硫化氢调控PI3K/AKT介导的Nrf2/HO-1通路减轻缺氧/复氧HL-1心肌细胞损伤  

Alleviation of hypoxia/reoxygenation-induced HL-1 cardiomyocyte injury with hydrogen sulfide through regulating Nrf2/HO-1 signaling pathway mediated by PI3K/AKT

作　　者：郑晓斌 王朝 武海燕 姚冰琪 候书贤 Zheng Xiaobin;Wang Chao;Wu Haiyan;Yao Bingqi;Hou Shuxian(Department of Cardiology,Hospital of Cardiovascular Medicine of Shanxi Province,Taiyuan 030000,China;不详)

机构地区：[1]山西省心血管病医院心内科,太原030000

出　　处：《中国循证心血管医学杂志》2025年第1期32-38,共7页Chinese Journal of Evidence-Based Cardiovascular Medicine

基　　金：山西省心血管病医院科研激励计划(XYS20210109)。

摘　　要：目的探讨硫化氢(H2S)对缺氧/复氧(H/R)HL-1心肌细胞的保护作用,并分析其潜在机制。方法首先,利用RNA干扰技术筛选并验证核因子红系2相关因子2(Nrf2)的选择性靶向小干扰RNA(siRNA)。随后,建立H/R损伤的HL-1心肌细胞模型,并分为Control、H/R、H/R+硫氢化钠(NaHS)、H/R+NaHS+Nrf2 siRNA和H/R+Nrf2 siRNA五组进行相应处理。利用CCK-8法检测各组细胞活力,试剂盒测定H2S水平、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力,TUNEL染色观察各组细胞凋亡情况。最后,蛋白质印迹(Western blot)方法分析各组细胞中凋亡、炎症、Nrf2/血红素氧合酶-1(HO-1)通路及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)通路相关蛋白的表达。结果成功筛选出针对Nrf2的有效siRNA片段,并验证了其对Nrf2表达的显著抑制作用,随后按分组要求转染Nrf2 siRNA。与Control组相比,H/R组细胞活力,H2S与SOD水平,B细胞淋巴瘤2(Bcl-2)、胞核Nrf-2、HO-1蛋白表达水平、p-PI3K/PI3K、p-AKT/AKT、磷酸化的糖原合成酶激酶-3β(p-GSK-3β)/GSK-3β比值均显著降低,MDA含量、细胞凋亡程度、剪切型半胱天冬酶-3(cle-capase-3)、胞浆Nrf-2、胞核酪氨酸蛋白激酶Fyn(Fyn)、白细胞介素-6(IL-6)蛋白表达水平及磷酸化的核因子κB p65亚基(pp65)/p65比值均显著升高(P<0.05);NaHS预处理能够显著逆转上述变化,然而,转染Nrf2 siRNA则部分抵消了NaHS的保护作用。结论H2S通过激活PI3K/AKT/GSK-3β信号通路增加Nrf2表达,减轻HL-1心肌细胞缺氧/复氧诱导的氧化应激损伤。Objective To investigate the protective effect of hydrogen sulfide(H2S)on hypoxia/reoxygenation(H/R)-inducedHL-1 cardiomyocyte injury,and analyze its potential mechanism.Methods Firstly,selective targeting siRNA of nuclear factor E2 related factor 2(Nrf2)was screened and validated by using RNA interference technology.Then,an H/R injury model of HL-1 cardiomyocytes was established and divided into 5 groups:control group,H/R group,H/R+NaHS group,H/R+NaHS+Nrf2 siRNA group and H/R+Nrf2 siRNA group.The viability of cardiomyocytes was detected by using CCK-8 assay,levels of H2S and malondialdehyde(MDA)and activity of superoxide dismutase(SOD)were detected by using assay kits,and cardiomyocyte apoptosis was observed by using TUNEL staining.Finally,apoptosis,inflammation and protein expressions of Nrf2/heme oxygenase 1(Nrf2/HO-1)signaling pathway and PI3K/AKT signaling pathway were analyzed by using Western blotting assay in all groups.Results The effective siRNA fragments targeting Nrf2 were successfully screened,and their significant inhibitory effects on Nrf2 expression were validated,followed by transfection of Nrf2 siRNA according to grouping requirements.The viability of cardiomyocytes,levels of H2S and SOD,protein expressions of Bcl-2,Nrf2,HO-1,p-PI3K/PI3K,p-AKT/AKT,and ratio of p-GSK-3β/GSK-3βdecreased significantly,and MDA,apoptosis severity,protein expressions of cleaved capase-3,Nrf-2,nucleus Fyn and IL-6,and ratio of p-p65/p65 increased significantly in H/R group compared with control group.The pretreatment of NaHS reversed significantly the above changes,but Nrf2 siRNA transfection counteracted partially NaHS protective effect.Conclusion H2S can improve Nrf2 expression and relieve oxidative stress injury induced by H/R inHL-1 cardiomyocytes through activating PI3K/AKT/GSK-3βsignaling pathway.

关 键 词：硫化氢 缺血再灌注损伤 氧化应激 PI3K/AKT信号通路 Nrf2/HO-1信号通路 

分 类 号：R318.11[医药卫生—生物医学工程]