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.

参考文献

[1] Singbartl K, Kellum JA. AKI in the ICU: definition, epidemiology, risk stratification, and outcomes [J]. Kidney Int, 2012, 81(9): 819-825.
[2] Xu X, Nie S, Liu Z, et al. Epidemiology and clinical correlates of AKI in Chinese hospitalized adults [J]. Clin J Am Soc Nephrol, 2015, 10(9): 1510-1518.
[3] Liu KD, Goldstein SL, Vijayan A, et al. AKI!Now initiative: recommendations for awareness, recognition, and management of AKI [J]. Clin J Am Soc Nephrol, 2020, 15(12): 1838-1847.
[4] Farrar A. Acute kidney injury [J]. Nurs Clin North Am, 2018, 53(4): 499-510.
[5] He L, Wei Q, Liu J, et al. AKI on CKD: heightened injury, suppressed repair, and the underlying mechanisms [J]. Kidney Int, 2017, 92(5): 1071-1083.
[6] Sharma N, Anders HJ, Gaikwad AB. Fiend and friend in the renin angiotensin system: an insight on acute kidney injury [J]. Biomed Pharmacother, 2019, 110: 764-774. doi: 10.1016/j.biopha.2018.12.018.
[7] Verano-Braga T, Martins ALV, Motta-Santos D, et al. ACE2 in the renin-angiotensin system [J]. Clin Sci(Lond), 2020, 134(23): 3063-3078.
[8] Yamamoto K, Takeshita H, Rakugi H. ACE2, angiotensin 1-7 and skeletal muscle: review in the era of COVID-19 [J]. Clin Sci(Lond), 2020, 134(22): 3047-3062.
[9] Hikmet F, Méar L, Edvinsson Å, et al. The protein expression profile of ACE2 in human tissues [J]. Mol Syst Biol, 2020, 16(7): e9610. doi: 10.15252/msb.20209610.
[10] Nath KA, Singh RD, Grande JP, et al. Expression of ACE2 in the intact and acutely injured kidney [J]. Kidney360, 2021, 2(7): 1095-1106.
[11] Fang F, Liu GC, Zhou X, et al. Loss of ACE2 exacerbates murine renal ischemia-reperfusion injury [J]. PLoS One, 2013, 8(8): e71433. doi: 10.1371/journal.pone.0071433.
[12] Yan S, Ye P, Aleem MT, et al. Mesenchymal stem cells overexpressing ACE2 favorably ameliorate LPS-induced inflammatory injury in mammary epithelial cells [J]. Front Immunol, 2022, 12: 796744. doi: 10.3389/fimmu.2021.796744.
[13] Rodrigues Prestes TR, Rocha NP, Miranda AS, et al. The anti-inflammatory potential of ACE2/angiotensin-(1-7)/Mas receptor axis: evidence from basic and clinical research [J]. Curr Drug Targets, 2017, 18(11): 1301-1313.
[14] Kuba K, Yamaguchi T, Penninger JM. Angiotensin-converting enzyme 2(ACE2)in the pathogenesis of ARDS in COVID-19 [J]. Front Immunol, 2021, 12: 732690. doi: 10.3389/fimmu.2021.732690.
[15] Kaltenecker CC, Domenig O, Kopecky C, et al. Critical role of neprilysin in kidney angiotensin metabolism [J]. Circ Res, 2020, 127(5): 593-606.
[16] Imai Y, Kuba K, Ohto-Nakanishi T, et al. Angiotensin-converting enzyme 2(ACE2)in disease pathogenesis [J]. Circ J, 2010, 74(3): 405-410.
[17] Kumar R, Thomas CM, Yong QC, et al. The intracrine renin-angiotensin system [J]. Clin Sci(Lond), 2012, 123(5): 273-284.
[18] Herr D, Bekes I, Wulff C. Local renin-angiotensin system in the reproductive system [J]. Front Endocrinol(Lausanne), 2013, 4: 150. doi: 10.3389/fendo.2013.00150.
[19] Liu C, Chen K, Wang H, et al. Gastrin attenuates renal ischemia/reperfusion injury by a PI3K/Akt/Bad-mediated anti-apoptosis signaling [J]. Front Pharmacol, 2020, 11: 540479. doi: 10.3389/fphar.2020.540479.
[20] Jun W, Benjanuwattra J, Chattipakorn SC, et al. Necroptosis in renal ischemia/reperfusion injury: a major mode of cell death? [J]. Arch Biochem Biophys, 2020, 689: 108433. doi: 10.1016/j.abb.2020.108433.
[21] Daemen MA, de Vries B, Buurman WA. Apoptosis and inflammation in renal reperfusion injury [J]. Transplantation, 2002, 73(11): 1693-1700.
[22] Daemen MA, vant Veer C, Denecker G, et al. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation [J]. J Clin Invest, 1999, 104(5): 541-549.
[23] He F, Ru X, Wen T. NRF2, a transcription factor for stress response and beyond[J]. Int J Mol Sci, 2020, 21(13): 4777. doi: 10.3390/ijms21134777.
[24] Bellezza I, Giambanco I, Minelli A, et al. Nrf2-Keap1 signaling in oxidative and reductive stress[J]. Biochim Biophys Acta Mol Cell Res, 2018, 1865(5): 721-733.
[25] Gallorini M, Petzel C, Bblay C, et al. Activation of the Nrf2-regulated antioxidant cell response inhibits HEMA-induced oxidative stress and supports cell viability[J]. Biomaterials, 2015, 56: 114-128. doi: 10.1016/j.biomaterials.2015.03.047.
[26] Chiang SK, Chen SE, Chang LC. A dual role of heme oxygenase-1 in cancer cells[J]. Int J Mol Sci, 2018, 20(1): 39. doi: 10.3390/ijms20010039.
[27] Mansouri A, Reiner Ž, Ruscica M, et al. Antioxidant effects of statins by modulating Nrf2 and Nrf2/HO-1 signaling in different diseases[J]. J Clin Med, 2022, 11(5): 1313. doi: 10.3390/jcm11051313.

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