褪黑素对自然衰老大鼠肾脏保护作用的研究

Study on the protective effects of melatonin on the kidney of naturally aging rats

目的:探讨mTOR信号介导的自噬在褪黑素(melatonin,Mel)减轻自然衰老SD大鼠肾脏损害的作用及机制。方法:将40只8周龄SD大鼠随机分为4组,空白对照组(A组)、单纯褪黑素处理组(褪黑素10 mg·kg^(-1)·d^(-1),B组)、老年模型组(C组)和褪黑素干预老年模型组(褪黑素10 mg·kg^(-1)·d^(-1),D组)。A组与B组大鼠用普通饲料饲养4周后取材,C组与D组大鼠以普通饲料饲养18个月后取材,测定血清肌酐(Scr)、尿素氮(UN)以检测肾功能,HE染色观察肾脏组织形态变化,TUNEL染色检测各组细胞凋亡情况,Western-blot检测肾脏组织自噬相关蛋白LC3B、Beclin1、p62和磷酸化mTOR(p-mTOR)的表达,免疫荧光染色法检测肾脏组织LC3B蛋白的表达。结果:与A组相比,B组各项指标均无明显变化;C组肾脏组织肾小球萎缩,Scr和UN水平显著升高(P<0.05),TUNEL阳性细胞数量明显增加(P<0.05),LC3B和Beclin1表达显著升高(P<0.05),而p62和p-mTOR的表达降低(P<0.05),免疫荧光染色法检测结果显示肾脏组织中LC3B蛋白表达增加(P<0.05);D组大鼠的肾小球形态明显改善,Scr和UN血清水平降低(P<0.05),TUNEL阳性细胞数减少(P<0.05),LC3B和Beclin1的表达减少(P<0.05),免疫荧光染色法检测到LC3B的表达降低(P<0.05)。结论:褪黑素可通过调节mTOR信号介导的自噬减轻老年SD大鼠肾脏损伤程度。

Objective:To investigate the protective effects and mechanism of mTOR signal-mediated autophagy of melatonin(Mel)in alleviating renal injury in aged SD rats.Methods:Forty 8-week-old SD rats were randomly divided into four groups:the blank control group(group A),the melatonin treatment group(melatonin 10 mg·kg^(-1)·d^(-1),group B),the aged model group(group C)and the melatonin intervention aged model group(melatonin 10 mg·kg^(-1)·d^(-1),group D).Rats in group A and B were fed with common diet for 4 weeks,while the rats in group C and D received common diet for 18 months.Serum creatinine(Scr)and urea nitrogen(UN)were measured to detect renal function.Morphological changes in the kidney tissue were observed by HE staining.Cell apoptosis in each group was detected by TUNEL staining.Autophagy-related proteins,such as LC3B,Beclin1,p62 and phosphorylated mTOR(p-mTOR)were detected by Western blot,and the expression of LC3B in the kidney tissue was detected by immunofluorescence staining.Results:Compared with those of group A,no significant changes were noted in all the indexes of group B.Glomerulus atrophy of the kidney tissue could obviously be seen in group C,and the Scr and UN levels were significantly elevated(P<0.05).TUNEL positive cells were significantly increased(P<0.05),and the expressions of LC3B and Beclin1 were significantly increased(P<0.05),while the expression of p62 and p-mTOR were significantly decreased(P<0.05).Immunofluorescence staining results showed that the expression of LC3B was also increased in the kidney tissue(P<0.05).There was significant improvement in glomerulus morphology for the rats of group D and the levels of Scr and UN decreased considerably(P<0.05).Moreover,the number of TUNEL positive cells and the expression levels of LC3B and Beclin1 also decreased by immunofluorescence staining(P<0.05).Conclusion:Melatonin could alleviate renal injury in aged SD rats by regulating mTOR signal-mediated autophagy.