SIRT1/Nrf2信号通路在小檗碱减轻小鼠肾缺血再灌注损伤中的作用

Role of SIRT1/Nrf2 signaling pathway in berberine-induced reduction of renal ischemia-reperfusion injury in mice

目的:评价沉默信息调节因子1(SIRT1)/核因子E2相关因子2(Nrf2)信号通路在小檗碱减轻小鼠肾缺血再灌注损伤中的作用。方法:SPF级健康雄性C57BL/6小鼠30只,6~8周龄,体重18~22 g,采用随机数字表法分为5组(n=6):假手术组(S组)、肾缺血再灌注组(RIR组)、小檗碱+缺血再灌注组(B组)、小檗碱+缺血再灌注+SIRT1抑制剂EX527组(BE组)和小檗碱+缺血再灌注+Nrf2抑制剂ATRA组(BA组)。通过切除右肾,夹闭左肾肾动脉45 min后恢复血流灌注的方法建立小鼠肾缺血再灌注模型。B组、BE组和BA组术前14 d给予小檗碱100 mg·kg^(-1)·d^(-1)灌胃,BE组和BA组术前3 d分别进行腹腔注射EX5275 mg·kg^(-1)·d^(-1)、ATRA 10 mg·kg^(-1)·d^(-1)。S组和RIR组连续14 d给予等容量生理盐水,于再灌注24 h时眶静脉采集血标本,检测血清Cr和BUN浓度,ELISA法检测IL-1β和TNF-α含量,光镜下观察肾小管病理学结果并进行肾小管损伤评分,采用Western blot法检测SIRT1、Nrf2、凋亡相关微粒蛋白(ASC)、caspase-1和NOD样受体蛋白3(NLRP3)的表达。结果:与S组比较,RIR组、B组、BE组和BA组血清Cr和BUN浓度、肾脏IL-1β和TNF-α含量、肾小管损伤评分升高,RIR组、BE组和BA组肾脏SIRT1、Nrf2、ASC、caspase-1和NLRP3、B组SIRT1、Nrf2、caspase-1和NLRP3表达上调(P<0.05);与RIR组比较,B组、BE组和BA组血清Cr和BUN浓度、肾脏IL-1β和TNF-α含量、肾小管损伤评分降低,B组SIRT1和Nrf2、BE组Nrf2和ASC、BA组SIRT1、ASC和caspase-1表达上调,B组ASC、caspase-1和NLRP3、BE组SIRT1和NLRP3、BA组Nrf2表达下调(P<0.05);与B组比较,BE组和BA组血清Cr、BUN浓度、IL-1β、TNF-α含量、肾小管损伤评分升高,BE组和BA组ASC、caspase-1和NLRP3表达上调,BE组SIRT1和BA组Nrf2表达下调(P<0.05)。结论:SIRT1/Nrf2信号通路参与了小檗碱减轻小鼠肾缺血再灌注损伤的过程,与抑制细胞焦亡有关。

Objective To evaluate the role of silencing information regulator 1(SIRT_(1))/nuclear factors E2-related factor2(Nrf2)signaling pathway in berberine-induced reduction of renal ischemia-reperfusion(I/R)injury in mice.Methods Thirty SPF healthy male C57BL/6 mice,aged 6-8 weeks,weighing 18-22 g,were divided into 5 groups(n=6 each)using a random number table method:sham operation group(S group),renal I/R group(RIR group),berberine+I/R group(B group),berberine+I/R+SIRT_(1) inhibitor EX527 group(BE group)and berberine+I/R+Nrf2 inhibitor ATRA group(BA group).After the right kidney was removed,the left renal artery was clamped for 45 min followed by reperfusion to establish the model of renal I/R injury.In B,BE,and BA groups,berberine 100 mg·kg^(-1)·d^(-1) was given for intragastric administration at 14 days before surgery.In group BE and group BA,EX5275 mg·kg^(-1)·d^(-1) and ATRA 10 mg·kg^(-1)·d^(-1) were injected intraperitoneally at 3 days before surgery,respectively.The equal volume of normal saline was given for 14 consecutive days in group S and group RIR.Blood samples were collected from orbital vein at 24 h of reperfusion for measurement of serum blood urea nitrogen(BUN)and creatinine(Cr)concentrations,for determination of the interleukin-1 beta(IL-1β)and tumor necrosis factor-alpha(TNF-α)contents(by enzyme-linked immunosorbent assay)and expression of SIRT_(1),Nrf2,apoptosis-associated speck-like protein containing CARD(ASC),caspase-1,nucleotide-binding oligomerization domain-like receptor containing pyrin domain(NLRP3)(by Western blot)and for examination of the pathological changes of renal tubules(with a light microscope).The damage to the renal tubules was scored.Results Compared with group S,the concentrations of serum Cr and BUN,the contents of renal IL-1βand TNF-αand renal tubular injury score were significantly increased in RIR,B,BE and BA groups,the expression of SIRT_(1),Nrf2,ASC,caspase-1 and NLRP3 was up-regulated in RIR,BE and BA groups,and the expression of SIRT_(1),Nrf2,caspase-1 and NLRP3 was up-regulated in group B(P<0.05).Compared with group RIR,the concentrations of serum Cr and BUN,the contents of renal IL-1βand TNF-αand renal tubular injury score were significantly decreased in B,BE and BA groups,the expression of SIRT_(1) and Nrf2 in group B,Nrf2 and ASC in BE group and SIRT_(1),ASC and caspase-1 in BA group was up-regulated,and the expression of ASC,caspase-1 and NLRP3 in group B,SIRT_(1) and NLRP3 in BE group and Nrf2 in BA group was down-regulated(P<0.05).Compared with group B,the serum concentrations of Cr and BUN,the contents of IL-1βand TNF-αand renal tubular injury score were significantly increased in BE and BA groups,the expression of ASC,caspase-1 and NLRP3 in BE and BA groups was up-regulated,and the expression of SIRT_(1) in BE and Nrf2 in BA groups was down-regulated(P<0.05).Conclusion SIRT_(1)/Nrf2 signaling pathway is involved in the process of berberine-induced reduction of renal I/R,which is related to inhibiting pyroptosis in mice.