Hippo/YAP通路在大鼠脑动脉瘤形成过程中的作用及机制研究

Study on the Mechanism of Hippo/YAP Pathway in the Formation of Cerebral Aneurysm in Rats

目的:探究Hippo/Yes相关蛋白(YAP)通路在脑动脉瘤(CA)形成过程中的作用及机制。方法:SPF级雄性SD大鼠随机分为对照组(n=10)、模型组(n=30)和YAP抑制剂组(n=10),模型组和YAP抑制剂组大鼠采用结扎左颈总动脉和双侧肾动脉联合高盐饮食建立CA模型。根据样本采样时间(造模后1周、造模后1个月、造模后3个月),将模型组分为A、B、C共3个亚组,每组10只。ELISA检测血清炎性因子[单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-10]及血管内皮损伤标志物[血管内皮生长因子(VEGF)、内皮素1(ET-1)]水平;光学显微镜下观察CA形态学变化;免疫荧光染色检测动脉瘤组织中巨噬细胞极化与浸润情况;Western blot检测动脉瘤血管组织Hippo/YAP通路相关蛋白[哺乳动物Ste-20样激酶(MST)1/2、p-MST1/2、大型肿瘤抑制因子(LATS)1/2、p-LATS1/2、YAP]表达。结果:对照组大鼠均无CA形成,无巨噬细胞浸润,与对照组相比,A、B、C组大鼠Willis环中出现大量F4/80阳性细胞,M1型巨噬细胞比例和M2型巨噬细胞比例、动脉瘤形成的级别、血清VEGF、ET-1、MCP-1、TNF-α、IL-6水平、动脉瘤血管组织胞核YAP的表达依次显著增加(P<0.05),血清IL-10水平及动脉瘤血管组织p-MST1/2/MST1/2、p-LATS1/2/LATS1/2比值依次显著降低(P<0.05),且随着时间的增加,M1/M2型巨噬细胞比值增加(P<0.05);YAP抑制剂组胞核YAP的表达明显降低,M1/M2型巨噬细胞比值降低,CA的形成和进展受到抑制(P<0.05)。结论:Hippo/YAP信号通路可能通过介导巨噬细胞极化参与CA的形成和进展。

Objective:To explore the mechanism of Hippo/Yes-associated protein(YAP)pathway in the formation and progression of cerebral aneurysm(CA).Method:SPF male SD rats were randomly divided into control group(n=10),model group(n=30)and YAP inhibitor group(n=10).The model group was divided into three subgroups A,B and C based on the sample sampling time(1 week after modeling,1 month after modeling,3 months after modeling),with 10 animals in each subgroup.Rats in the model group and YAP inhibitor group used ligation of the left common carotid artery and bilateral renal arteries in combination with a high-salt diet to establish a CA model.ELISA was used to detect the levels of serum inflammatory factors[monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-10]and markers of vascular endothelial damage[vascular endothelial growth factor(VEGF),endothelin-1(ET-1)];an optical microscope was used to observe the morphological changes of CA;immunofluorescence staining was used to detect the polarization and infiltration of macrophages in the aneurysm tissue;Western blot was used to detect the expression of Hippo/YAP pathway related proteins[mammalian sterile 20-like kinase(MST)1/2,p-MST1/2,large tumor suppressor(LATS)1/2,p-LATS1/2,YAP]in aneurysm vascular tissue.Results:The rats in the control group had no CA formation and macrophage infiltration.Compared with the control group,there were a large number of F4/80 positive cells in the Willis circle of the rats in groups A,B and C,the proportion of M1 macrophages and the proportion of M2 macrophages,the level of aneurysm formation,the levels of serum VEGF,ET-1,MCP-1,TNF-α,IL-6 and the expression of YAP in the nucleus of aneurysm vascular tissue were increased significantly(P<0.05),the level of serum IL-10 and the ratios of p-MST1/2/MST1/2 and p-LATS1/2/LATS1/2 in the aneurysm vascular tissue were decreased significantly(P<0.05),and with the increase of time,the ratio of M1/M2 type macrophages was increased;the use of YAP inhibitor,Verteporfin,could significantly reduce the expression of nuclear YAP,reduce the ratio of M1/M2 macrophages,and inhibit the formation and progression of CA.Conclusion:The Hippo/YAP signaling pathway may participate in the formation and progression of CA by mediating macrophage polarization.