating MFN2 and inhibiting MFN2 protein-zymosomal degradation.Conclusion During IRI-induced renal fibrosis,deubiquitinating enzyme USP36 inhibits MFN2 protein-enzymic degradation through deubiquitinating MFN2 protein modification,thereby stabilizing MFN2 protein,promoting mitochondrial fusion,and ultimately alleviating renal fibrosis.