ACE2基因通过调控Nrf2/HO-1通路改善肾缺血再灌注损伤

doi:10.6040/j.issn.1671-7554.0.2022.1226

摘要/Abstract

摘要： 目的探讨血管紧张素转化酶2(ACE2)对缺氧复氧诱导的肾小管上皮细胞HK-2氧化应激、炎症、凋亡及核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路的影响。方法将ACE2慢病毒转染HK-2细胞,按照实验需要分为常氧组(Control组)、缺氧复氧模型组(H/R组)、缺氧复氧转染阴性对照慢病毒组(H/R-NC组)和缺氧复氧转染ACE2慢病毒组(H/R-ACE2组)。细胞经H/R处理后,通过CCK-8法检测细胞活力;RT-PCR及ELISA法检测炎症因子白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)水平;比色法检测超氧化物歧化酶(SOD)、丙二醛(MDA)表达水平;Western blotting法检测胱天蛋白酶3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2关联X蛋白(Bax)、Nrf2、HO-1的蛋白水平。采用Nrf2抑制剂ML385以及HO-1抑制剂SnPPIX抑制Nrf2/HO-1通路,Western blotting法检测Caspase-3、Bcl-2、Bax、Nrf2、HO-1的蛋白表达水平变化,比色法检测SOD和MDA表达变化。结果与Control组相比,H/R组细胞活力降低(t=7.58,P<0.001),MDA含量和炎症因子IL-6、TNF-α和IL-1β表达水平以及细胞凋亡相关蛋白Caspase-3、Bax蛋白水平均增加(t_MDA=11.08,P_MDA<0.001;t_PCR-IL-6=5.82,P_PCR-IL6<0.001;t_PCR-TNF-α=7.69,P_PCR-TNF-α<0.001;t_PCR-IL-1β=4.80,P_PCR-IL-1β=0.001;t_ELISA-IL-6=34.11,P_ELISA-IL-6<0.001;t_ELISA-TNF-α=14.12,P_ELISA-TNF-α<0.001;t_ELISA-IL-1β=9.63,P_ELISA-IL-1β<0.001;t_Caspase-3=2.73,P_Caspase-3=0.026;t_Bax=27.75,P_Bax<0.001),SOD活性、Bcl-2和ACE2蛋白水平下降(t_SOD=7.74,P_SOD<0.001;t_Bcl-2=75.49,P_Bcl-2<0.001;t_ACE2=11.41,P_ACE2<0.001)。与H/R组相比,H/R-ACE2组细胞活力增加(t=3.61,P=0.002),MDA含量和炎症因子IL-6、TNF-α和IL-1β表达水平以及细胞凋亡相关蛋白Caspase-3、Bax蛋白水平均下降(t_MDA=6.15,P_MDA<0.001;t_PCR-IL-6=3.34,P_PCR-IL-6=0.006;t_PCR-TNF-α=3.65,P_PCR-TNF-α=0.007;t_PCR-IL-1β=4.06,P_PCR-IL-1β=0.004;t_ELISA-IL-6=14.62,P_ELISA-IL-6<0.001;t_ELISA-TNF-α=10.42,P_ELISA-TNF-α<0.001;t_ELISA-IL-1β=8.65,P_ELISA-IL-1β<0.001;t_Caspase-3=3.74,P_Caspase-3=0.006;t_Bax=30.52,P_Bax<0.001),SOD活性、Bcl-2和ACE2蛋白水平增加(t_SOD=3.58,P_SOD=0.007;t_Bcl-2=63.86,P_Bcl-2<0.001;t_ACE2=58.72,P_ACE2<0.001),Nrf2/HO-1信号通路被激活蛋白水平增加(t_Nrf2=44.55,P_Nrf2<0.001;t_HO-1=14.19,P_HO-1<0.001)。然而ML385和SnPPIX处理会抑制ACE2基因过表达在H/R中HK-2细胞的保护作用(F_Bax=11.02,P_Bax=0.003;F_Bcl-2=21.48,P_Bcl-2<0.001;F_Caspase-3=20.80,P_Caspase-3<0.001;F_SOD=133.49,P_SOD<0.001;F_MDA=14.06,P_MDA=0.001)。结论 ACE2在HK-2细胞缺氧复氧损伤中具有抑制氧化应激、调节炎症、改善凋亡的作用,Nrf2/HO-1信号通路发挥重要作用。

