JMJD3通过TLR4/NF-κB信号通路调控脓毒症诱导的肾小管上皮细胞铁死亡  

JMJD3 regulating sepsis-induced ferroptosis of renal tubular epithelial cells via TLR4/NF-κB signaling pathway

作　　者：金光军 张建成 潘旭鸣 徐步海 陈嘉安 刘佳丽 何煜舟 JIN Guangjun;ZHANG Jiancheng;PAN Xuming;XU Buhai;CHEN Jiaan;LIU Jiali;HE Yuzhou(Department of Emergency Medicine,the Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310005,China)

机构地区：[1]浙江中医药大学附属第二医院急诊医学科,杭州310005

出　　处：《浙江医学》2025年第3期235-242,共8页Zhejiang Medical Journal

基　　金：国家中医药管理局科技司-浙江省中医药管理局共建科技计划项目(GZY-ZJ-KJ-24070);浙江省中医药科技计划项目(2022ZA079)。

摘　　要：目的探究Jumonji结构域蛋白3(JMJD3)通过Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路调控脓毒症诱导的人肾小管上皮细胞HK-2铁死亡的作用机制。方法将HK-2细胞分为对照组、脂多糖(LPS)组、LPS+空白对照的小干扰RNA(si-NC)组、LPS+JMJD3的小干扰RNA(si-JMJD3)组、LPS+si-JMJD3+铁死亡激动剂(erastin)组、LPS+si-JMJD3+空白对照的过表达载体(oe-NC)组和LPS+si-JMJD3+TLR4的过表达载体(oe-TLR4)组。对照组细胞正常培养,LPS组细胞用LPS处理,LPS+si-NC组细胞经LPS处理后转染si-NC,LPS+si-JMJD3组细胞经LPS处理后转染si-JMJD3,LPS+si-JMJD3+erastin组细胞经LPS处理并转染si-JMJD3后用erastin处理,LPS+si-JMJD3+oe-NC组细胞经LPS处理并转染si-JMJD3后再转染空载腺病毒,LPS+si-JMJD3+oe-TLR4组细胞经LPS处理并转染si-JMJD3后再转染过表达TLR4的腺病毒。采用蛋白质印迹法检测JMJD3、谷胱甘肽过氧化物酶4(Gpx4)、铁蛋白重链1(FTH-1)、TLR4、NF-κB和磷酸化NF-κB(p-NF-κB)蛋白表达水平,细胞计数试剂盒-8法检测细胞活性,流式细胞术检测细胞凋亡率,比色法检测细胞中Fe^(2+)水平。结果与对照组比较,LPS组JMJD3蛋白表达水平升高,细胞活性降低,细胞凋亡率和细胞中Fe^(2+)水平均升高(均P<0.05)。与LPS+si-NC组比较,LPS+si-JMJD3组JMJD3、Gpx4、TLR4和p-NF-κB蛋白表达水平均降低,FTH-1蛋白表达水平升高,细胞活性升高,细胞凋亡率和细胞中Fe^(2+)水平均降低(均P<0.05)。与LPS+si-JMJD3组相比,LPS+si-JMJD3+erastin组Gpx4蛋白表达水平升高,FTH-1蛋白表达水平降低,细胞活性降低,细胞凋亡率和细胞中Fe^(2+)水平均升高(均P<0.05)。与LPS+si-JMJD3+oe-NC组相比,LPS+si-JMJD3+oe-TLR4组TLR4和p-NF-κB蛋白表达水平均升高,细胞活性降低,细胞凋亡率和细胞中Fe^(2+)水平均升高(均P<0.05)。结论JMJD3能够通过激活TLR4/NF-κB信号通路,进而促进脓毒症诱导的肾小管上皮细胞的铁死亡。Objective To explore the mechanism of Jumonji domain-containing protein 3(JMJD3)regulating ferroptosis of human renal tubular epithelial cells HK 2 induced by sepsis through Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB)signaling pathway.Methods HK-2 cells were divided into the control group,lipopolysaccharide(LPS)group,LPS+transfection with empty small interfering RNA(siRNA)(si-NC)group,LPS+transfection with JMJD3 siRNA(si-JMJD3)group,LPS+si-JMJD3+ferroptosis inducer(erastin)group,LPS+si-JMJD3+transfection with empty adenovirus(oe-NC)group and LPS+si-JMJD3+transfection with adenovirus overexpressing TLR4(oe-TLR4)group.Cells in the control group were cultured normally,while those in the LPS,LPS+si-NC,LPS+si-JMJD3,LPS+si-JMJD3+erastin,LPS+si-JMJD3+oe-NC,and LPS+si JMJD3+oe-TLR4 groups were treated with LPS,empty siRNA after LPS treatment,transfection with si-JMJD3 after LPS treatment,LPS and transfection with si-JMJD3 followed by erastin,LPS and transfection with si JMJD3 followed by transfection with empty adenovirus,and LPS and transfection with si JMJD3 followed by transfection with adenovirus overexpressing TLR4.Western blot was used to detect the expression levels of JMJD3,glutathione peroxidase 4(Gpx4),ferritin heavy chain 1(FTH-1),TLR4,phosphorylated(p)-NF-κB and NF-κB proteins.Cell counting kit-8 method was used to detect cell activity.Flow cytometry was used to detect cell apoptosis rate.Colorimetry was used to detect the level of Fe^(2+)in cells.Results Compared with the control group,the expression level of JMJD3 protein in LPS group was significantly increased,the cell activity was significantly decreased,and the cell apoptosis rate and the level of Fe^(2+)in cells were significantly increased(all P<0.05).Compared with LPS+si-NC group,the expression levels of JMJD3,Gpx4,TLR4 and p-NFκB protein in LPS+si-JMJD3 group were significantly decreased,the expression level of FTH 1 protein was significantly increased,the cell activity was significantly increased,and the cell apoptosis rate and the

关 键 词：脓毒症 急性肾损伤 Jumonji结构域蛋白3 Toll样受体4/核因子-κB信号通路 铁死亡 

分 类 号：R73[医药卫生—肿瘤]