摘要
目的观察高糖刺激对人肾小球系膜细胞(HMC)A类清道夫受体(SR-A)表达的影响,并初步探讨SR-A介导HMC在高糖环境下发生炎症损伤的相关机制。方法将体外培养的HMC按照其培养基中D-葡萄糖浓度分为正常糖组和高糖组(葡萄糖浓度分别为5.5 mmol/L和30 mmol/L),并以甘露醇组作为高渗对照,在高糖组瞬时转染SR-A小干扰RNA(siSR-A)并设置转染对照组(siNC)。运用蛋白免疫印迹法检测各组SR-A、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、白细胞介素(IL)1β蛋白含量,免疫荧光技术检测SR-A,实时荧光定量PCR检测各组NLRP3、半胱氨酸天冬氨酸特异性蛋白水解酶-1(Caspase-1)、IL-1β、纤连蛋白(FN)、Ⅳ型胶原(ColⅣ)、α-平滑肌肌动蛋白(α-SMA)、内质网应激标志物葡萄糖调节蛋白(GRP)78基因mRNA,酶法检测Caspase-1相对活性,酶联免疫吸附法检测细胞培养基中IL-1β浓度,流式细胞术检测细胞周期。运用单因素方差分析和SNK q检验进行统计分析。结果高糖组HMC中SR-A蛋白水平高于正常糖组和甘露醇组(1.23±0.21比0.68±0.10,1.23±0.21比0.78±0.13,均P<0.05),高糖组SR-A蛋白平均荧光强度,NLRP3和IL-1β的蛋白水平,NLRP3、Caspase-1、IL-1β的mRNA水平,Caspase-1相对活性和IL-1β浓度均高于正常糖组和甘露醇组(均P<0.05)。沉默SR-A基因后,高糖siNC组SR-A蛋白水平高于高糖siSR-A组和正常糖siNC组(1.23±0.10比0.20±0.01,1.23±0.10比0.87±0.01,均P<0.01)。高糖siNC组NLRP3、IL-1β蛋白水平和NLRP3、Caspase-1、IL-1β、FN、ColⅣ、α-SMA、GRP78 mRNA水平和HMC细胞周期中DNA合成期占比也均明显高于高糖siSR-A组和正常糖siNC组(均P<0.05)。结论高糖可以通过上调SR-A表达促进HMC异常增殖、系膜基质产生增加和发生氧化应激,加重细胞炎症损伤,这一过程可能与SR-A调节NLRP3-Caspase-1-IL-1β通路相关。
Objective To investigate the effect of high glucose on scavenger receptor-A(SR-A)in human glomerular mesangial cells(HMC)and explore the mechanism of inflammatory injury mediated by SR-A in HMC cultured in high-glucose medium.Methods According to the concentration of D-glucose in culture medium,HMC were divided into normal glucose group(5.5 mmol/L)and high glucose group(30 mmol/L),with mannitol group as hypertonic control.High glucose group was transfected with SR-A small interfering RNA(siSR-A)and the transfection control(siNC)group were set up.Western blotting technology was used to detect the levels of SR-A,NOD-like receptor family pyrin domain-containing 3(NLRP3),interleukin-1β(IL-1β)protein.Immunofluorescent staining was applied to measure the SR-A in HMC.The mRNA of NLRP3,Caspase-1,IL-1β,FN,ColⅣ,α-SMA and GRP78 were detected by real-time quantitative PCR.The relative activity of Caspase-1 was detected by enzyme method and the concentration of IL-1βin culture medium was detected by enzyme linked immunosorbent assay.Flow cytometry was used to measure the cell cycles of HMC.One-way ANOVA and SNK-q test were used for statistical analysis.Results The protein level of SR-A in high glucose group was higher than that in normal glucose group and mannitol group(1.23±0.21 vs.0.68±0.10,1.23±0.21 vs.0.78±0.13,all P<0.05).In addition,mean fluorescence intensity of SR-A,protein levels of NLRP3 and IL-1β,mRNA of NLRP3,Caspase-1 and IL-1β,relative activity of Caspase-1 as well as the concentration of IL-1βin high glucose group were all significantly higher than those in normal glucose group and mannitol group(all P<0.05).After transfection induced silencing,SR-A protein in high glucose siNC group was higher than that in high glucose siSR-A group and normal glucose siNC group(1.23±0.10 vs.0.20±0.01,1.23±0.10 vs.0.87±0.01,all P<0.01).In high glucose siNC group,the NLRP3,IL-1βproteins,the NLRP3,Caspase-1 and IL-1βmRNA,all of the mRNA levels of FN,ColⅣ,α-SMA,GRP78 and the proportion of DNA synthesis phase
作者
肖寒
张高福
阳海平
陈压西
王墨
李秋
Xiao Han;Zhang Gaofu;Yang Haiping;Chen Yaxi;Wang Mo;Li Qiu(Department of Nephrology,Children’s Hospital of Chongqing Medical University,National Clinical Research Center for Child Health and Disorders,Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of Child Infection and Immunity,Chongqing 400014,China;Key Laboratory of Metabolism on Lipid and Glucose,Center for Lipid Research,Chongqing Medical University,Chongqing 400014,China)
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2021年第5期393-399,共7页
Chinese Journal of Pediatrics
基金
国家自然科学基金(81770713)。
关键词
清道夫受体
A类
糖尿病肾病
肾小球系膜细胞
炎症
Scavenger receptors,class A
Diabetic nephropathy
Mesangial cells
Inflammation
