NLRP3抑制剂CY-09对小鼠肾缺血再灌注损伤后远隔器官肝脏的保护作用

The protective effect of NLRP3 inhibitor CY-09 on liver injury after renal ischemia reperfusion injury in mice

目的探讨NOD样受体热蛋白结构域相关蛋白3(NLRP3)特异性抑制剂(CY-09)的预处理对小鼠肾缺血再灌注(IR)损伤后远隔器官肝脏功能的保护作用。方法将32只Bal/C小鼠随机平均分成四组,分别为假手术组(Sham组)、Sham+CY-09预处理组(Sham+CY-09组)、肾缺血再灌注组(IR组)、IR+CY-09组,每组8只。Sham+CY-09组和IR+CY-09组于术前30 min腹腔注射CY-0950μg/kg,Sham组和IR组注射等体积生理盐水。所有组均切除小鼠右肾,Sham组和Sham+CY-09组充分暴露左肾但不夹闭,IR组和IR+CY-09组暴露左肾并夹闭45 min,建立左肾IR损伤模型并再灌注24 h。检测血肌酐(sCr)、谷丙转氨酶(ALT)浓度变化,采用苏木精-伊红(HE)染色观察肝、肾组织病理学改变,采用细胞凋亡(Tunel)染色检测肝脏组织凋亡,采用蛋白免疫印迹法(Western blot)测定NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)蛋白,酶联免疫吸附法(ELISA)检测炎症因子白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)浓度。结果与Sham组比较,IR组sCr、ALT浓度明显升高(P<0.0001),肝、肾组织病理性损伤明显,肝组织凋亡明显增加(P<0.0001),肝组织中NLRP3(P<0.01)、Caspase-1(P<0.05)、ASC(P<0.05)蛋白表达含量明显升高,炎症因子IL-1β(P<0.01)、IL-6(P<0.001)浓度明显升高。与IR组比较,IR+CY-09组sCr、ALT浓度明显下降(P<0.01),肝、肾组织病理性损伤有所减轻,肝组织凋亡减少(P<0.05),肝组织中NLRP3(P<0.01)、Caspase-1(P<0.05)、ASC(P<0.05)蛋白表达明显下降,炎症因子IL-1β浓度明显下降(P<0.05),而炎症因子IL-6浓度差异无统计学意义(P>0.05)。结论肾IR可激活小鼠肝组织中NLRP3炎症小体,进而促进炎症反应,诱发肝损伤,NLRP3抑制剂CY-09的预处理可减轻肾IR所致的肝组织损伤。

Objective To investigate the protective effect of NOD-like receptor thermal protein domain associated protein 3(NLRP3)inhibitor CY-09 on liver injury after renal ischemia reperfusion(IR)injury in mice.Methods Thirty two Bal/C mice were randomly divided into four groups:Sham group,Sham with NLRP3 inhibitor group(Sham+CY-09),IR group,IR with NLRP3 inhibitor group(IR+CY-09).Each group contained eight mice.Sham+CY-09 group and IR+CY-09 group were injected CY-09(50μg/kg)intraperitoneally at 30 min before surgery.Sham group and IR group were injected with saline with equal volume.The right kidney was resected in all groups.Sham group and Sham+CY-09 group fully exposed the left kidney without clamping,but the left kidney was fully exposed and clipped for 45 minutes in the IR group and IR+CY-09 group.Ischemia for 45 minutes and reperfusion for 24 hours was performed in the left renal.Serum creatinine(sCr)and alanine transaminase(ALT)concentration were detected by kits.Liver and kidney histopathological injury were detected by HE staining.Apoptosis of liver tissue was observed by Tunel staining.The proteins level of NLRP3,apoptosis-associated speck-like protein(ASC)and Caspase-1 were determined by Western blot.The content of IL-1βand IL-6 inflammatory factors were detected by ELISA kits.Results Compared with the Sham group,the levels of sCr and ALT in the IR group were significantly increased(P<0.0001).HE staining showed obvious pathological damage to liver and kidney tissue,and Tunel staining showed a significant increase in apoptosis of liver tissue(P<0.05).The content of NLRP3,Caspase-1 and ASC protein in liver tissue was significantly increased(P<0.01),and the level of inflammatory factor IL-1β(P<0.01),IL-6(P<0.001)was significantly increased.Compared with the IR group,the levels of sCr and ALT in the IR+CY-09 group decreased significantly(P<0.01).HE staining showed a reduction in pathological damage to liver and kidney tissue,and Tunel staining showed a decrease in liver tissue apoptosis(P<0.05).NLRP3(P<0.01),Caspase-1(P<0.05),ASC(P<0.05)protein content in liver tissue decreased significantly.The content of inflammatory factor IL-1βdecreased significantly(P<0.05),while the level of the inflammatory factor IL-6 showed no difference(P>0.05).Conclusions Renal ischemia reperfusion can activate the NLRP3 inflammasome in liver tissue to promote inflammation-induced liver damage,and pretreatment with NLRP3 inhibitor CY-09 can alleviate liver tissue damage.