褪黑素延缓糖尿病大鼠肾脏细胞衰老的机制研究

Study on the mechanism of melatonin to delay the aging of kidney cells in diabetic rats

目的观察褪黑素(MLT)对糖尿病肾病(DN)大鼠的影响,探究MLT延缓大鼠DN进程的作用机制。方法SD雄性大鼠15只随机分成正常组、模型组、MLT组,每组各5只。模型组及MLT组采用腹腔注射法给予链脲佐菌素(STZ)55 mg/kg,建立DN大鼠模型。成功建立模型后,MLT组采用腹腔注射给予MLT 10 mg/(kg·d),正常组与模型组给予等体积生理盐水。连续给药16周后,取大鼠腹主动脉血及肾脏组织,检测血清中尿素氮(BUN)、血肌酐(SCR);Masson染色观察肾脏组织形态学变化;β-半乳糖苷酶染色(SA-β-gal)检测肾脏细胞衰老变化;采用Western blot法检测肾脏组织中自噬标志物(p62、LC3B)、肌球蛋白轻链激酶(MLCK)及细胞周期蛋白依赖激酶(CDK)抑制因子(p16、p53和p21)的表达水平。结果与正常组比较,模型组血清中BUN、SCR水平升高,肾间质纤维化明显,肾脏组织自噬水平降低,MLCK、p16、p53、p21表达水平均升高。给予MLT后,与模型组比较,MLT组血清中BUN、SCR水平降低,肾脏组织衰老延缓,病理损伤明显减轻,肾脏组织自噬水平也得到改善,MLCK、p16、p53、p21表达水平均降低。结论MLT可以通过增强自噬、延缓衰老进而改善DN大鼠的DN进程。

Objective To observe the effect of melatonin(MLT)on diabetic nephropathy(DN)rats,and to explore the mechanism of MLT delaying the progression of DN in rats.Methods Fifteen male SD rats were randomly divided into the normal group,model group and MLT group with 5 rats in each group.Rats in the model group and the MLT group were given 55 mg/kg of streptozotocin(STZ)by intraperitoneal injection in order to establish a DN rat model.After the model was successfully established,rats in the MLT group were given 10 mg/(kg·d)of MLT by intraperitoneal injection,and rats in the normal group and model group were given the equal volume of normal saline.Abdominal aortic blood and kidney tissue of rats were taken 16 weeks after they were given,blood urea nitrogen(BUN)and serum creatinine(SCR)in serum were detected;massion staining was used to observe morphological changes of kidney tissue;β-galactosidase staining(SA-β-gal)was used to detect changes in kidney cell aging;Western blot was used to detect the expression levels of autophagy markers(p62,LC3B),myosin light chain kinase(MLCK)and cyclin-dependent kinase(CDK)inhibitors(p16,p53,and p21).Results Compared with the normal group,the levels of BUN and SCR in serum in the model group increased,renal interstitial fibrosis was obvious,the autophagy level of kidney tissue decreased,and the expression levels of MLCK,p16,p53,and p21 increased.Compared with the model group,the levels of BUN and SCR in serum in the MLT group decreased,and the aging of the kidney tissue was delayed.After MLT was administered,the pathological damage was significantly reduced,and the autophagy level of kidney tissue was also improved.The expression levels of MLCK,p16,p53 and p21 decreased.Conclusion MLT can improve the progress of DN in DN rats by increasing autophagy and delaying aging.