Expression and effect of RIP3,HMGB1 in necroptosis of HK-2 cells
Cao Yanwei
Zhang Shiqiu
Wang Chuanling
Yu Rong
Zhu Yongjun
Abstract：Objective To construct the model of HK-2 cell programmed necrosis of human renal tubular epithelium(HK-2),and observe the expression and role of receptor interaction protein 3(RIP3)and high mobility group protein 1(HMGB1)in HK-2 cell programmed necrosis.Methods The experiment will be conducted in the Laboratory of Hainan Med-ical University from May 2022 to January 2024.HK-2 cells were divided into control group,TNF-α group,TNF-α+Nec-ls group,TNF-α+GSK'872 group and TNF-α+NSA group.Flow cytometry was used to detect apoptosis and necrosis rates in each group of cells.TUNEL+RIP3 fluorescence double staining combined with laser confocal microscopy was used to detect the percentage of TUNEL+RIP3 positive cells.The expression of HMGB1 protein was detected by ELISA.The mR-NA expression levels of RIP3 and HMGB1 were detected by qRT-PCR.The expression levels ofRIP3 and HMGB1 were de-tected by Western blot.Results Compared with control group,the apoptosis and necrosis rate of HK-2 cells,the percent-age of TUNEL+RIP3 double positive cells,the expression level of HMGB1 protein and the mRNA and protein expression levels ofRIP3 and HMGB1 in TNF-α group were significantly increased(q/P=56.786＜0.001,47.963/＜0.001,24.186/＜0.001,5.020/0.034,4.708/0.047,46.495/＜0.001,26.837/＜0.001).Compared to the TNF-α group,apoptosis and necrosis rate of HK-2 cells,percentage of TUNEL+/RIP3+double positive cells,HMGB1 protein expression and mRNA and protein expression levels of RIP3,HMGB1 in TNF-α+Nec-1s group,TNF-α+GSK'872 group and TNF-α+NSA group were significantly de-creased(The apoptosis and necrosis rates of HK-2 cells:q/P=44243/＜0.001,37.666/＜0.001,30324/＜0.001;percentage of TUNEL+/RIP3+double positive cells:q/P=35.176/＜0.001,28.461/＜0.001,21.104/＜0.001;HMGB1 protein expression:q/P=39.043/＜0.001,39.412/＜0.001,41.510/＜0.001;RIP3 mRNA expression:q/P=13.982/＜0.001,5.386/0.022,8.811/0.001,HMGB1 mRNA expression:q/P=7219/0.003,6318/0.008,4.658/0.049,RIP3 protein expression:q/P=62.436/＜0.001,46.495/＜0.001,39.853/＜0.001,HMGB1 protein expression:q/P=20.982/＜0.001,20.006/＜0.001,28.301/＜0.001).There were statistical-ly significant differences in apoptosis and necrosis rate,percentage of TUNEL+/RIP3+double positive cells and expres-sion level of RIP3 protein in TNF-α+Nec-1s group,TNF-α+GSK'872 group and TNF-α+NSA group(P＜0.05).Conclu-sion TNF-α can induce RIP3-mediated necroptosis in HK-2 cells and release HMGB1 molecules.
Keywords：NecroptosisTumor necrosis factor-αReceptor-interacting protein 3High mobility group box-1 pro-tein
Publication Date：2025-02-17
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)