关键词: 血管紧张素转化酶2, 缺血再灌注损伤, 氧化应激, 凋亡, 信号通路

Abstract: Objective To investigate the effects of angiotensin-converting enzyme 2(ACE2)on the oxidative stress, inflammation, apoptosis and the nuclear factor E2-related factor 2(Nrf2)/ heme oxygenase 1(HO-1)signaling pathway in renal tubular epithelial cells(HK-2)induced by hypoxia/reoxygenation(H/R). Methods HK-2 cells were transfected with ACE2 lentivirus, and divided into the control group, H/R group, H/R-NC group, and H/R-ACE2 group. After H/R treatment, cell viability was measured with CCK-8 assay; the levels of inflammatory factors including interleukin-6(IL-6), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were measured with ELISA and RT-PCR; superoxide dismutase(SOD)and malondialdehyde(MDA)levels were measured with colorimetric assay; protein levels of Caspase-3, Bcl-2, Bax, Nrf2, and HO-1 were measured with Western blotting. After ML385 and SnPPIX were used to inhibit the Nrf2/HO-1 pathway, changes in the expressions of Caspase-3, Bcl-2, Bax, Nrf2 and HO-1 were detected with Western blotting, and changes in SOD and MDA levels were detected with colorimetry. Results Compared with the control group, the H/R group showed lower cell viability(t=7.58, P<0.001), higher expression levels of MDA, IL-1β, IL-6, TNF-α, Caspase-3 and Bax(t_MDA=11.08, P_MDA<0.001; t_PCR-IL-6=5.82, P_PCR-IL6<0.001; t_PCR-TNF-α=7.69, P_PCR-TNF-α<0.001; t_PCR-IL-1β=4.80, P_PCR-IL-1β=0.001; t_ELISA-IL-6=34.11, P_ELISA-IL-6<0.001; t_ELISA-TNF-α=14.12, P_ELISA-TNF-α<0.001; t_ELISA-IL-1β=9.63, P_ELISA-IL-1β<0.001; t_Caspase-3=2.73, P_Caspase-3=0.026; t_Bax=27.75, P_Bax<0.001), but lower levels of SOD, Bcl-2 and ACE2(t_SOD=7.74, P_SOD<0.001; t_Bcl-2=75.49, P_Bcl-2<0.001; t_ACE2=11.41, P_ACE2<0.001). Compared with the H/R group, the H/R-ACE2 group had higher cell viability(t=3.61, P=0.002), lower levels of MDA, IL-1β, IL-6, TNF-α, Caspase-3 and Bax(t_MDA=6.15, P_MDA<0.001; t_PCR-IL-6=3.34, P_PCR-IL6=0.006; t_PCR-TNF-α=3.65, P_PCR-TNF-α=0.007; t_PCR-IL-1β=4.06, P_PCR-IL-1β=0.004; t_ELISA-IL-6=14.62, P_ELISA-IL-6<0.001; t_ELISA-TNF-α=10.42, P_ELISA-TNF-α<0.001; t_ELISA-IL-1β=8.65, P_ELISA-IL-1β<0.001; t_Caspase-3=3.74, P_Caspase-3=0.006; t_Bax=30.52, P_Bax<0.001), higher levels of SOD, Bcl-2, ACE2, Nrf2, and HO-1(t_SOD=3.58, P_SOD=0.007; t_Bcl-2=63.86, P_Bcl-2<0.001; t_ACE2=58.72, P_ACE2<0.001; t_Nrf2=44.55, P_Nrf2<0.001; t_HO-1=14.19, P_HO-1<0.001). However, ML385 and SnPPIX inhibited the protective effects of ACE2 gene overexpression on HK-2 cells under H/R(F_Bax=11.02, P_Bax=0.003; F_Bcl-2=21.48, P_Bcl-2<0.001; F_Caspase-3=20.80, P_Caspase-3<0.001; F_SOD=133.49, P_SOD<0.001; F_MDA=14.06, P_MDA=0.001). Conclusion ACE2 inhibits the oxidative stress, regulates inflammation, and ameliorates apoptosis in HK-2 cells under H/R, and the Nrf2/HO-1 signaling pathway may play an important role in this progress.

Key words: Angiotensin-converting enzyme 2, Ischemia-reperfusion injury, Oxidative stress, Apoptosis, Signaling pathway

中图分类号:

R574

张嘉颖,宿荣允,王英惠,王洪刚,柳刚. ACE2基因通过调控Nrf2/HO-1通路改善肾缺血再灌注损伤[J]. 山东大学学报 (医学版), 2023, 61(4): 1-9.

ZHANG Jiaying, SU Rongyun, WANG Yinghui, WANG Honggang, LIU Gang. ACE2 gene protects against renal ischemia-reperfusion injury by regulating the Nrf2/HO-1 signaling pathway[J]. Journal of Shandong University (Health Sciences), 2023, 61(4): 1-9.

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