梅花鹿鹿茸总蛋白对糖尿病肾病大鼠肾损伤的治疗作用及分子机制

Therapeutic effect and molecular mechanism of Sika deer velvet antler protein on renal injury of rats with diabetes nephropathy

目的探讨梅花鹿鹿茸总蛋白提取物(SVPr)对糖尿病肾病(DN)大鼠肾损伤的治疗作用及分子机制。方法将120只Sprague Dawley大鼠按照简单随机数字表法分为对照组、模型组、二甲双胍组、低剂量SVPr组、中剂量SVPr组、高剂量SVPr组,每组20只。模型组、二甲双胍组、低剂量SVPr组、中剂量SVPr组、高剂量SVPr组大鼠造模前禁食12 h,单次腹腔注射链脲佐菌素50 mg•kg^(-1)制备DN大鼠模型;造模成功后,二甲双胍组大鼠给予二甲双胍500 mg•kg^(-1)•d^(-1)灌胃,低剂量SVPr组、中剂量SVPr组、高剂量SVPr组大鼠分别经尾静脉注射质量浓度为1.47、2.94、4.41 g•L^(-1)的SVPr溶液0.1 mL,对照组及模型组大鼠注射等量生理盐水,每日1次,持续给药4周。采用生物化学法检测各组大鼠血尿素氮(BUN)、血肌酐(SCr)、血糖、24 h尿蛋白水平,酶联免疫吸附试验法检测各组大鼠肾组织中活性氧(ROS)、核因子-κB(NF-κB)水平,Western blot法检测各组大鼠肾组织中沉默调节蛋白1(SIRT1)、核因子E2相关因子2(Nrf-2)、血红素加氧酶-1(HO-1)蛋白相对表达量。结果模型组大鼠BUN、SCr、24 h尿蛋白、血糖水平均显著高于对照组(P<0.05);低剂量SVPr组、中剂量SVPr组、高剂量SVPr组和二甲双胍组大鼠BUN、SCr、24 h尿蛋白、血糖水平显著低于模型组(P<0.05);高剂量SVPr组大鼠SCr、24 h尿蛋白水平显著低于二甲双胍组(P<0.05),高剂量SVPr组与二甲双胍组大鼠BUN、血糖水平比较差异无统计学意义(P>0.05);中剂量SVPr组大鼠SCr水平显著低于二甲双胍组,BUN、血糖水平显著高于二甲双胍组(P<0.05);中剂量SVPr组与二甲双胍组大鼠24 h尿蛋白水平比较差异无统计学意义(P>0.05);低剂量SVPr组大鼠BUN、24 h尿蛋白、血糖水平显著高于二甲双胍组(P<0.05),低剂量SVPr组与二甲双胍组大鼠SCr水平比较差异无统计学意义(P>0.05)。高剂量SVPr组大鼠BUN、血糖、24 h尿蛋白、SCr水平显著低于中剂量SVPr和低剂量SVPr组,中剂量SVPr组BUN、血糖、24 h尿蛋白、SCr水平显著低于低剂量SVPr组(P<0.05)。模型组大鼠肾组织中ROS、NF-κB水平显著高于对照组(P<0.05);低剂量SVPr组、中剂量SVPr组、高剂量SVPr组和二甲双胍组大鼠肾组织中ROS、NF-κB水平显著低于模型组(P<0.05);高剂量SVPr组大鼠肾组织中ROS、NF-κB水平显著低于二甲双胍组(P<0.05);中剂量SVPr组大鼠肾组织中ROS水平显著高于二甲双胍组(P<0.05),中剂量SVPr组与二甲双胍组大鼠肾组织中NF-κB水平差异无统计学意义(P>0.05);低剂量SVPr组大鼠肾组织中ROS、NF-κB水平显著高于二甲双胍组(P<0.05);高剂量SVPr组大鼠肾组织中ROS、NF-κB水平显著低于中剂量SVPr组和低剂量SVPr组;中剂量SVPr组大鼠肾组织中ROS水平显著低于低剂量SVPr组(P<0.05),中剂量SVPr组与低剂量SVPr组大鼠肾组织中NF-κB水平比较差异无统计学意义(P>0.05)。模型组大鼠肾组织中SIRT1、Nrf-2、HO-1相对表达量均显著低于对照组(P<0.05);低剂量SVPr组、中剂量SVPr组、高剂量SVPr组和二甲双胍组大鼠肾组织中SIRT1、Nrf-2、HO-1相对表达量显著高于模型组(P<0.05);高剂量SVPr组大鼠肾组织中SIRT1、Nrf-2相对表达量显著高于二甲双胍组(P<0.05),高剂量SVPr组与二甲双胍组大鼠肾组织中HO-1相对表达量比较差异无统计学意义(P>0.05);中剂量SVPr组大鼠肾组织中SIRT1、Nrf-2相对表达量显著高于二甲双胍组,HO-1相对表达量显著低于二甲双胍组(P<0.05);低剂量SVPr组大鼠肾组织中SIRT1相对表达量显著高于二甲双胍组(P<0.05),低剂量SVPr组与二甲双胍组大鼠肾组织中Nrf-2、HO-1相对表达量比较差异无统计学意义(P>0.05);高剂量SVPr组大鼠肾组织中SIRT1、Nrf-2、HO-1相对表达量显著高于中剂量SVPr组和低剂量SVPr组,中剂量SVPr组大鼠肾组织中SIRT1、Nrf-2、HO-1相对表达量显著高于低剂量SVPr组(P<0.05)。结论SVPr可通过上调SIRT1表达来抑制NF-κB转录活性、激活HO-1/Nrf-2抗氧化应激信号,减轻DN大鼠肾损伤。

Objective To investigate the therapeutic effect and molecular mechanism of Sika deer velvet antler protein(SVPr)on renal injury in diabetes nephropathy(DN)rats.Methods A total of 120 Sprague Dawley rats were divided into control group,model group,metformin group,low-dose of SVPr group,medium-dose of SVPr group and high-dose of SVPr group according to the simple random number table,with 20 rats in each group.The rats in the model group,metformin group,low-dose of SVPr group,medium-dose of SVPr group and high-dose of SVPr group were fasted for 12 hours before modeling,and the rats were single intraperitoneally injected with streptozocin 50 mg•kg^(-1) to prepare the DN model.After successful modeling,the rats in the metformin group were given metformin by gavage with a dose of 500 mg•kg^(-1)•d^(-1);the rats in the low-dose of SVPr