微小RNA-23靶向线粒体分裂蛋白1调控炎症反应和氧化应激保护脓毒症肾小管上皮细胞损伤

张利; 葛亮; 马小力; 马菁菁; 张阳; 孙林春

doi:10.3969/j.issn.1671-2390.2024.03.006

微小RNA-23靶向线粒体分裂蛋白1调控炎症反应和氧化应激保护脓毒症肾小管上皮细胞损伤

miR-23 regulates inflammation and oxidative stress by targeting FIS1 for protecting renal tubular epithelial cell damage in sepsis

摘要

摘要:
目的探讨微小RNA-23（microRNA-23，miR-23）和线粒体分裂蛋白1（fission protein 1，FIS1）在脓毒症肾小管上皮细胞中的作用关系以及miR-23靶向FIS1对细胞炎症反应和氧化应激的调控。
方法体外培养人肾小管上皮细胞（human kidney-2，HK-2），用脂多糖（lipopolysaccharide，LPS）诱导建立脓毒症模型；利用双荧光素酶报告基因验证miR-23和FIS1的相互作用。将HK-2细胞采用随机数字表法随机分组，用miR-23的模拟物/抑制物和FIS1的真核表达载体/小干扰RNA单独或联合处理，细胞计数试剂检测细胞增殖，流式细胞技术检测细胞凋亡和活性氧（reactive oxygen species，ROS）水平、丙二醛（malondialdehyde，MDA）试剂盒检测细胞MDA水平，酶联免疫吸附试验检测肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素（interleukin， IL）1β、IL-6和IL-10水平。
结果 LPS诱导的HK-2细胞miR-23 mRNA表达量为对照组的（0.60±0.12）倍（P＜0.05），FIS1 mRNA表达量为对照组的（2.16±0.21）倍（P＜0.05），FISI蛋白表达水平是对照组的（2.69±0.28）倍（P＜0.05）。miR-23可以与FIS1的3 '-UTR区形成互补结合，负向调控FIS1。miR-23 mimic组的TNF-α、IL-1、IL-6分别为（0.021±0.003）、（0.0310±0.007）和（0.017±0.006）ng/ml，FIS1 siRNA组的TNF-α、IL-1、IL-6分别为（0.015±0.004）、（0.043±0.007）和（0.020±0.008）ng/ml，均低于空白对照组（P＜0.05），两组的IL-10分别为（0.325±0.011）和（0.376±0.018）ng/ml，高于空白对照组（P＜0.05）。miR-23 inhibitor组的TNF-α和IL-6分别为（0.117±0.015）和（0.096±0.007）ng/ml，pcDNA3.1-FIS1组TNF-α和IL-6分别为（0.168±0.024）和（0.147±0.030）ng/ml，均高于空白对照组（P均＜0.05），miR-23 inhibitor组和pcDNA3.1-FIS1组的IL-10分别为（0.149±0.012）和（0.121±0.024） ng/ml，均低于空白对照组（P＜0.05）。miR-23 mimic+pcDNA3.1-FIS1组TNF-α、IL-1、IL-6分别为（0.060±0.010）、（0.084±0.009）和（0.048 ±0.008）ng/ml，均高于miR-23 mimic组的（0.021±0.003）、（0.0310±0.007）和（0.017±0.006）ng/ml（P＜0.05），IL-10为（0.149±0.025）ng/ml，低于miR-23 mimic组的（0.325±0.011）ng/ml（P＜0.05）。