人重组骨形态发生蛋白7通过下调半胱氨酸蛋白酶-1/焦孔素D信号通路抑制高糖诱导的近曲小管上皮细胞的焦亡  

Recombinant human bone morphogenetic protein 7 suppressed high glucose-induced pyroptosis of proximal tubular epithelial cells through down-regulating caspase-1/GSDMD signaling pathway

作　　者：魏凯悦 米焱[1] 王彩丽[1] 吴雅茹 张丁予 高和平 Wei Kai-yue;Mi Yan;Wang Cai-li;Wu Ya-ru;Zhang Ding-yu;Gao He-ping(Department of Nephrology,First Affiliated Hospital,Baotou Medical College,Inner Mongolia University of Science&Technology,Baotou 014010,China)

机构地区：[1]内蒙古科技大学包头医学院第一附属医院肾内科,包头014010

出　　处：《临床肾脏病杂志》2023年第5期403-411,共9页Journal Of Clinical Nephrology

基　　金：国家自然科学基金(82160414)。

摘　　要：目的 观察人重组骨形态发生蛋白-7(recombinant human bone morphogenetic protein-7,rhBMP-7)对高糖诱导的人肾皮质近曲小管上皮细胞(human kidney-2,HK-2)的焦亡和炎症反应的作用。方法 体外培养高糖(high glucose,HG)刺激的HK-2细胞模拟糖尿病肾病模型。分为正常浓度葡萄糖对照组(Control)、高浓度葡萄糖组(HG)、Control+rhBMP-7组、HG+rhBMP-7组。采用免疫荧光方法检测HK-2细胞中E-钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-smooth muscle actin, α-SAM)、焦孔素D(gasderminD, GSDMD)蛋白和焦孔素E(gasderminE, GSDME)蛋白;TUNEL染色检测细胞死亡情况;蛋白质印迹法检测各组HK-2细胞中焦亡相关蛋白半胱氨酸蛋白酶-1(caspase-1)、半胱氨酸蛋白酶-4(caspase-4)、半胱氨酸蛋白酶-5(caspase-5)、 N-GSDMD和GAPDH的表达;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测各组HK-2细胞培养上清液中炎症因子白细胞介素-18(interleukin-18,IL-18)和白细胞介素-1β(interleukin-1β,IL-1β)的表达。结果 与Control组相比,高糖组HK-2细胞Tunel染色的阳性率明显增加,差异具有统计学意义(P<0.000 1),α-SMA蛋白和α-SMA mRNA表达量也显著增加(P<0.000 1),E-cadherin蛋白和Ecadherin mRNA表达量显著下降(P=0.000 6,P<0.000 1);HG组添加rhBMP-7后α-SMA蛋白和m RNA水平显著下降(P=0.011 7, P=0.002), E-cadherin蛋白和m RNA水平明显升高(P=0.001 5,P<0.000 1)。与对照组比较,HG组HK-2细胞GSDME/GSDMD蛋白的荧光信号明显增强。进而,HG组添加rhBMP-7后GSDME/GSDMD蛋白荧光信号显著减弱。同样,HG组HK-2细胞中焦亡相关蛋白caspase-1、caspase-4、caspase-5以及GSDMD的表达量显著高于对照组,差异明显(均P<0.001)。HG组添加rhBMP-7后焦亡相关蛋白的表达明显下降(分别P<0.001,P=0.002,P<0.001)。与对照组比较,HG组HK-2细胞培养上清液中IL-1β和IL-18蛋白浓度显著增加(均P<0.001)。进而导致rhBMP-7干预后HG组细胞上清液中IL-10和IL-1β蛋白�Objective To observe the effect of recombinant human bone morphogenetic protein-7(rhBMP-7)on pyroptosis and inflammatory response of human kidney-2(HK-2)cell after a stimulation of high glucose(HG).Methods HK-2 cell stimulated by high glucose(HG)was cultured in vitro for diabetic nephropathy modeling.There were four groups of normal glucose control(control),HG,control+rhBMP-7 and HG+rhBMP-7.The levels of E-cadherin,a-smooth muscle actin(a-SAM),Gasdermin D(GSDMD)and Gasdermin E(GSDME)were detected by immunofluorescence.Cell death was detected by TdT-mediated dUTP nick end labeling(TUNEL)stain.Western blot was utilized for detecting the expressions of pyroptosis-related proteins caspase-1/4/5,N-GSDMD and GAPDH.And the expressions of inflammatory factors of interleukin-18(IL-18)and interleukin-1β(IL-1β)in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Results Compared with control group,positive rate of TUNEL stain spiked sharply in HG group and the difference was statistically significant(P<0.0001).The expressions ofα-SMA protein/mRNA spiked significantly(P<0.0001)while the expression of E-cadherin protein/RNA dropped markedly(P=0.0006,P<0.0001);Furthermore,the level ofα-SMA protein/mRNA declined significantly in HG group after adding rhBMP-7(P=0.0117,P=0.002)while the level of E-cadherin protein/mRNA jumped significantly(P=0.0015,P<0.0001).Compared with control group,fluorescent signal of GSDME/GSDMD protein became significantly strengthened and then weakened in HG group after an addition of rhBMP-7.Similarly,the expression levels of caspase-1/4/5 and GSDMD of HG group were significantly higher than those of control group(P<0.001).The expressions of pyroptosis-related proteins were significantly down-regulated in HG group after adding rhBMP-7(P<0.001,P=0.002,P<0.001).Compared with control group,protein concentrations of IL-1βand IL-18 in supernatant of HG group jumped significantly(both P<0.001).Furthermore,after adding rhBMP-7,the protein concentrations of IL-10 and IL-1βin super

关 键 词：人肾皮质近曲小管上皮细胞 人重组骨形态发生蛋白-7 细胞焦亡 半胱氨酸蛋白酶-1/焦孔素D 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]