GPR35致主动脉夹层的分子机制研究

Molecular mechanism of aortic dissection induced by GPR35

目的探讨G蛋白偶联受体35(GPR35)在主动脉夹层(AD)发生发展中的作用及机制。方法将30只野生型(WT)雄性小鼠随机均分为WT+Sham组和WT+AD组,15只GPR35基因敲除(GPR35-/-)小鼠纳入GPR35-/-+AD组,建立AD模型:WT+AD组和GPR35-/-+AD组使用血管紧张素Ⅱ[4.5 mg/(kg·24 h)]微型渗透泵埋于小鼠背部皮下,WT+Sham组使用等量生理盐水,术后14d主动脉取材。将小鼠主动脉平滑肌细胞系(MOVAS)分为3组:对照组、AD组及GPR35抑制剂组(CID组)。采用q-PCR及Western blotting检测主动脉和MOVAS细胞中GPR35的表达;采用HE染色和VVG染色检测主动脉夹层形成和弹力纤维结构情况;采用免疫组化和q-PCR检测主动脉内单核巨噬细胞(CD68阳性细胞)浸润及炎性因子白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α的表达水平;采用TUNEL染色和流式技术检测主动脉平滑肌细胞凋亡情况。结果与WT+Sham组相比,WT+AD组GPR35 mRNA表达水平升高(1.88±0.07)倍,GPR35蛋白表达水平升高(1.34±0.05)倍,在细胞模型中亦得到类似结果;与WT+AD组小鼠的AD发生率(53.3%)相比,GPR35-/-+AD组小鼠AD发生率(13.3%)明显降低(P<0.05);与WT+AD组相比,GPR35-/-+AD组的主动脉扩张和中层增厚情况明显减轻,弹性纤维结构排列也较规则;WT+AD组小鼠CD68阳性细胞率高于WT+Sham组和GPR35-/-+AD组[(32.8±1.1)%vs.(5.1±0.9)%vs.(16.0±1.1)%],差异有统计学意义(P<0.05)。WT+AD组的IL-6、TNF-αmRNA表达水平(2.6±0.1、1.8±0.1)均明显高于WT+Sham组(均为1.00±0.06),GPR35-/-+AD组的IL-6、TNF-αmRNA表达水平(1.6±0.1、1.4±0.1)均明显低于WT+AD组,差异均有统计学意义(P<0.05)。WT+AD组凋亡细胞数明显高于WT+Sham组和GPR35-/-+AD组[(24.0±0.7)%vs.(6.6±0.5)%vs.(11.2±0.9)%],差异均有统计学意义(P<0.05)。AD组的MOVAS凋亡率高于对照组和CID组[(11.6±0.5)%vs.(4.7±0.4)%vs.(7.6±0.4)%],差异有统计学意义(P<0.05)。结论抑制GPR35可通过阻止主动脉炎症和平滑肌凋亡来发挥预防主动脉夹层生成的作用。

Objective To investigate the function of G-protein-coupled receptor 35(GPR35) in aortic dissection(AD)and its mechanism. Methods Thirty wild-type(WT) male mice were randomly divided into the WT+Sham group and WT+AD group(15 each), and 15 GPR35 knockout mice(GPR35-/-) were set as GPR35-/-+AD group. Mice in WT+AD group and GPR35-/-+AD group were back-subcutaneously implanted with angiotensin Ⅱ[4.5 mg/(kg·24 h)] by miniosmotic pump, while in WT+Sham group received equivalent saline. Mice were sacrificed and the aortas were removed on the 14 th postoperative day for further experiments. The mice aortic vascular smooth muscle cell lines(MOVAS) were divided into control group, aortic dissection group(AD group), GPR35 inhibitor group(CID group). The expressions of GPR35 in aorta and MOVAS were determined by q-PCR and Western blotting. The aortas were stained with HE and VVG to detect the formation of aortic dissection and elastic fiber structure.The infiltration of monocyte macrophage(CD68 positive cell) and expression of inflammatory factors(IL)-6 and(TNF)-α in aortawere detected with immunohistochemistry and q-PCR. The apoptosis of aortic smooth muscle was measured by TUNEL staining and flow cytometer. Results Compared with the WT+Sham group, the expression of GPR35 mRNA increased by(1.88±0.07)times and of GPR35 protein increased by(1.34±0.05) times in WT+AD group(P<0.05), and similar results were obtained in cell model;Compared with the WT+AD group, the incidence of AD decreased obviously in GPR35-/-+AD group(53.3% vs. 13.3%,P<0.05), meanwhile the aortic dilatation, media thickness and elastic fiber structure were attenuated evidently;Furthermore, the mononuclear macrophage infiltration(CD68 positive cells) were elevated in WT+AD group than in WT+Sham group and GPR35-/-+AD group [(32.8±1.1)% vs.(5.1±0.9)% vs.(16.0±1.1)%] with statistical significance(P<0.05). The expression levels of IL-6 and TNF-α mRNA were higher in WT+AD group(2.6±0.1, 1.8±0.1) than in WT+Sham group(both 1.00±0.06) and GPR35-/-+AD group(1.6±0.1, 1.4±0.1) with statistical significance(P<0.05);The number of apoptotic aortic smooth muscle cells in WT+AD group [(24.0±0.7)%] was more than that in WT+Sham group and GPR35-/-+AD group [(6.6±0.5)% and(11.2±0.9)%]with statistical significance(P<0.05). Meanwhile, the apoptosis rate of MOVAS was also significantly higher in AD group[(11.6±0.5)%] than in control group and CID group [(4.7±0.4)% and(7.6±0.4)%, P<0.05]. Conclusion Inhibition of GPR35 can protect aortic dissection by preventing aortic inflammation and smooth muscle apoptosis.