NLRP3炎症体信号通路在促进慢性肾脏病相关性血管新生内膜增生中的作用  

Role of the NLRP3 inflammatory signaling pathway in promoting neointimal hyperplasia associated with chronic renal disease

作　　者：卢健 郭丽丽 薛福平 张婷婷[4] 李媛 王艳勤 李爱中 李亚峰 李荣山 Lu Jian;Guo Lili;Xue Fuping;Zhang Tingting;Li Yuan;Wang Yanqin;Li Aizhong;Li Yafeng;Li Rongshan(Department of Nephrology,Shanxi Provincial People's Hospital Affiliated to Shanxi Medical University,Taiyuan 030012,China;Shanxi Precision Medical Diagnosis and Treatment Center,Taiyuan 030012,China;Department of Nephrology,Shanxi Armed Police Corps Hospital,Taiyuan 030000,China;Institute of Applied Biology,Shanxi University,Taiyuan 030006,China)

机构地区：[1]山西医科大学附属人民医院肾内科,太原030012 [2]山西省精准医学诊断治疗中心,太原030012 [3]武警山西省总队医院肾病科,太原030000 [4]山西大学应用生物学研究所,太原030006

出　　处：《中华肾脏病杂志》2021年第3期198-208,共11页Chinese Journal of Nephrology

基　　金：国家自然科学基金(8187021561、81400751);山西省研究生教育创新项目(2019SY288)。

摘　　要：目的探讨Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)在慢性肾脏病相关性血管新生内膜增生(neointimal hyperplasia,NH)中的作用及其机制。方法野生型C57BL/6J雄性小鼠被随机分为正常对照组(n=6)和实验组(n=18),实验组小鼠用5/6肾脏切除和左颈总动脉结扎的方法建立NH模型。建模成功后,小鼠被随机分为NH模型组、NLRP3抑制剂组和药物对照组(n=6/组)。NLRP3基因敲除组C57BL/6J雄性小鼠建立NH模型后不做任何处理。3周后收集小鼠血清和颈动脉结扎处血管组织样本。苏木素-伊红(HE)染色观察小鼠血管组织病理改变;免疫荧光染色观察NLRP3相关蛋白的表达和定位;实时荧光定量PCR法检测小鼠血管组织NLRP3 mRNA的表达水平;比色法测定血管组织半胱氨酸天冬氨酸蛋白水解酶1(caspase-1)活性水平。用10%尿毒症患者血清干预人主动脉血管平滑肌细胞(HASMCs)模拟尿毒症期机体内环境。转染NLRP3小分子干扰RNA(siRNA)或加入NLRP3抑制剂格列苯脲进行干预。实时荧光定量PCR法检测HASMCs的NLRP3 mRNA表达水平;比色法测定HASMCs caspase-1活性水平。结果NH模型组小鼠血清血肌酐、尿素氮水平较正常对照组显著升高(均P<0.01)。HE染色结果显示,NH模型组小鼠血管内膜较对照组明显增厚,血管平滑肌细胞明显增生肥大,细胞核深染,细胞排列杂乱并向血管内膜面迁徙。NH模型组新生内膜与管腔比值较对照组显著增加(P<0.01)。免疫荧光染色结果显示,与对照组相比,NH模型组小鼠血管组织NLRP3、caspase-1、IL-18、IL-1β和增殖细胞核抗原蛋白表达增加,α平滑肌肌动蛋白(α-SMA)表达下降(P<0.01),NLRP3主要定位于血管平滑肌细胞,血管平滑肌细胞表现为合成表型。与NH模型组相比,NLRP3抑制剂组和NLRP3基因敲除组小鼠NLRP3、caspase-1、IL-18、IL-1β和PCNA蛋白表达减少,α-SMA蛋白表达增加(均P<0.01),血管病理改变减轻。尿毒症血清刺激组细胞NObjective To investigate the role and mechanism of Nod-like receptor protein 3(NLRP3)in chronic kidney disease(CKD)-related neointimal hyperplasia(NH)of vessels.Methods Wild type C57BL/6J male mice were randomly divided into normal control group(n=6)and experimental group(n=18),by removal of 5/6 kidney and ligation of left common carotid artery to establish a NH model.After established successfully,the mice in NH experimental group were randomly divided into NH model group,NLRP3 inhibitor group,and drug control group(n=6/group).C57BL/6J male mice with NLRP3 gene knockout group did not do any treatment after the establishment of NH model.After 3 weeks of feeding,the blood and vascular tissue samples of mice were collected.The pathological changes of vascular tissue samples in mice were observed by hematoxylin-eosin staining.The expressions and localization of NLRP3-related protein were observed by immunofluorescence staining.The expression of NLRP3 mRNA in vascular tissue was detected by quantitative real-time PCR.The activity of caspase-1 in vascular tissue was measured by colorimetric method.Human aortic smooth muscle cells(HASMCs)were treated with 10%uremic serum to simulate the body's internal environment during the uremic phase.NLRP3 small interfering RNA(siRNA)was transfected or NLRP3 inhibitor glibenclamide was added to the cell cultures.The expression of NLRP3 mRNA in HASMCs was detected by quantitative real-time PCR.The activity of caspase-1 in HASMCs was detected by colorimetric method.Results Compared with the control group,the levels of serum creatinine and blood urea nitrogen were significantly increased in the NH model group(both P<0.01).The vascular histopathology showed that vascular intima thickened,vascular smooth muscle cells proliferated and hypertrophied,nuclei were deeply stained,and cells arranged disorderly and migrated to vascular intima in the experimental group.Quantitative analysis showed that the ratio of neointima to lumen increased significantly in the NH model group than that in con

关 键 词：NLR家族 热蛋白结构域包含蛋白3 肾功能不全 慢性 格列本脲 血管炎症 新生内膜增生 血管平滑肌细胞 

分 类 号：R692[医药卫生—泌尿科学]