group,medium-dose of SVPr group and high-dose of SVPr group were injected via tail vein with 0.1 mL SVPr solution with concentrations of 1.47,2.94 and 4.41 g•L^(-1),respectively;the rats in the control group and model group were injected with the same amount of normal saline,once a day for 4 weeks.The levels of blood urea nitrogen(BUN),serum creatinine(SCr),blood glucose and 24 h urine protein of rats in each group were detected by biochemical method,and the levels of reactive oxygen species(ROS)and nuclear factor-κB(NF-κB)in kidney tissue of rats in each group were detected by enzyme-linked immunosorbent assay,the relative expressions of silencing regulatory protein 1(SIRT1),nuclear factor erythroid-2 related factor 2(Nrf-2),heme oxygenase-1(HO-1)protein in renal tissues of rats in each group were detected by Western blot.Results The levels of BUN,SCr,24 h urine protein and blood glucose of rats in the model group were significantly higher than those in the control group(P<0.05);the levels of BUN,SCr,24 h urine protein and blood glucose of rats in the low-dose of SVPr group,medium-dose of SVPr group,high-dose of SVPr group and metformin group were significantly lower than those in the model group(P<0.05);the levels of SCr and 24 h urine protein of rats in the high-dose of SVPr group were significantly lower than those in the metformin group(P<0.05),and there was no significant difference in the levels of BUN and blood glucose of rats between the high-dose of SVPr group and metformin group(P>0.05);the level of SCr of rats in the medium-dose of SVPr group was significantly lower than that in the metformin group,the levels of BUN and blood glucose of rats were significantly higher than those in the metformin group(P<0.05);there was no significant difference in the level of 24 h urine protein of rats between the medium-dose of SVPr group and metformin group(P>0.05);the levels of BUN,SCr and 24 h urine protein of rats in the low-dose of SVPr group were significantly higher than those in the metformin group(P<0.05),and there was no significant difference in the level of SCr of rats between the low-dose of SVPr group and metformin group(P>0.05).The levels of BUN,blood glucose,24 h urine protein and SCr of rats in the high-dose of SVPr group were significantly lower than those in the medium-dose of SVPr group and low-dose of SVPr group(P<0.05);and the levels of BUN,blood glucose,24 h urine protein and SCr of rats in the medium-dose of SVPr group were significantly lower than those in the low-dose of SVPr group(P<0.05).The levels of ROS and NF-κB in kidney tissue of rats in the model group were significantly higher than those in the control group(P<0.05);the levels of ROS and NF-κB in renal tissue of rats in the low-dose of SVPr group,medium-dose of SVPr group,high-dose of SVPr group and metformin group were significantly lower than those in the model group(P<0.05);the levels of ROS and NF-κB in renal tissue of rats in