达拉菲尼对小鼠肾脏缺血再灌注损伤的保护作用

Protective effects of dabrafenib on mouse renal ischemia reperfusion injury

目的:探讨达拉菲尼对小鼠肾脏缺血再灌注损伤(IRI)的保护作用及机制。方法:将24只C57BL/6小鼠随机分为4组:假手术组(sham组);缺血再灌注组(IRI组),夹闭双侧肾蒂30 min再灌注24h;达拉菲尼组(DAB组),仅与IRI组在相同时间灌胃给予相同量的达拉菲尼;达拉菲尼预处理+缺血再灌注组(DAB+IRI组),灌胃给予达拉菲尼2h后进行IRI造模,每组6只。收集小鼠IRI 24h后的血清及肾脏组织。采用相应试剂盒检测各组肾功能血清肌酐(SCr)和血尿素氮(BUN)水平,HE及PAS染色比较各组肾脏病理学改变,Western Blot及qRT-PCR检测肾小管损伤标志物中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子1(KIM-1)的蛋白及mRNA水平,qRT-PCR检测炎症因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的mRNA水平,免疫组织化学染色检测肾脏组织中TNF-α蛋白表达,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测肾组织细胞死亡情况,Western Blot检测肾脏组织中程序性坏死(necroptosis)通路相关的受体相互作用蛋白1(RIP1)、受体相互作用蛋白3(RIP3)、磷酸化的受体相互作用蛋白3(pRIP3)、混合系列蛋白激酶复合物(MLKL)、磷酸化的混合系列蛋白激酶复合物(pMLKL)蛋白水平。结果:与sham组相比,IRI组SCr、BUN水平升高(P<0.05),肾组织病理损伤严重,小管损伤标志物NGAL和KIM-1蛋白和mRNA水平升高,炎症因子IL-6以及TNF-αmRNA含量增加,肾脏组织TNF-α蛋白表达升高,肾脏组织细胞死亡增多,RIP1、RIP3、pRIP3、MLKL以及pMLKL蛋白表达增加;与IRI组比较,DAB+IRI组减轻肾功能、肾组织损伤,减少NGAL、KIM-1的蛋白和mRNA表达水平,下调IL-6和TNF-αmRNA水平,减少肾脏组织中TNF-α蛋白表达,抑制RIP3、pRIP3及pMLKL的蛋白表达(P均<0.05),而RIP1和MLKL蛋白改变不明显。DAB组与sham组间各指标无统计学差异。结论:达拉菲尼通过抑制RIP3介导的程序性坏死从而对小鼠肾脏缺血再灌注损伤起保护作用。

Objective:To investigate the protective effects of dabrafenib(DAB) on mouse renal ischemia reperfusion injury(IRI). Methodology:Mice were randomly divided into four groups(n=6) including sham group,IRI group,DAB group and dabrafenib preconditioning(DAB)+IRI group.Mice in sham group and DAB group received no treatment of IRI.Serum and kidney samples were collected 24 hours after IRI.The level of serum creatitine(SCr) and blood urea nitrogen(BUN) were measured.Renal injuries were evaluated by HE and PAS staining.The protein leves of NGAL and KIM-1 were detected by Western Blot.The mRNA expressions of NGAL,KIM-1,IL-6 and TNF-α were analyzed by qRT-PCR.The expression of TNF-α was observed by immunohistochemistry.Cell death was assessed by TUNEL.And the protein expressions of RIP1,RIP3,pRIP3,MLKL and pMLKL were detected by Western Blot. Results:Compared with sham group,SCr,BUN and renal damages were increased in IRI group(all P<0.05).Moreover,renal NGAL and KIM-1 protein levels and mRNA expressions,IL-6 、TNF-α mRNA expressions and TNF-α expression in renal tissue,RIP3,pRIP3,MLKL,pMLKL protein expressions were both up-regulated(all P<0.05).In addition,TUNEL-positive cells were more in IRI group.The effects of IRI were inhibited by dabrafenib.Compared to that in IRI group,dabrafenib precondition reversed IRI-induced damages of renal fuction and renal tissues,decreased the mRNA expressions of NGAL,KIM-1,IL-6 and TNF-α,lowered the expression of TNF-α in renal tissues,and reduced NGAL and KIM-1 proteion levels(all P<0.05).Moreover,dabrafenib reduced TUNEL-positive cells induced by IRI,together with RIP3、pRIP3 and pMLKL protein expressions(all P<0.05).However,RIP1 and MLKL protein levels had no changes between IRI and DAB+IRI group.All of the parameters above had no differences between sham and DAB group. Conclusion:Pretreatment with dabrafenib has protective effects on renal ischemia reperfusion injury of mice,which may be associated with inhibiting RIP3-mediated necroptosis.