摘要目的 评价转录因子E2相关因子2(Nrf2)信号通路在富氢保存液减轻大鼠供肾低温保存期缺血损伤中的作用.方法 健康成年雄性Wistar大鼠40只,体重200～ 250 g,采用随机数字表法将其分为4组(n=10):正常对照组(C组)、保存液(UW液)组(UW组)、富氢UW液组(HRUW组)、Nrf2抑制剂全反式维甲酸组(ATRA组).ATRA组腹腔注射全反式维甲酸7 mg/kg,1次/d,连续2d,最后1次给药后8h时制备肾游离及冷保存期缺血损伤模型,肾脏在4℃富氢UW液中保存48h.UW组和HRUW组腹腔注射等容量生理盐水替代,肾脏分别在4℃UW液和富氢UW液中保存48 h.取肾组织,HE染色观察病理学结果,行肾小管损伤评分;采用ELISA法测定肾组织TNF-α、IL-1β、高迁移率族蛋白1(HMGB1)、IL-10及8-异前列腺素F2α(8-iso-PGF2α)的含量;采用分光光度法测定SOD和过氧化氢酶(CAT)活性;采用Western blot法测定肾组织Nrf2、血红素氧合酶-1(HO-1)、Bcl-2、Bax和caspase-3的表达.结果 与C组比较,UW组肾小管损伤评分升高,肾组织TNF-α、IL-1β、HMGB1和8-iso-PGF2α含量升高,IL-10含量降低,Nrf2和HO-1表达上调,SOD和CAT活性降低,Bcl-2表达下调,Bax和caspase-3表达上调(P＜0.05或0.01);与UW组比较,HRUW组肾小管损伤评分降低,肾组织TNF-α、IL-1β、HMGB1和8-iso-PGF2α含量降低,IL-10含量升高,Nrf2和HO-1表达上调,SOD和CAT活性升高,Bcl-2表达上调,Bax和caspase-3表达下调,ATRA组肾组织Nrf2和Bcl-2表达上调(P＜0.05),其余指标差异无统计学意义(P＞0.05).与HRUW组比较,ATRA组肾小管损伤评分升高,肾组织TNF-α、IL-1β3、HMGB1和8-iso-PGF2α含量升高,IL-10含量降低,HO-1和Bcl-2表达下调,SOD和CAT活性降低,Bax和easpase-3表达上调(P＜0.05).结论 富氢保存液减轻大鼠供肾低温保存期炎性反应、氧化损伤和细胞凋亡,从而减轻缺血损伤的机制与激活Nrf2信号通路有关.

abstractsObjective To evaluate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in attenuation of ischemia injury by hydrogen-rich University of Wisconsin (HRUW) solution during cold storage of rat donor kidneys.Methods Forty healthy adult male Wistar rats,weighing 200-250 g,were divided into 4 groups (n =10 each) using a random number table:control group (group C),University of Wisconsin (UW) solution group (group UW),HRUW solution group (group HRUW) and Nrf2 inhibitor all-trans retinoic acid (ATRA) group (group ATRA).ATRA 7 mg/kg was intraperitioneally injected once a day for 2 consecutive days,kidneys were isolated and underwent cold storage at 8 h after the last administration,and kidneys were stored in HRUW solution for 48 h at 4 ℃C in group ATRA.In UW and HRUW groups,the equal volume of normal saline was intraperitioneally injected instead,and isolated kidneys were stored in UW solution and HRUW solution for 48 h at 4 ℃C,respectively.Kidney specimens were obtained for microscopic examination and for determination of tumor necrosis factoralpha (TNF-α),interleukin-lbeta (IL-1β3),high-mobility group box 1 protein (HMGB1),IL-10 and 8-iso-prostaglandin F2α (8-iso-PGF2α) contents (by enzyme-linked immunosorlbent assay),superoxide dismutase (SOD) and catalase (CAT) activities (using spectrophotometry),and expression of Nrf2,heme oxygenase-1 (HO-1),Bcl-2,Bax and caspase-3 in renal tissues (by using Western blot).The damage to the renal tubules was scored.Results Compared with group C,renal tubular damage scores were signifieantly increased,TNF-α,IL-1β,HMGB1 and 8-iso-PGF2α contents were increased,IL-10 contents were decreased,the expression of Nrf2 and HO-1 was up-regulated,SOD and CAT activities were decreased,the expression of Bcl-2 was down-regulated,and the expression of Bax and caspase-3 was upregulated in group UW (P＜0.05 or 0.01).Compared w,ith group UW,renal tubular damage scores were significantly decreased,TNF-α,IL-1β,HMGB1 and 8-iso-PGF2α contents were decreased,IL-10 contents were increased,the expression of Nrf2 and HO-1 was up-regulated,SOD and CAT activities were increased,the expression of Bcl-2 was up-regulated,and the expression of Bax and caspase-3 was down-regulated in group HRUW,and the expression of Nrf2 and Bcl-2 was up-regulated (P＜0.05),and no significant change was found in the other parameters in group ATRA (P＞0.05).Compared witb group HRUW,renal tubular damage seores were significantly increased,TNF-α,IL-1β,HMGB1 and 8-iso-PGF2α contents were increased,IL-10 contents were decreased,the expression of HO-1 and Bcl-2 was down-regulated,SOD and CAT activities were decreased,and the expression of Bax and caspase-3 was up-regulated in group ATRA.Conclusion HRUW solution reduces inflammatory responses,oxidative damage and cell apoptosis during cold storage of rat donor kidneys,and the mechanism by which HRUW solution attenuates ischemia injury is related to activation of Nrf2 signaling pathway.