EPO对糖尿病肾病大鼠NK细胞活化性受体及HMGB1/Beclin-1信号通路的影响

Effects of EPO on NK cell activation receptor and HMGB1/Beclin-1 signaling pathway in diabetic nephropathy rats

目的:基于HMGB1/Beclin-1信号通路探究EPO对糖尿病肾病大鼠NK细胞活化性受体的作用机制。方法:40只SPF级SD雄性大鼠随机分为正常(A)组、模型(B)组、二甲双胍(C)组、EPO(D)组,每组10只,对B、C、D组采用高脂饲料及链脲佐菌素进行糖尿病肾病建模。建模成功后,对C组灌胃100 mg/kg二甲双胍,对D组腹腔内注射100 U/kg EPO,A组、B组同期灌胃等体积生理盐水;另取20只大鼠建模,建模成功后,随机分为E组、F组,E组灌胃给予30 mg/kg HMGB1抑制剂,F组灌胃给予30 mg/kg HMGB1抑制剂和腹腔内注射100 U/kg EPO;取HK-2细胞,分为高糖+HK-2细胞(HH)组、EPO+高糖+HK-2细胞(EH)组、甘草酸苷L+高糖+HK-2细胞(LH)组,葡萄糖培养后进行细胞实验。血糖仪检测大鼠血糖,全自动生化分析仪检测大鼠生化指标,PAS染色法检测大鼠肾组织病理形态,免疫印迹法检测HMGB1/Beclin-1蛋白表达,RT-PCR及免疫组化法检测NK细胞活化性受体表达。结果:与A组比较,B组血糖及血糖曲线、血液及尿液中BUN、Scr、UAlb含量、NKp30、NKp44、NKp46的mRNA及蛋白表达明显升高(P<0.05),体质量明显降低(P<0.05);与B组比较,C组、D组血糖及24 h尿量、血糖曲线、血液及尿液中BUN、Scr、UAlb含量、NKp30、NKp44、NKp46 mRNA及蛋白表达均明显降低(P<0.05),体质量明显升高(P<0.05),肾脏病理学明显改善,且D组较C组变化显著(P<0.05);与A组比较,B组大鼠肾组织中HMGB1/Beclin-1蛋白表达显著升高(P<0.05),与B组相比,C、D、E、F组大鼠肾组织中HMGB1/Beclin-1蛋白表达均显著降低(P<0.05),且D组HMGB1/Beclin-1蛋白表达与C组相比降低明显(P<0.05),E组与D组无明显差异,F组较E组降低明显;HMGB1/Beclin-1蛋白表达与NKp30、NKp44、NKp46mRNA均呈正相关(P<0.05);与HH组相比,EH、LH组细胞增殖率显著升高(P<0.05),凋亡率及HMGB1/Beclin-1蛋白表达均显著降低(P<0.05),而LH组与EH组相比差异无统计学意义(P>0.05)。结论:EPO可有效降低糖尿病肾病大鼠NK细胞活化性受体表达,抑制HMGB1/Beclin-1信号通路,提示EPO可能通过调控NK细胞活化性受体及HMGB1/Beclin-1信号通路发挥作用,而NK细胞活化性受体表达与HMGB1/Beclin-1信号通路呈正相关。

Objective:To explore the mechanism of EPO on activating receptors of NK cells in rats with diabetic nephropathy based on HMGB1/Beclin-1 signaling pathway.Methods:Forty SPF male SD rats were randomly divided into normal group(A),model group(B),metformin group(C)and EPO group(D),with 10 rats in each group.Diabetic nephropathy modeling was performed on groups B,C and D using high-fat diet and streptozotocin.After successful modeling,group C was given metformin 100 mg/kg by intragastric administration,and group D was intraperitoneal injection of EPO 100 U/kg.Group A and B were given normal saline at the same volume by intragastric administration simultaneously.After successful modeling,the remaining 20 rats were randomly divided into group E and group F.Group E was given 30 mg/kg HMGB1 inhibitor by intragastric administration,group F was given 30 mg/kg HMGB1 inhibitor by intragastric administration and 100 U/kg EPO by intraperitoneal injection.HK-2 cells were taken and divided into high glucose+HK-2 cells(HH)group,EPO+high glucose+HK-2 cells(EH)group,glycyrrhizin L+high glucose+HK-2 cells(LH)group,and cultured with glucose for cell experiment.Blood glucose was detected by glucose analyzer,biochemical indexes were detected by automatic biochemical analyzer,and renal pathological morphology of rats was detected by PAS staining.Expression of HMGB1/Beclin-1 protein was detected by Western blot,and expressions of NK cell activated receptors were detected by RT-PCR and immunohistochemistry.Results:Compared with group A,blood glucose,24 h urine volume,blood glucose curve,BUN,Scr,UAlb contents in blood and urine,mRNA and protein expressions of NKp30,NKp44,NKp46 in group B were significantly increased(P<0.05),while body weight was significantly decreased(P<0.05).Compared with group B,blood glucose,24 h urine volume,blood glucose curve,BUN,Scr,UAlb contents in blood and urine,mRNA and protein expression of NKp30,NKp44 and NKp46 in group C and group D were significantly decreased(P<0.05),while body weight was significantly increased(P<0.05),renal pathology was also significantly improved,the changes of group D was significantly higher than that of group C(P<0.05).Compared with group A,expression of HMGB1/Beclin-1 protein in renal tissues of rats in group B was significantly increased(P<0.05),and expression of HMGB1/Beclin-1 protein in renal tissues of rats in groups C,D,E and F was significantly decreased(P<0.05),expression of HMGB1/Beclin-1 protein in group D was significantly decreased compared with group C(P<0.05),there was no significant difference between group E and group D,and expression of HMGB1/Beclin-1 protein in group F was significantly decreased compared with group E.HMGB1/Beclin-1 protein expression was positively correlated with NKp30,NKp44 and NKp46 mRNA(P<0.05).Compared with HH group,proliferation rates of EH and LH groups were significantly increased(P<0.05),while apoptosis rate and HMGB1/Beclin-1 protein expression were significantly decreased(P<0.05),there was no significant difference between LH group and EH group(P>0.05).Conclusion:EPO can effectively reduce expressions of NK cell activated receptors and inhibit HMGB1/Beclin-1 signaling pathway in diabetic nephropathy rats,suggesting that EPO may exert its effect by regulating NK cell activated receptors and HMGB1/Beclin-1 signaling pathway.NK cell activation receptors expressions are positively correlated with HMGB1/Beclin-1 signaling pathway.