miR-23 inhibitor+FIS1 siRNA组TNF-α、IL-1、IL-6分别为（0.049±0.007）、（0.056±0.008）和（0.037± 0.006）ng/ml，均低于miR-23 inhibitor组的（0.117±0.015）、（0.085±0.015）和（0.096±0.007）ng/ml（P＜0.05），IL-10为（0.325±0.027）ng/ml，高于miR-23 inhibitor组的（0.149±0.012）ng/ml（P＜0.05）。MiR-23 mimic组和FIS1 siRNA组ROS分别为（1 328±84）和（1 300±70），均低于空白对照组（P＜0.05），miR-23 inhibitor组和pcDNA3.1-FIS1组ROS分别为（1825±80）和（1930±105），均高于空白对照组（P＜0.05）。miR-23 mimic+pcDNA3.1-FIS1组ROS为（1538±96），高于miR-23 mimic组的（1328±84）（P＜0.05）；miR-23 inhibitor+FIS1 siRNA组ROS为（1490±70），低于miR-23 inhibitor组的（1825±80）（P＜0.05）。MiR-23 mimic组和FIS1 siRNA组MDA分别为（17.34±2.18）mol/L和（12.05±2.47）mol/L，均低于空白对照组（P＜0.05）；miR-23 inhibitor组和pcDNA3.1-FIS1组MDA分别为（52.20±7.48） mol/L和（46.25±6.50）mol/L，均高于空白对照组（P＜0.05）；miR-23 mimic+pcDNA3.1-FIS1组MDA为（35.44±3.03）mol/L，高于miR-23 mimic组的（17.34±2.18） mol/L（P＜0.05）；miR-23 inhibitor+FIS1 siRNA组MDA为（40.26±4.87）mol/L，低于miR-23 inhibitor组的（52.20±7.48）mol/L（P＜0.05）。miR-23 mimic组和FIS1 siRNA组细胞增殖率分别为（183±14）%和（171±11）%，均高于空白对照组（P＜0.05），两组凋亡率分别为（7.89±1.21）%和（11.22±2.98）%，均低于空白对照组（P＜0.05）；miR-23 inhibitor组和pcDNA3.1-FIS1组细胞增殖率分别为（134±10）%和（127±9）%，均低于空白对照组（P＜0.05），凋亡率分别为（23.22±2.54）%和（28.15±3.12）%，均高于空白对照组（P＜0.05）；miR-23 mimic+pcDNA3.1-FIS1组细胞增殖率为（148±12）%，低于miR-23 mimic组的（183±14）%（P＜0.05），凋亡率为（17.02±2.29）%，高于miR-23 mimic组的（7.89±1.21）%（P＜0.05）；miR-23 inhibitor+FIS1 siRNA组细胞增殖率为（160±8）%，高于miR-23 inhibitor组的（134±10）%（P＜0.05），凋亡率为（14.27±2.00）%，低于miR-23 inhibitor组的（23.22±2.54）%（P＜0.05）。
结论 LPS诱导的肾小管上皮细胞miR-23和FIS1异常表达与细胞的炎症反应和氧化应激过程有关。miR-23通过靶向负调控FIS1，减轻HK-2细胞的炎症反应和氧化应激水平，对脓毒症肾小管上皮细胞损伤起保护作用。

