基于NLRP3炎症小体介导的巨噬细胞焦亡探究黄芩-黄连对动脉粥样硬化的干预机制

Scutellariae Radix-Coptidis Rhizoma Treats Atherosclerosis via NLRP3 Inflammasome-mediated Pyroptosis of Macrophages

目的:探究黄芩-黄连(SRCR)对动脉粥样硬化(AS)小鼠的治疗作用及通过NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体对斑块内巨噬细胞焦亡的影响。方法:载脂蛋白敲除(Apo E^(-/-))小鼠通过高脂饲料喂养构建AS模型,随机分为模型组、立普妥组(5 mg·kg^(-1))、SRCR低、中、高剂量组(1.95、3.9、7.8 g·kg^(-1)),正常C57BL/6J小鼠作为正常组。给药8周后,苏木素-伊红(HE)染色观察主动脉斑块病理情况;油红O染色观察主动脉斑块中脂质含量;检测小鼠血清中总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平;免疫荧光双染检测小鼠主动脉根部斑块组织中小鼠含生长因子样模体黏液样激素样受体(EMR1/F4/80)/NLRP3与F4/80/gasdermin D(GSDMD)共定位表达情况;酶联免疫吸附测定法(ELISA)检测血清白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)水平;蛋白免疫印迹法(Western blot)检测NLRP3、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(Caspase-1)、剪切(cleaved) Caspase-1、GSDMD、GSDMD蛋白N端(GSDMD-NT)、IL-1β前体(pro IL-1β)、IL-1β、IL-18蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测NLRP3、ASC、Caspase-1、GSDMD、IL-1β、IL-18 mRNA表达水平。结果:与正常组比较,模型组出现明显斑块,小鼠血清TG、TC、LDL-C、IL-1β及IL-18水平显著升高(P<0.01),HDL-C水平显著降低(P<0.01),主动脉组织中NLRP3炎症小体及焦亡相关分子表达显著升高(P<0.01);与模型组比较,SRCR各剂量组尤其是中、高剂量组,显著改善AS小鼠斑块病理情况,减少斑块脂质含量(P<0.05,P<0.01),有效改善小鼠血脂水平(P<0.05),减少主动脉根部斑块中巨噬细胞的募集(P<0.01)、NLRP3炎症小体的激活及焦亡(P<0.05),降低血清中IL-1β、IL-18的水平(P<0.01),降低主动脉组织中NLRP3、ASC、Caspase-1、cleaved Caspase-1、GSDMD、GSDMD-NT、pro IL-1β、IL-1β及IL-18的蛋白表达(P<0.05),降低主动脉组织中NLRP3、ASC、Caspase-1、GSDMD、IL-1β及IL-18的mRNA表达水平(P<0.05)。结论:SRCR对高脂饲料喂养诱导的AS小鼠具有治疗作用,其机制与抑制NLRP3炎症小体的激活,降低斑块内巨噬细胞焦亡水平有关。

Objective:To investigate the therapeutic effect of Scutellariae Radix-Coptidis Rhizoma(SRCR) on atherosclerosis (AS) in mice and the effect of SRCR on macrophage pyroptosis in plaques via NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasomes.Method:Apo E^(-/-)mice were fed with a high-fat diet for the modeling of AS and randomized into model,atorvastatin (5 mg·kg^(-1)),and low-,medium-,and high-dose (1.95,3.9,7.8 g·kg^(-1),respectively) SRCR groups.Normal C57BL/6J mice were selected as the control group.After 8 weeks of administration,hematoxylin-eosin staining was used to observe the pathological status of the aortic plaque.The lipid accumulation in aortic plaque was observed by oil red O staining.The serum levels of total cholesterol (TC),triglyceride (TG),high-density lipoprotein cholesterol (HDL-C),and low-density lipoprotein cholesterol (LDL-C) in mice were measured.Immunofluorescence double staining was employed to detect the co-localized expression of EGF-like modulecontaining mucin-like hormone receptor-like 1 (EMR1)/NLRP3 and EMR1/gasdermin D (GSDMD).The serum levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were determined by enzyme-linked immunosorbent assay (ELISA).The protein levels of NLRP3,apoptosis-associated speck-like protein (ASC),Caspase-1,cleaved Caspase-1,GSDMD,N-terminus of GSDMD (GSDMD-NT),pro-IL-1β,IL-1β,and IL-18 were determined by Western blot,and the mRNA levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,and IL-18were determined by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR).Result:Compared with the control group,the model group showed obvious plaques,elevated serum levels of TG,TC,LDL-C,IL-1β,and IL-18 (P<0.01),lowered serum level of HDL-C (P<0.01),and up-regulated expression of NLRP3 inflammasomes and molecules related to pyroptosis in the aortic plaques (P<0.01).Compared with the model group,SRCR,especially at the medium and high doses,alleviated the plaque pathology,reduced the lipid content in plaques (P<0.05,P<0.01),recovered the serum lipid levels (P<0.05),reduced the macrophage recruitment (P<0.01),activation of NLRP3 inflammasomes,and pyroptosis in aortic root plaques (P<0.05),lowered the serum IL-1β and IL-18 levels (P<0.01),and down-regulated the protein levels of NLRP3,ASC,Caspase-1,cleaved Caspase-1,GSDMD,GSDMD-NT,pro-IL-1β,IL-1β,and IL-18 (P<0.05) and the mRNA levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,and IL-18 in the aortic tissue (P<0.05).Conclusion:SRCR exerts a therapeutic effect on high-fat diet-induced AS in mice by inhibiting the activation NLRP3inflammasomes and reducing the pyroptosis of macrophages in plaques.