基于NF-κB/NLRP3/Caspase-1通路介导的巨噬细胞焦亡探究葛根芩连汤对动脉粥样硬化易损斑块的干预机制

Mechanism of Gegen Qinliantang against Vulnerable Plaque of Atherosclerosis:Based on Macrophage Pyroptosis Mediated by NF-κB/NLRP3/Caspase-1 Pathway

目的:探究葛根芩连汤(GQL)通过调控核转录因子(NF)-κB/NOD样受体蛋白3(NLRP3)/胱天蛋白酶(Caspase)-1通路介导的巨噬细胞焦亡对动脉粥样硬化(AS)易损斑块的作用。方法:12只正常C57BL/6CNC小鼠作为空白组,60只同品系的载脂蛋白E基因敲除(ApoE^(-/-))小鼠随机分为5组,即模型组、葛根芩连汤低、中、高剂量组(GQL-D、Z、G组)、立普妥组,以高脂饲料喂养造模。空白组、模型组予等体积无菌蒸馏水灌胃;GQL-D、Z、G、立普妥组分别予对应浓度的药物灌胃8周。苏木素-伊红(HE)染色观测主动脉区域斑块情况,酶联免疫吸附测定法(ELISA)检测血清白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)水平,ELISA检测巨噬细胞甘露糖受体(MMR/CD206)/凋亡相关斑点样蛋白(ASC)、CD206/NLRP3蛋白表达水平,蛋白免疫印迹法(Western blot)检测各组小鼠gasdermin D蛋白C端(C-terminal GSDMD)、gasdermin D蛋白N端(N-terminal GSDMD)、NLRP3、含胱天蛋白酶-1前体(pro-Caspase-1)和NF-κB p65蛋白表达水平。结果:与空白组比较,模型小鼠AS病变程度严重,血清IL-1β、IL-18、组织ASC、NLRP3、C-terminal GSDMD、N-terminal GSDMD、pro-Caspase-1和NF-κB p65表达水平明显升高(P<0.05),CD206水平明显下降(P<0.05);与模型组比较,给药各组小鼠主动脉壁AS病变程度有所减轻,血清IL-1β、IL-18、组织ASC、NLRP3、C-terminal GSDMD、N-terminal GSDMD、pro-Caspase-1和NF-κB p65表达水平有不同程度下降,CD206水平不同程度上升,部分组结果差异有统计学意义(P<0.05)。结论:GQL对AS易损斑块的干预作用可能是通过调控NF-κB/NLRP3/Caspase-1通路,减轻其介导的巨噬细胞焦亡来实现的。

Objective:To explore the effect of Gegen Qinliantang(GQL)on vulnerable plaque of atherosclerosis based on the macrophage pyroptosis mediated by nuclear factor(NF)-κB/NOD-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease(Caspase)-1 pathway.Method:A total of 12 normal C57BL/6CNC mice were used as the control group,and 60 ApoE^(-/-)mice of the same line were randomized into 5groups:model group,low-dose,medium-dose,and high-dose GQL groups(GQL-D,GQL-Z,GQL-G groups,respectively),and western medicine group.The control group and model group were given(ig)equal volume sterile distilled,and GQL-D,GQL-Z,GQL-G and western medicine groups received(ig)corresponding concentration of drugs for 8 weeks.Aortic plaques were observed based on hematoxylin and eosin(HE)staining.Serum levels of interleukin(IL)-1βand IL-18 were detected by enzyme-linked immunosorbent assay(ELISA),protein levels of macrophage mannose receptor(CD206)/apoptosis-associated speck-like protein containing a CARD(ASC)and CD206/NLRP3 by double-labeling immunofluorescence,and C-terminal gasdermin D(GSDMD),N-terminal GSDMD,NLRP3,pro-cysteinyl aspartate specific proteinase 1(proCaspase-1)and NF-κB p65 by Western blot.Result:Compared with the control group,model group demonstrated serious pathological changes,rise of the levels of serum IL-1βand IL-18 and tissue ASC,NLRP3,C-terminal GSDMD,N-terminal GSDMD,pro-Caspase-1,and NF-κB p65,and decrease of CD206 level(P<0.05).As compared with model group,the administration groups showed alleviation of the lesions in aortic wall,decrease in levels of serum IL-1βand IL-18 and tissue ASC,NLRP3,C-terminal GSDMD,N-terminal GSDMD,pro-Caspase-1,and NF-κB p65,and rise of CD206 level,with significant difference between some groups(P<0.05).Conclusion:Gegen Qinliantang alleviates vulnerable plaque of atherosclerosis by regulating NF-κB/NLRP3/Caspase-1 pathway and further relieving macrophage pyroptosis.