泛素E3连接酶CHIP抑制血管紧张素Ⅱ诱导心脏重塑的作用机制

发布时间：2018-08-09 14:35

【摘要】：研究背景和目的：热休克蛋白70羰基末端反应蛋白(CHIP)是一个分子伴侣蛋白和含有U-盒子(U-box)的泛素E3连接酶,可促进多种蛋白底物被泛素-蛋白酶体系统降解和清除,在心肌细胞损伤、细胞凋亡、肿瘤、热应急等方面起着重要的作用。但是,CHIP在Ang Ⅱ诱导的心肌纤维化、凋亡和炎症反应中的作用及机制尚不清楚。因此,本研究主要探讨了CHIP在Ang Ⅱ诱导心肌重塑中的作用及机制。方法：用10周龄野生型小鼠(WT)和心脏特异性过表达CHIP的转基因小鼠(CHIP-TG)各16只,随机分为4组,每组8只：(1)生理盐水(NS)+WT组；(2)NS+CHIP-TG组；(3) Ang Ⅱ+WT组；(4) Ang Ⅱ+CHIP-TG组。采用植入式微量缓释泵给小鼠灌注NS和Ang Ⅱ (1500ng/kg/min),一周后用B超仪检测动物的心脏功能；心脏组织切片进行HE、Masson染色,分别观察不同组别心脏组织中炎性细胞浸润和胶原沉积的情况；应用免疫组织化学染色检测不同组别心脏中巨噬细胞(Mac2-阳性细胞)数量、单核细胞趋化因子(MCP-1)、血管细胞间粘附分子(ICAM-1)、α-平滑肌肌动蛋白(α-SMA)、胶原Ⅰ型(Collage Ⅰ)和转化生长因子-β (TGF-β)的表达水平。应用RT-PCR检测不同处理组MCP-1、ICAM-1、 Collage Ⅰ、 TGF-β、白细胞介素-1β(IL-1β)及IL-6的表达水平。应用TUNEL染色方法观察心肌细胞的凋亡情况。应用siRNA-GFP和siRNA-CHIP腺病毒感染原代培养的新生大鼠心肌细胞24小时,然后用Ang Ⅱ (100nM)处理6-24小时,应用RT-PCR及TUNEL染色方法检测不同处理组炎症因子(如MCP-1、ICAM-1、 IL-1β和IL-6)的表达水平及心肌细胞的凋亡情况。另外,原代培养新生大鼠心肌细胞,用丝裂原激活的蛋白激酶(MAPK) p38、c-Jun氨基末端激酶(JNK)的抑制剂刺激0.5小时后再用Ang Ⅱ(100nM)处理6-24小时,分别用RT-PCR和TUNEL染色方法检测IL-1β、IL-6和心肌细胞凋亡的情况。最后,用免疫印记(Western blot)法检测心脏组织及原代培养心肌细胞中p38MAPK、JNK和NF-κB的表达水平。结果： 1)在NS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。 2) Ang Ⅱ处理7天后,与NS+WT组相比,Ang Ⅱ+WT组的心脏功能、纤维化面积、细胞凋亡情况和炎性细胞的浸润程度均明显增加。 3)与Ang Ⅱ+WT组相比,Ang Ⅱ+CHIP-TG组小鼠心脏组织中纤维化面积(Masson染色),α-SMA.Collage Ⅰ和TGF-β表达水平(α-SMA、Collage Ⅰ、 TGF-p免疫组化染色)明显减少；心脏组织中巨噬胞浸润(Mac-2染色)及炎症因子表达水平(MCP-1, ICAM-1IL-1β和IL-6)均明显减少；心肌细胞的凋亡情况(TUNEL)减轻。 4)原代培养新生大鼠心肌细胞经Ang Ⅱ处理后显示,与siRNA-GFP对照组相比,siRNA-CHIP感染后可明显上调炎症因子(如MCP-1、ICAM-1、IL-1β和IL-6)的表达及增加心肌细胞的凋亡数量。 5) CHIP对Ang Ⅱ引起心肌重塑的保护作用是通过抑制p38MAPK.JNK及NF-κB信号通路的活性而实现的。结论： CHIP通过抑制p38MAPK、JNK及NF-κB信号通路的激活,抑制Ang Ⅱ引起的心肌纤维化、细胞凋亡和炎症反应。
[Abstract]:Research background and purpose:
The heat shock protein 70 carbonyl terminal reaction protein (CHIP) is a molecular chaperone protein and a ubiquitin E3 ligase containing the U- box (U-box). It can promote the degradation and removal of a variety of protein substrates by the ubiquitin proteasome system, and play an important role in myocardial cell damage, apoptosis, tumor, heat emergency and so on. But CHIP is induced by Ang II. The role and mechanism of myocardial fibrosis, apoptosis and inflammatory response are not clear. Therefore, this study mainly discusses the role and mechanism of CHIP in the remodeling of myocardium induced by Ang II.