Abstract:
Objective To explore the interaction between microRNA-23 （miR-23） and mitochondrial fission protein 1 （FIS1） in human kidney-2 （HK-2） cells and examine the effect of miR-23 on inflammatory response and oxidative stress by targeting FIS1.
Methods HK-2 cells were cultured in vitro and induced by lipopolysaccharide （LPS） for modeling of sepsis. Dual luciferase reporter assay was utilized for verifying the interaction between miR-23 and FIS1. HK-2 cells were randomized into groups of miR-23 mimic/inhibitor and eukaryotic expression vector and small interfering RNA of FIS1 alone or in a combination. Cell proliferation was detected by cell counting kit-8 （CCK-8） assay and apoptosis and reactive oxygen species （ROS） were examined by flow cytometry. Malondialdehyde （MDA） was detected by MDA kit and the levels of tumor necrosis factor t （TNF-α）, interleukin （IL）-1β and IL-6/10 were measured by enzyme-linked immunosorbent assay（ELISA）.
Results The miR-23 mRNA in HK-2 cells induced by LPS was (0.60±0.12) folds that of control group(P<0.05) while FIS1 mRNA was (2.16±0.21) folds that of control group(P<0.05), FISI protein level was (2.69±0.28) folds that of control group(P<0.05). And miR-23 could form complementary binding to 3'-UTR of FIS1 and negatively regulate FIS1. TNF-α, IL-1 and IL-6 of miR-23 mimic group were (0.021±0.003), (0.0310±0.007) and (0.017±0.006) mg/g protein and (0.015±0.004), (0.043±0.007) and (0.020±0.008) mg/g protein in FIS1 siRNA group. TNF-α, IL-1 and IL-6 in miR-23 mimic and FIS1 siRNA groups were lower than those of blank control group (P<0.05). The levels of IL-10 in miR-23 mimic and FIS1 siRNA groups were (0.325±0.011) and (0.376±0.018) mg/g protein and both were higher than those of control group (P<0.05). TNF-α and IL-6 of miR-23 inhibitor group were (0.117±0.015) and (0.096±0.007) mg/g protein versus (0.168±0.024) and (0.147±0.030) mg/g protein in pcDNA3.1-FIS1 group. TNF-α and IL-6 of miR-23 inhibitor and pcDNA3.1-FIS1 groups were higher than those of blank control group (P<0.05). IL-10 of miR-23 inhibitor and pcDNA3.1-FIS1 groups was (0.149±0.012) and (0.121±0.024) mg/g protein and both were lower than those of blank control group (P<0.05). TNF-α, IL-1 and IL-6 of miR-23 mimic+pcDNA3.1-FIS1 group were (0.060±0.010), (0.084±0.009) and (0.048±0.008) mg/g protein and they were higher than (0.021±0.003), (0.0310±0.007) and (0.017±0.006) mg/g protein of miR-23 mimic group(P<0.05) while IL-10 (0.149±0.025) mg/g protein was lower than (0.325±0.011) mg/g protein of miR-23 mimic group(P<0.05). TNF-α, IL-1 and IL-6 were (0.049±0.007), (0.056±0.008) and (0.037±0.006) mg/g protein in miR-23 inhibitor+FIS1 siRNA group and they were lower than (0.117±0.015), (0.085±0.015) and (0.096±0.007) mg/g protein of miR-23 inhibitor group(P<0.05). And IL-10 (0.325±0.027) mg/g protein was higher than (0.149±0.012) mg/g protein of miR-23 inhibitor group(P<0.05). ROS of miR-23 mimic and FIS1 siRNA groups were (1328±84) and (1300±70) and both were lower than those of blank control group(P<0.05). ROS of miR-23 inhibitor and pcDNA3.1-FIS1 group were (1825±80) and (1930±105) and both were higher than those of blank control group(P<0.05). ROS of miR-23 mimic+pcDNA3.1-FIS1 group was (1538±96) was higher than (1 328±84) of miR-23 mimic group(P<0.05). ROS of miR-23 inhibitor+FIS1 siRNA group was (1490±70) and it was lower than (1825±80) of miR-23 inhibitor group(P<0.05). MDA of miR-23 mimic and FIS1 siRNA groups were (17.34±2.18) mol/L and (12.05±2.47) mol/L and both were lower than those of blank control group(P<0.05). MDA of miR-23 inhibitor and pcDNA3.1-FIS1 groups were (52.20±7.48) mol/L and (46.25±6.50) mol/L and both were higher than those of blank control group(P<0.05). MDA of miR-23 mimic+pcDNA3.1-FIS1 group was (35.44±3.03) mol/L and it was higher than (17.34±2.18) mol/L of miR-23 mimic group(P<0.05). MDA of miR-23 inhibitor+FIS1 siRNA group was (40.26±4.87) mol/L and it was lower than (52.20±7.48) mol/L of miR-23 inhibitor group(P<0.05). Proliferation rate of miR-23 mimic and FIS1 siRNA group were (183±14)% and (171±11)% and both were higher than those of blank control group (P<0.05). Apoptotic rates were (7.89±1.21) % and (11.22±2.98)% and both were lower than those of blank control group(P<0.05). Proliferation rate of miR-23 inhibitor and pcDNA3.1-FIS1 groups were (134±10)% and (127±9)% and both were lower than those of blank control group(P<0.05). Apoptotic rate was (23.22±2.54)% and (28.15±3.12)% and it was higher than that of blank control group(P<0.05). Proliferation rate of miR-23 mimic+pcDNA3.1-FIS1 group was (148±12)% and it was lower than (183±14)% of miR-23 mimic group(P<0.05). Apoptotic rate was (17.02±2.29)% and it was higher than (7.89±1.21)% of miR-23 mimic group(P<0.05). Proliferation rate of miR-23 inhibitor+FIS1 siRNA group was (160±8)% and it was higher than (134±10)% of miR-23 inhibitor group(P<0.05). Apoptotic rate was (14.27±2.00)% and it was lower than (23.22±2.54)% of miR-23 inhibitor group (P<0.05).
Conclusion Abnormal LPS-induced expression of miR-23/FIS1 is relevant to inflammatory response and oxidative stress process in renal tubular epithelial cells. And miR-23 may reduce the inflammation and oxidative stress by negatively regulating FIS1 in HK-2 cells. It plays a protective role in renal tubular epithelial cell injury in sepsis.

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