Nrf2／HO-1信号通路在饱和氢盐水减轻大鼠原位肝移植术后急性肾损伤中的作用

Role of Nrf2/HO-1 signaling pathway in reduction of acute kidney injury following orthotopic liver transplantation by hydrogen-rich saline in rats

目的探讨核因子E2相关因子2，血红素加氧酶-1（Nrf2／HO-1）信号通路在饱和氢盐水减轻大鼠原位肝移植术后急性肾损伤中的作用。方法清洁级健康成年雄性SD大鼠32只，体重220—250g，采用随机数字表法分为4组（n=8）：假手术组（S组）：行单纯开关腹操作，游离相应血管；原位肝移植组（OLT组）：建立大鼠自体原位肝移植模型，并于门静脉阻断前5min经肝下下腔静脉缓慢注射生理盐水6ml／kg；饱和氢盐水组（HS组）：于门静脉阻断前5min经肝下下腔静脉缓慢注射饱和氢盐水6ml／kg；全反式维甲酸组（ATRA组）连续2d天腹腔注射Nrf2抑制剂全反式维甲酸7mg／kg，最后一次给药后16h建立自体原位肝移植模型，其他处理同HS组。于肝脏再灌注6h时取血样，测定血清BUN、Cr、IL-10和TNF-α浓度；取血结束后，取肾组织，测定MDA含量和SOD活性，并观察肾组织病理学结果，进行损伤评分；采用RT-PCR法检测肾组织HO-1、Bcl-2和Bax的mR-NA的表达水平，采用Westernblot法检测肾组织HO-1的表达水平。结果与S组比较，OLT组血清BUN、Cr和TNF-α浓度升高，血清IL-10浓度降低，肾组织MDA含量和肾小管损伤评分升高，SOD活性降低，肾组织HO-1及其mRNA、Bcl-2和Bax的mRNA表达上调（P〈0．05）；与OLT组比较，HS组血清BUN、Cr和TNF-α浓度降低，血清IL-10浓度升高，肾组织MDA含量和肾小管损伤评分降低，SOD活性升高，HO-1及其mRNA和Bcl-2mRNA表达上调，BaxmRNA表达下调（P〉0．05）；与HS组比较，ATRA组血清BUN、Cr和TNF-α浓度升高，血清IL-10浓度降低，肾组织MDA含量和肾小管损伤评分升高，SOD活性降低，HO-1及其mRNA、Bcl-2mRNA表达下调，BaxmRNA表达上调（P〈0．05）。结论饱和氢盐水减轻大鼠原位肝移植术后急性肾损伤的机制可能与激活Nrf2／HO-1信号通路有关。

Objective To investigate the role of nuclear factor erythroid 2-related factor 2 （Nrf2） / heme oxygenase-1 （HO-1） signaling pathway in reduction of acute kidney injury following orthotopic liver transplantation （OLT） by hydrogen-rich saline in rats. Methods Thirty-two healthy adult male SpragueDawley rats, weighing 220-250 g, were randomly assigned into 4 groups （n= 8 each） using a random number table： sham operation group （ S group） , OLT group, hydrogen-rich saline group （ HS group） , and all-trans retinoic acid （ATRA） group. Laparotomy was performed, and the related blood vessels were isolated in group S. The model of orthotopic autologous liver transplantation was established in OLT, HS and ATRA groups. Normal saline and hydrogen-rich saline 6 ml/kg were injected through the inferior vena cava at 5 rain before the portal vein was clamped in OLT and HS groups, respectively. In group ATRA, Nrf2 inhibitor ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days, the model of orthotopic autologous liver transplantation was established at 16 h after the last injection of ATRA, and the other treatments were similar to those previously described in group HS. At 6 h of reperfusion, blood samples were collected for determination of serum blood urea nitrogen （BUN） , creatinine （Cr）, interleukin- 10 （IL-10） and tumor necrosis factor-alpha （TNF-α） concentrations. After blood sampling, the lungs were removed for determination of malondialdehyde （MDA） content, superoxide dismutase （SOD） activity, expression of HO-1, Bcl-2 and Bax mRNA （by using real-time reverse transcriptase polymerase chain reaction） , and HO-1 protein expression in lung tissues （by Western blot） and for microscopic examination. The damage to the renal tubules was scored. Results Compared with group S, the serum BUN, Cr and TNF-α concentrations were significantly increased, the serum IL-10 concentrations were decreased, the MDA content and renal tubular damage score were increased, the SOD activity was decreased, and the expression of HO-1 protein and mRNA, and Bcl-2 and Bax mRNA was up-regulated in group OLT （P〈 0.05）. Compared with group OLT, the serum BUN, Cr and TNF-a concentrations were significantly decreased, the serum IL-10 concentrations were increased, the MDA content and renal tubular damage score were decreased, the SOD activity was increased, the expression of HO-1 protein and mRNA and Bcl-2 mRNA was up-regulated, and the expression of Bax mRNA was down-regulated in group HS （P〈0.05）. Compared with group HS, the serum BUN, Cr and TNF-α concentrations were significantly increased, the serum IL-10 concentrations were decreased, the MDA content and renal tubular damage score were increased, the SOD activity was decreased, the expression of HO-1 protein and mRNA and Bcl-2 mRNA was down-regulated, and the expression of Bax mRNA was up-regulated in group ATRA （P〈0.05）. Conclusion The mechanism by which hydrogen-rich saline reduces acute kidney injury following OLT is probably associated with activation of Nrf2/HO-1 signaling pathway in rats.