Method:
10 weeks old wild type mice (WT) and 16 transgenic mice (CHIP-TG) with heart specific overexpression of CHIP were randomly divided into 4 groups, 8 rats in each group: (1) physiological saline (NS) +WT group; (2) NS+CHIP-TG group; (3) Ang II +WT group; (4) Ang II +CHIP-TG group. The cardiac function of animals was detected by B ultrasound, and HE and Masson staining of cardiac tissue sections were used to observe the infiltration of inflammatory cells and collagen deposition in different groups of cardiac tissues, and the number of macrophages (Mac2- positive cells), monocyte chemoattractant factor (MCP-1) and blood in different groups of hearts were detected by immunohistochemical staining. The expression level of intercellular adhesion molecule (ICAM-1), alpha smooth muscle actin (alpha -SMA), collagen type I (Collage I) and transforming growth factor beta (TGF- beta). The expression of MCP-1, ICAM-1, Collage I, TGF- beta, interleukin -1 beta (IL-1 beta) and IL-6) in different treatment groups was detected by RT-PCR. SiRNA-GFP and siRNA-CHIP adenovirus infected the primary cultured neonatal rat cardiomyocytes for 24 hours, and then treated with Ang II (100nM) for 6-24 hours. The expression level of inflammatory factors (such as MCP-1, ICAM-1, IL-1 beta and IL-6) in different treatment groups and the apoptosis of cardiac myocytes were detected by RT-PCR and TUNEL staining. Primary cultured neonatal rat cardiomyocytes were stimulated with mitogen activated protein kinase (MAPK) p38, c-Jun amino terminal kinase (JNK) inhibitor for 0.5 hours and then treated with Ang II (100nM) for 6-24 hours. RT-PCR and TUNEL staining methods were used to detect the apoptosis of IL-1 beta, IL-6 and cardiac myocytes. Finally, the immunoimprint (Western blot) method was used. The expression levels of p38 MAPK, JNK and NF-kappa B in cardiac tissue and primary cultured cardiomyocytes were detected.
Result:
1) There was no significant difference in cardiac function, pathological changes and inflammatory cell infiltration between WT and CHIP-TG groups during NS treatment.
2) after 7 days of Ang II treatment, compared with the NS+WT group, the cardiac function, the area of fibrosis, the cell apoptosis and the infiltration degree of inflammatory cells in the group Ang II +WT were significantly increased.
3) compared with the Ang II +WT group, the area of fibrosis in the Ang II +CHIP-TG group (Masson staining), the level of the expression of alpha -SMA.Collage I and TGF- beta (alpha -SMA, Collage I, TGF-p immunohistochemical staining) were significantly reduced, and the expression of macrophage infiltration (Mac-2 staining) and inflammatory factors in the cardiac tissue (MCP-1, beta and +CHIP-TG) were all clear The apoptosis of cardiomyocytes (TUNEL) was reduced.
4) the primary cultured neonatal rat cardiomyocytes were treated with Ang II. Compared with the siRNA-GFP control group, siRNA-CHIP infection could obviously increase the expression of inflammatory factors (such as MCP-1, ICAM-1, IL-1 beta and IL-6) and increase the number of apoptosis in cardiac myocytes.
5) The protective effect of CHIP on Ang II-induced myocardial remodeling is achieved by inhibiting the activity of p38MAPK.JNK and NF-kappa B signaling pathways.
Conclusion:
CHIP inhibits myocardial fibrosis, apoptosis and inflammation induced by Ang II by inhibiting the activation of p38 MAPK, JNK and NF-kappa B signaling pathways.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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