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目的明确E3泛素连接酶CHIP在血管紧张素Ⅱ(AngⅡ)引起心肌纤维化过程中的作用以及分子机制。方法用10周龄野生型小鼠(WT)和CHIP心脏特异表达转基因小鼠(CHIP-TG)各16只,随机分为生理盐水+WT组、生理盐水+CHIP-TG组、AngⅡ+WT组和AngⅡ+CHIP-TG组。采用植入式胶囊渗透压泵对小鼠灌注生理盐水和AngⅡ[1500 ng/(kg.min)]1周,然后用B超仪检测动物心脏功能;心脏组织切片进行HE、Masson染色分别观察心脏组织中炎性细胞浸润和胶原沉积情况。应用免疫组织化学染色检测不同组别心脏中巨噬细胞(Mac-2阳性细胞)数量和CollagenⅠ的表达水平。另外,用siRNA-GFP和siRNA-CHIP腺病毒感染培养的胎鼠心肌细胞24 h,然后用AngⅡ(100 nmol/L)处理6 h,应用RT-PCR检测不同处理组炎症因子[如白细胞介素1β(IL-1β)和IL-6]的表达水平。结果在生理盐水处理时,WT和CHIP-TG组心脏功能、病理改变和炎性细胞浸润情况均无明显差别。AngⅡ处理7天后,与生理盐水+WT组相比,AngⅡ+WT组的心脏功能、纤维化面积和炎性细胞的浸润程度均明显增加(P<0.05)。与AngⅡ+WT组相比,AngⅡ+CHIP-TG组小鼠的心脏功能明显增强(心脏B超:FS为81.18%±4.09%比72.25%±3.78%;EF为48.57%±4.66%比40.27%±3.02%),心脏组织中纤维化面积(Masson染色)和CollagenⅠ表达水平明显减少,心脏组织中巨噬细胞浸润(Mac-2染色)明显减少。胎鼠心肌细胞经AngⅡ处理后,与siRNA-GFP对照组相比,siRNA-CHIP感染后可明显上调炎症因子(如IL-1β和IL-6)的表达。结论 CHIP过表达抑制AngⅡ引起的炎症反应和心肌纤维化。
Objective To clarify the role and molecular mechanism of EIP ubiquitin ligase CHIP in myocardial fibrosis induced by angiotensin Ⅱ (Ang Ⅱ). Methods Twenty-six mice of WT (10 weeks old) and CHIP-TG (CHIP-TG) were randomly divided into saline + WT group, saline + CHIP-TG group and AngⅡ + WT group And AngⅡ + CHIP-TG group. The rats were perfused with saline and AngⅡ [1500 ng / (kg · min)] for 1 week using an implanted capsule osmotic pressure pump. The heart function of the animals was measured by B-mode ultrasonography. HE and Masson staining were used to observe the heart Inflammatory cell infiltration and collagen deposition in tissues. Immunohistochemical staining was used to detect the number of macrophages (Mac-2 positive cells) and the expression of Collagen I in different groups of hearts. In addition, the cultured fetal rat cardiomyocytes were infected with siRNA-GFP and siRNA-CHIP adenovirus for 24 h and then treated with AngⅡ (100 nmol / L) for 6 h. RT-PCR was used to detect the expression of inflammatory cytokines 1β (IL-1β) and IL-6]. Results There was no significant difference in cardiac function, pathological changes and infiltration of inflammatory cells in WT and CHIP-TG groups when treated with saline. After AngⅡ treatment for 7 days, the cardiac function, the area of fibrosis and the infiltration of inflammatory cells in AngⅡ + WT group were significantly increased compared with saline + WT group (P <0.05). Compared with AngⅡ + WT group, the cardiac function of AngⅡ + CHIP-TG group was significantly enhanced (B ultrasound: FS was 81.18% ± 4.09% vs 72.25% ± 3.78%; EF was 48.57% ± 4.66% vs 40.27% ± 3.02%). The area of fibrosis (Masson staining) and Collagen Ⅰ expression in heart tissue were significantly decreased, while the macrophage infiltration (Mac-2 staining) in heart tissue was significantly decreased. Compared with siRNA-GFP control group, the expression of inflammatory factors (such as IL-1β and IL-6) was significantly up-regulated after siRNA-CHIP infection in fetal rat cardiomyocytes treated with AngⅡ. Conclusion Overexpression of CHIP can inhibit Ang Ⅱ-induced inflammation and myocardial fibrosis.