FNDC5基因过表达或沉默对动脉粥样硬化斑块形成中焦亡相关因子的影响及机制研究  

Effect of FNDC5 gene overexpression or silencing on pyroptosis-related factors during atherosclerotic plaque formation and its mechanism

作　　者：龚才伟 王尧 刘大男 欧航君 赵权威 李辉 李芸芸 陈龙 GONG Caiwei;WANG Yao;LIU Danan;OU Hangjun;ZHAO Quanwei;LI Hui;LI Yunyun;CHEN Long(Department of Cardiology,Affiliated Hospital of Guizhou Medical University,Institute of Medical Sciences,Guizhou Medi-cal University,Guiyang 550004,China)

机构地区：[1]贵州医科大学附属医院心内科,贵州医科大学医学科学研究所,贵州贵阳550004

出　　处：《中国病理生理杂志》2023年第6期961-972,共12页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81660083);贵州省科技创新人才团队项目[黔科合平台人才(2020)5014号];贵州省“百”层次创新型人才培养计划项目[黔科合人才(2015)4026号]。

摘　　要：目的:探索含Ⅲ型纤连蛋白结构域蛋白5(FNDC5)基因过表达或沉默对高脂饮食喂养的载脂蛋白E基因敲除(ApoE^(-/-))小鼠动脉粥样硬化斑块形成中焦亡相关因子的影响及其机制。方法:建立ApoE^(-/-)小鼠动脉粥样硬化模型;构建FNDC5基因过表达或沉默载体并转染动物模型,分组干预;采用酶法检测血清脂质含量;主动脉行油红O染色、HE染色和Masson染色检测斑块面积;采用免疫组化检测主动脉壁FNDC5、磷酸化核因子κB(pNF-κB)p65、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、caspase-1和消皮素D N端片段(GSDMD-N)的定位表达;采用TUNEL+caspase-1荧光双染检测主动脉壁细胞焦亡及其特征;采用ELISA法检测主动脉组织乳酸脱氢酶(LDH)活性;采用RT-qPCR和Western blot分别检测主动脉组织FNDC5、NLRP3、NF-κB p65、cleaved caspase-1、含caspase募集结构域的凋亡相关斑点样蛋白(ASC)、GSDMD-N、白细胞介素1β(IL-1β)和IL-18的mRNA及蛋白表达水平。结果:成功构建FNDC5腺病毒过表达和沉默载体。过表达FNDC5降低高脂饮食喂养的ApoE^(-/-)小鼠血清低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)和总胆固醇(TC)水平,减少主动脉斑块面积,降低内膜/中膜比值,降低主动脉LDH活性,降低主动脉NF-κB p65、NLRP3、cleaved caspase-1、ASC、GSDMD-N、IL-1β和IL-18的mRNA及蛋白表达水平,减少细胞焦亡(P<0.05);而沉默FNDC5基因使高脂饮食喂养的ApoE^(-/-)小鼠呈现相反的结果(P<0.05)。结论:FNDC5可调控动脉粥样硬化进程中的细胞焦亡,其机制可能是:通过降低NF-κB活性和表达,抑制NLRP3激活及降低其下游焦亡信号通路相关因子caspase-1、GSDMD、IL-1β和IL-18表达,抑制细胞焦亡,从而延缓动脉粥样硬化斑块形成。AIM:To investigate the effect of fibronectin type III domain-containing protein 5(FNDC5)gene overexpression or silencing on the expression of pyroptosis-related factors during atherosclerotic plaque formation in apolipoprotein E gene knockout(ApoE^(-/-))mice fed with high-fat diet,and to explore its mechanism.METHODS:An atherosclerotic model in ApoE^(-/-)mice was established,and the mice were transfected with overexpression or silencing vectors of FNDC5 gene,followed by group intervention.Serum lipid levels were measured by enzymatic assays.Plaque area was detected by oil red O staining,HE staining and Masson staining of the aorta.Immunohistochemistry was used to detect the expression and localization of FNDC5,phosphorylated nuclear factor-κB(p-NF-κB)p65,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1 and N-terminal fragment of gasdermin D(GSDMD-N)in the aor‐tic wall.TUNEL+caspase-1 fluorescence double staining were used to detect the pyroptosis and the characteristic of pyrop‐tosis in the aortic wall.Lactate dehydrogenase(LDH)activity in the aortic tissue was detected by ELISA.RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of FNDC5,NLRP3,NF-κB p65,cleaved cas‐pase-1,apoptosis-associated speck-like protein containing a caspase recruitment domian(ASC),GSDMD-N,interleukin-1β(IL-1β)and IL-18 in the aorta.RESULTS:Adenoviral vectors for overexpression and silencing of FNDC5 were suc‐cessfully constructed.Overexpression of FNDC5 reduced serum levels of low-density lipoprotein cholesterol(LDL-C),tri‐glyceride(TG)and total cholesterol(TC),decreased atherosclerotic plaque area,intima/media ratio and LDH activity in the arota,inhibited the mRNA and protein expression of NF-κB p65,NLRP3,cleaved caspase-1,ASC,GSDMD-N,IL-1βand IL-18,and attenuated cell pyroptosis(P<0.05)in ApoE^(-/-)mice fed with high-fat diet.In contrast,silencing of FNDC5 gene led to the opposite effects(P<0.05).CONCLUSION:FNDC5 regulates cell pyroptosis in the process

关 键 词：动脉粥样硬化 含Ⅲ型纤连蛋白结构域蛋白5 鸢尾素 细胞焦亡 

分 类 号：R541.4[医药卫生—心血管疾病] R363.2[医药卫生—内科学]