the high-dose of SVPr group were significantly lower than those in the metformin group(P<0.05);the level of ROS in kidney tissue of rats in the medium-dose of SVPr group was significantly higher than that in the metformin group(P<0.05),there was no significant difference in NF-κB level in kidney tissue of rats between the medium-dose of SVPr group and metformin group(P>0.05);the levels of ROS and NF-κB in kidney tissue of rats in the low-dose of SVPr group were significantly higher than those in the metformin group(P<0.05);the levels of ROS and NF-κB in renal tissue of rats in the high-dose of SVPr group were significantly lower than those in the medium-dose of SVPr group and low-dose of SVPr group(P<0.05);the level of ROS in kidney tissue of rats in the medium-dose of SVPr group was significantly lower than that in the low-dose of SVPr group(P<0.05),there was no significant difference in NF-κB level in kidney tissue of rats between the medium-dose of SVPr group and low-dose of SVPr group(P>0.05).The relative expression levels of SIRT1,Nrf-2 and HO-1 in kidney tissue of rats in the model group were significantly lower than those in the control group(P<0.05);the relative expression levels of SIRT1,Nrf-2 and HO-1 in kidney tissue of rats in the low-dose of SVPr group,medium-dose of SVPr group,high-dose of SVPr group and metformin group were significantly higher than those in the model group(P<0.05);the relative expression levels of SIRT1 and Nrf-2 in kidney tissue of rats in the high-dose of SVPr group were significantly higher than those in the metformin group(P<0.05).There was no significant difference in the relative expression level of HO-1 in kidney tissue of rats between the high-dose of SVPr group and metformin group(P>0.05);the relative expression levels of SIRT1 and Nrf-2 in kidney tissue of rats in the medium-dose of SVPr group were significantly higher than those in the metformin group,and the relative expression level of HO-1 was significantly lower than that in the metformin group(P<0.05);the relative expression level of SIRT1 in kidney tissue of rats in the low-dose of SVPr group was significantly higher than that in the metformin group(P<0.05),and there was no significant difference in the relative expression levels of Nrf-2 and HO-1 in kidney tissue of rats between the low-dose of SVPr group and metformin group(P>0.05);the relative expression levels of SIRT1,Nrf-2 and HO-1 in kidney tissue of rats in the high-dose of SVPr group were significantly higher than those in the medium-dose of SVPr group and low-dose of SVPr group(P<0.05);the relative expression levels of SIRT1,Nrf-2 and HO-1 in kidney tissue of rats in the medium-dose of SVPr group were significantly higher than those in the low-dose of SVPr group(P<0.05).Conclusion SVPr can reduce kidney damage of DN rats through inhibiting NF-κB transcriptional activity and activating HO-1/Nrf-2 antioxidant stress signal by up-regulating SIRT1 expression.