S100A11对主动脉夹层的影响及其机制

Role of S100A11 in aortic dissection and its mechanism

目的探讨S100A11在主动脉夹层(AD)中的作用及可能的机制。方法在体内实验中,构建携带S100A11的慢病毒质粒Lv-S100A11-shRNA和阴性对照质粒Lv-NC-shRNA,分别转染至HEK293T细胞,获得病毒上清。将40只SD大鼠随机分为5组:对照组(不做任何干预)、假手术组(尾静脉注射生理盐水)、AD组(连续3周在饮水中加入0.25%的β-氨基丙腈建立AD模型)、AD+Lv-NC-shRNA组(AD模型大鼠予尾静脉注射Lv-NC-shRNA)和AD+Lv-S100A11-shRNA组(AD模型大鼠予尾静脉注射Lv-S100A11-shRNA),每组8只。通过H-E染色观察主动脉的组织病理学变化,TUNEL染色观察细胞凋亡情况,免疫组织化学染色检测S100A11和下游信号通路相关蛋白糖基化终末产物受体(RAGE)和磷酸化p38(p-p38)的表达,蛋白质印迹法检测迁移蛋白基质金属蛋白酶(MMP)2和MMP9、凋亡相关蛋白Bcl-2和Bcl-2相关X蛋白(Bax)、细胞增殖相关蛋白增殖细胞核抗原(PCNA)和Ki-67的表达水平。在体外实验中,构建S100A11过表达质粒OV-S100A11,将人主动脉平滑肌细胞(HASMC)分为3组:对照组(未进行任何干预)、EV组(转染pIRES2-GFP空载体)和OV-S100A11组(转染pIRES2-GFP-S100A11过表达S100A11)。通过流式细胞术检测细胞凋亡情况,然后用RAGE抑制剂FPS ZM1和p38磷酸化抑制剂SB203580处理转染pIRES2-GFP-S100A11的细胞,采用蛋白质印迹法检测S100A11、RAGE、p38、p-p38、MMP2、MMP9、Bax、Bcl-2、PCNA、Ki-67的表达。结果动物实验结果显示,与假手术组相比,AD组大鼠主动脉血管形成充满血液的夹层,凋亡细胞增多,S100A11、RAGE、p-p38、MMP2、MMP9和Bax蛋白水平均升高(P均<0.01),Bcl-2、PCNA和Ki-67蛋白水平均降低(P均<0.01);与AD组相比,Lv-S100A11-shRNA组主动脉病变得到缓解,凋亡细胞减少,S100A11、RAGE、p-p38、MMP2、MMP9和Bax蛋白水平均降低(P均<0.01),Bcl-2、PCNA和Ki-67蛋白水平均升高(P均<0.01)。细胞实验结果显示,与对照组相比,OV-S100A11组细胞凋亡率升高(P<0.01),细胞中RAGE、p-p38、MMP2、MMP9、Bax蛋白水平均升高(P均<0.01),Bcl-2、PCNA、Ki-67蛋白水平均降低(P均<0.01),而在FPS ZM1和SB203580处理后上述蛋白质的变化趋势相反。结论S100A11在AD大鼠中高表达,其可通过RAGE-p38 MAPK通路促进细胞凋亡从而参与AD的形成。

Objective To investigate the role of S100 calcium binding protein A11(S100A11)in aortic dissection(AD)and its possible mechanism.Methods In vivo,lentivirus plasmid Lv-S100A11-shRNA and negative control plasmid Lv-NC-shRNA were constructed and transfected into HEK293T cells respectively to obtain viral supernatant.Forty SD rats were randomly divided into 5 groups:control group(without any intervention),sham operation group(with physiological saline injected into the tail vein),AD group(with 0.25% β-aminopropionitrile added into drinking water for 3 consecutive weeks to establish AD model),AD+Lv-NC-shRNA group(AD model rats were injected with Lv-NC-shRNA via tail vein)and AD+Lv-S100A11-shRNA group(AD model rats were injected with Lv-S100A11-shRNA via tail vein),with 8 rats in each group.Hematoxylin-eosin(H-E)staining was used to observe the histopathological changes of the aorta,terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)staining was used to observe cell apoptosis,immunohistochemistry staining was used to detect the protein expression levels of S100A11 and downstream signaling pathway related proteins receptor for advanced glycation endproducts(RAGE)and phosphorylated p38(p-p38),and Western blotting was used to detect the protein expression levels of migration proteins matrix metalloproteinase(MMP)2 and MMP9,apoptosis proteins B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X(Bax),proliferation proteins proliferating cell nuclear antigen(PCNA)and Ki-67.In vitro,the S100A11 overexpression plasmid OV-S100A11 was constructed,and human aortic smooth muscle cells(HASMC)were divided into 3 groups:control group(without any intervention),EV group(transfected with pIRES2-GFP empty vector)and OV-S100A11 group(transfected with pIRES2-GFP-S100A11 overexpression S100A11).Flow cytometry was used to detect apoptosis,and then the cells transfected with pIRES2-GFP-S100A11 were treated with RAGE inhibitor FPS ZM1 and p38 phosphorylation inhibitor SB203580.Western blotting was used to detect the protein expression of S100A11,RAGE,p38,p-p38,MMP2,MMP9,Bax,Bcl-2,PCNA and Ki-67.Results The results of animal experiments showed that compared with the sham operation group,the aortic vessels of rats in the AD group formed a bloodfilled dissection,the apoptotic cells were increased,the protein levels of S100A11,RAGE,p-p38,MMP2,MMP9 and Bax were increased(all P<0.01),while the protein levels of Bcl-2,PCNA and Ki-67 were decreased(all P<0.01).Compared with the AD group,the aortic lesions in the Lv-S100A11-shRNA group were relieved,the apoptotic cells were decreased,the levels of S100A11,RAGE,p-p38,MMP2,MMP9 and Bax were decreased(all P<0.01),while the protein levels of Bcl-2,PCNA and Ki-67 were increased(all P<0.01).The results of cell experiments showed that the apoptosis rate of OV-S100A11 group was increased compared with the control group(all P<0.01),the protein levels of RAGE,p-p38,MMP2,MMP9 and Bax in the cells were increased(all P<0.01),the protein levels of Bcl-2,PCNA and Ki-67 were decreased(all P<0.01),but the change of the above proteins was reversed after the treatment with FPS ZM1 and SB203580.Conclusion S100A11 is highly expressed in AD formation rats,and it can promote cell apoptosis through RAGE-p38 mitogen activated protein kinase pathway,participating in AD formation.