应用基因缺失小鼠研究TLR4在LPS诱导的急性肾衰竭中的作用

Role of Toll Like Receptor 4 in Gene-Deficient Mice with LPS-Induced Actue Renal Failure

目的:探讨Toll样受体4(TLR4)在脂多糖(LPS)诱导的小鼠急性肾衰竭(ARF)肾脏中的作用机制。方法:以TLR4基因缺失的C3H/HeJ小鼠和其野生正常对照的C3H/HeN小鼠为实验对象。实验分TLR4+(C3H/HeN)组、TLR4-(C3H/HeJ)组,单次性腹腔注射LPS(15 mg/kg)诱导小鼠ARF模型。24 h后检测血清肌酐(Cr)和尿素氮(BUN)值来评估肾功能;Nomura评分法对肾组织病理改变进行半定量评分;免疫组织化学SABC法检测TLR4在两组小鼠肾组织中的表达、RT-PCR法检测肾组织总TLR4 mRNA的表达变化、免疫蛋白印迹检测肾组织总TLR4蛋白水平;TUNEL法检测肾脏组织的凋亡情况。结果:TLR4+(C3H/HeN)组的Cr、BUN分别为(202.26±11.08)μmol/L(、20.36±1.52)mmol/L,TLR4-(C3H/HeJ)组的Cr、BUN分别为(109.67±13.32)μmol/L(、6.42±0.41)mmol/L,差异有显著性(P<0.01);TLR4-(C3H/HeJ)组的肾组织结构正常,无明显的病理改变,而TLR4+(C3H/HeN)组小鼠肾小管呈轻中度的病理改变,主要表现为近端肾小管管腔扩大、空泡形成等;与TLR4-(C3H/HeJ)组相比,TLR4+(C3H/HeN)组的TLR4蛋白及mRNA表达明显上调;TUNEL法显示凋亡小体主要出现在TLR4+(C3H/HeN)组小鼠肾脏的近端小管及管周围,肾小球无明显凋亡小体出现,TLR4-(C3H/HeJ)组小鼠肾组织中未检测到凋亡小体,二者平均吸光度值分别为(0.117±0.008)和(0.038±0.003),差异有显著性(P<0.001)。结论:TLR4在LPS诱导的小鼠急性肾功能衰竭的发病中起一定作用。LPS可能通过激活TLR4信号转导途径引起肾组织中肾小管上皮细胞凋亡过度,肾小管上皮细胞数量减少,从而导致急性肾功能衰竭的发生。

Objective. To identify the expression and its role of Toll like receptor 4 （TLR4） in endotoxin-acute renal failure （ARF） mice. Methods： The TLR4-deficient mice（C3H/HeJ） and wild normal contrast mice（C3H/HeN） were injected by ARF model. Mice were sacrificed at 24 h to Lipopolysaccharide （LPS, 15 mg/kg） to establish the collect the sample of blood and renal tissue. The value of serum creatinine （Cr） and blood urea nitrogen （BUN） were measured to judge renal function. Nomura scoring was adopted to evaluate renal morphologic pathological changes. Immuno-histochemistry （IHC）, RT-PCR and Western blot were applied to detected expression of TLR4 mRNA and TLR4 protein respectively. Terminal deoxynucleotidyhransferase mediated X-dUTP nick end labeling （TUNEL） was applied to monitor apoptosis of renal tissue. Results： The values of Cr and BUN of TLR4^＋ （C3H/HeN） mice were higher than those of TLR4- （C3H/HeJ） mice respectively （Cr：（202.26±11.08）μmol/L versus （109.67±13.32）μmol/L, P〈0.01;BUN： （20.36±1.52） mmol/L versus （6.42±0.41） mmol/L, P〈0. 01）. Compared with that in TLR4- （C3H/HeJ） mice, the level of renal TLR4 mRNA and protein expression was up-regulated in TLR4＋ （C3H/HeN） mice. The apoptotic bodies were mainly detected on the renal tubule. There was significant difference in apoptotic body optical density value （OD） between TLR4^＋（C3H/HeN） mice and TLR4-（C3H/HeJ） mice, （0.117±0.008） versus （0.038±0.003）, P〈0.001. Conclusion. TLR4 may play a role in LPS-induced mice ARF. LPS may cause the over apoptosis of renal tubular cell via TLR4 signal transduct pathway, decrease the number of renal tubular cell, and result in ARF.