缬沙坦联合霉酚酸酯对糖尿病大鼠肾脏TRAIL及NF-KB的影响

Effects of valsartan, mycophenolate mofetil and their combined application on TRAIL and nuclear factor-kB expression in the kidneys of diabetic rats

目的探讨肿瘤坏死因子相关的凋亡诱导配体（TRAIL）及其核因子-kB（NF-kB）在糖尿病大鼠肾组织的表达及其联合应用缬沙坦（valsartan）和霉酚酸酯（MMF）对糖尿病大鼠肾组织TRAIL及其NF-kB的影响。方法采用一侧肾切除腹腔注射STZ（55mg／kg）建立糖尿病大鼠模型，将80只Wistar大鼠随机分为：对照组（NC）、糖尿病组（DM）、缬沙坦治疗组（DM＋V）、霉酚酸酯治疗组（DM＋M）、联合治疗组（DM＋V＋M）。取材，检测各项生化指标，并采用荧光实时定量RQ—PCR方法检测肾皮质TRAIL及其NF-kB的mRNA的表达。免疫组织化学测定肾组织TRAIL及其NF-kB蛋白的表达。结果①DM组24h尿蛋白和肾重／体重比均高于NC组（均P〈0．05），并随糖尿病时程延长逐渐增高，于DM12周时达到最大值；血白蛋白在DM8周组开始下降（P〈0，01），并逐渐降低；血肌酐、尿素氮于DM16周组开始下降（P〈0．01）。各治疗组与DM8周组相比较，肾重／体重比变小（P〈0.05），24h尿蛋白总量降低（均P〈0．05），血白蛋白回升（P〈0．05）。以联合组最为明显。②荧光定量PCR显示TRAIL在12周前表达降低（均P〈0．01），16周时其表达量明显升高（P〈0．01）。各治疗组与DM8周组比较表达增加（P〈0．05），以霉酚酸酯组和联合组最为明显（P〈0．01）。DM组NF-kB的mRNA表达随时间延长逐渐增强（P〈0．01），各治疗组与DM8周组比较表达均降低（均P〈0．05），以联合组最为明显。③免疫组化显示TRAIL主要在肾曲小管表达，肾小球和脉管系统没有表达。DM各组在12周前表达均降低（均P〈0．05），差异有统计学意义；16周后表达明显增强（均P〈0．01）。各治疗组阳性细胞数明显增加（均P〈0．05）。NF-kB在肾小球和肾小管均有表达，DM各组NF-kB表达量随时间逐渐增高，各治疗组可显著降低NF-kB表达。结论TRAIL作为自身免疫系统的一个重要监控因子，其在糖尿病大鼠肾脏中的表达说明其密切参与了糖尿病肾病的发生发展，机制可能由NF-kB调节。早期缬沙坦和霉酚酸酯联合干预治疗可通过来增强TRAIL的表达，对肾脏产生保护作用。

Objective To evaluate the expression of TNF-related apoptosis inducing ligand （TRAIL） and nuclear factor （NF）-KB in the kidney tissues of diabetic rats and the effects of valsartan, mycophenolate mofetil （MMF） , and their combined application on the renal TRAIL and NF-kB expression. Methods Eighty uninephrectomized male Wistar rats were randomly divided into 2 groups： normal control （NC） group （ n = 28 ）, undergoing intraperitoneal injection of citric acid buffer, and diabetes mellitus （DM） group, undergoing intraperitoneal injection of streptozotocin （STZ） to establish DM models. The 52 DM rats were randomly divided into 7 equal subgroups： DM without treatment for 4 weeks （ DM4）, DM without treatment for 8 weeks （ DM8 ） DM without treatment for 12 weeks （ DM12 ） , and DM without treatment for 16 weeks （DM16） , valsartan treatment （ DM ＋ V） , MMF treatment （ DM ＋ M） , and combined treatment （ DM ＋ V ＋ M）. The treatment subgroups were treated for 8 weeks immediately after the diabetic models ere established. Twenty-four hour urine was collected to measure the amount of protein 4, 8, 12, and 16 weeks after the induction of DM respectively. The rats were sacrificed. Blood samples were collected from the abdominal aorta to detect the blood urea nitrogen （BUN）, serum creatinine （sCr）,albumin, and glucose. The kidneys were taken out. Hypertrophy index （left kidney weight/body weight） was determined. Quantitative real time RT-PCR was performed to detect the expression of TRAIL and NF-KB mRNA. Immunohistochemistry was used to detect the protein expression of TRAIL and NF-kB. Results （1） The 24 h urine protein levels and hypertrophy indexes of all DM subgroups were significantly higher than those of the NC groups （ all P 〈 0.05 ）. 24 h urine protein and hypertrophy index increased gradually and peaked in thel2th week ; blood albumin gradually decreased since the 8th week（ P 〈 0.01 ）, and BUN and sCr began to decrease only since the 16th week（ both P 〈 0.01 ）. Compared with the DM 8 subgroup, the hypertrophy index and 24 h urine protein of the different treatment subgroups, especially the DM ＋ V ＋ M subgroup, were significantly lower （ all P 〈 0.05 ）. （2） Quantitative real time RT-PCR showed that compared with the NC group , the TRAIL expression levels of the DM subgroups were significantly lower before the 12th week after induction of DM model（ all P 〈 0.01 ） , and then significantly higher in the 16th week（ all P 〈 0. 01 ）. The TRAIL expression of the treatment groups, especially that of the DM ＋ M subgroup, were significantly higher than that of the DM8 group （ all P 〈 0.05 ）. Compared with the NC group, the NF-kB expression levels of the DM subgroups were significantly higher time-dependently （ all P 〈 0.01 ）. Compared with the DM8 group , the NF-KB expression levels of the treatment subgroups, especially that of the DM ＋ V ＋ M subgroup were significantly lower（ all P 〈0.05）. （3） The expression of TRAIL was mainly located in the convoluted tubule of kidney, and no TRAIL protein expression was detected in the glomeruli or renal vasculature. The levels of NF-KB protein expression, shown in glomeruli and convoluted tubules, of all DM subgroups were all higher than that of the NC group. The NF-KB protein expression level of the DM ＋ V ＋ M subgroup was significantly lower. The number of NF-KB positive cells was significantly related to the mononuclear macrophage infiltration , kidney function, and structural lesion. Condusion An important monitoring factor in the autoimmune system, TRAIL closely participates in the pathogenesis of diabetic nephropathy,possibly controlled by NF-KB. In the early stage combination of valsartan and MMF may upregulate the expression of TRAIL, thus protecting the kidney function.