金匮肾气汤促进糖尿病小鼠肾脏足细胞自噬
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摘要
目的探讨金匮肾气汤对db/db糖尿病小鼠肾脏足细胞自噬的影响及机制。方法将6周龄db/db糖尿病小鼠随机分为生理盐水组、金匮肾气汤低、高剂量组、二甲双胍组,均灌胃给药;♂db/m小鼠作为正常对照组。于第18周龄处死动物,留24 h尿检测尿白蛋白、肌酐;取血,检测血糖、肌酐;留取肾脏组织,分别进行PAS染色观察肾小球内细胞外基质增生情况,免疫组织化学检测WT1在肾组织的表达,透射电子显微镜观察足细胞内自噬体情况,Western blot检测LC3Ⅰ/Ⅱ、p62、p-mTOR/mTOR、p-AMPK/AMPK在肾组织的表达。结果与db/db糖尿病小鼠组比较,金匮肾气汤治疗后,24 h尿白蛋白减少、尿白蛋/肌酐下降、血肌酐下降(P<0.05),肾小球内系膜增宽程度减轻,足细胞内自噬体增加,WT1阳性细胞核数量明显增加(P<0.05),肾皮质内LC3Ⅱ表达明显增强、p62表达明显降低(P<0.05),AMPK磷酸化明显增加,mTOR磷酸化受到抑制(P<0.05)。结论金匮肾气汤通过AMPK-mTOR途径,促进足细胞自噬,保护足细胞,延缓糖尿病肾病的进展。
Aim To investigate the effect of Jinguishenqi decoction on renal podocyte autophagy in db/db diabetic mice and its mechanism.Methods Twenty 6-week old db/db diabetic mice were randomly divided into four groups, with five mice in each group: saline group, Jinguishenqi decoction low dose group, Jinguishenqi decoction high dose group, metformin positive control group, and 5 male db/m mice as normal control group. The animals were sacrificed at 18 weeks old, and urinary albumin and creatinine were detected in 24 hours′ urine. Blood glucose and creatinine were measured. The glomerular extracellular matrix proliferation in renal tissues was observed by PAS staining. The expression of WT1 in renal tissues was detected by immunohistochemistry, and autophagy in podocyte was observed by transmission electron microscopy. The protein expressions of LCⅠ/Ⅱ, p62, p-mTOR/mTOR and p-AMPK/AMPK in renal tissues were detected by Western blot.Results Compared with saline group of db/db diabetic mice, urinary albumin, urinary albumin/creatinine in 24 hours urine, serum creatinine and glomerular mesangial width all decreased after the treatment of Jinguishenqi decoction. Autophagosome in podocyte and the number of WT1 positive cell increased(P<0.05), and the expression of LC3Ⅱ in renal cortex decreased significantly(P<0.05) after the treatment of Jinguishenqi decoction. The phosphorylation of AMPK increased significantly(P<0.05) and the phosphorylation of mTOR was inhibited(P<0.05) after the treatment of Jinguishenqi decoction.Conclusions Jinguishenqi decoction could promote autophagy, protect podocyte and delay the progression of diabetic nephropathy through AMPK-mTOR pathway.
Aim To investigate the effect of Jinguishenqi decoction on renal podocyte autophagy in db/db diabetic mice and its mechanism.Methods Twenty 6-week old db/db diabetic mice were randomly divided into four groups, with five mice in each group: saline group, Jinguishenqi decoction low dose group, Jinguishenqi decoction high dose group, metformin positive control group, and 5 male db/m mice as normal control group. The animals were sacrificed at 18 weeks old, and urinary albumin and creatinine were detected in 24 hours′ urine. Blood glucose and creatinine were measured. The glomerular extracellular matrix proliferation in renal tissues was observed by PAS staining. The expression of WT1 in renal tissues was detected by immunohistochemistry, and autophagy in podocyte was observed by transmission electron microscopy. The protein expressions of LCⅠ/Ⅱ, p62, p-mTOR/mTOR and p-AMPK/AMPK in renal tissues were detected by Western blot.Results Compared with saline group of db/db diabetic mice, urinary albumin, urinary albumin/creatinine in 24 hours urine, serum creatinine and glomerular mesangial width all decreased after the treatment of Jinguishenqi decoction. Autophagosome in podocyte and the number of WT1 positive cell increased(P<0.05), and the expression of LC3Ⅱ in renal cortex decreased significantly(P<0.05) after the treatment of Jinguishenqi decoction. The phosphorylation of AMPK increased significantly(P<0.05) and the phosphorylation of mTOR was inhibited(P<0.05) after the treatment of Jinguishenqi decoction.Conclusions Jinguishenqi decoction could promote autophagy, protect podocyte and delay the progression of diabetic nephropathy through AMPK-mTOR pathway.
引文
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[6] 李青, 韩宇博. 金匮肾气丸联合美卡素治疗Ⅲ期糖尿病肾病的临床研究[J]. 光明中医, 2014, 29(3): 576-7.[6] Li Q, Han Y B. Clinical study of Jinguishenqi Pill combined with mekacin in the treatment of stage III diabetic nephropathy[J]. Guangming J Chin Med, 2014, 29(3): 576-7.
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[8] 金智生,陈雪,李甜. 金匮肾气丸对实验性2型糖尿病肾病大鼠肾脏组织NO、NOS的影响[J]. 中医药学报,2012,40(1): 60-3.[8] Jin Z S, Chen X, Li T. Effect of Jinguishenqi Pill on NO, NOS in renal tissue of experimental type 2 diabetic nephropathy rats[J]. Acta Chin Med Pharmacol, 2012,40(1): 60-3.
[9] Weil E J, Lemley K V, Mason C C, et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy[J]. Kidney Int, 2012, 82(9): 1010-7.
[10] Shankland S J. The podocyte′s response to injury: role in proteinuria and glomerulosclerosis[J]. Kidney Int, 2006, 69(12): 2131-47.
[11] 丁海华,邱原野,王盈盈, 等. 小檗碱对糖尿病肾病大鼠nephrin、podocin和α3β1整合素表达的影响[J]. 中国药理学通报,2015,31(10): 1414-20.[11] Ding H H, Qiu Y Y, Wang Y Y, et al. Effect of berberine on the expression of nephrin, podocin and intergrin α3β1 in diabetic nephropathy rats[J]. Chin Pharmacol Bull,2015,31(10):1414-20.
[12] Lenoir O, Jasiek M, Henique C, et al. Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis[J]. Autophagy, 2015, 11(7): 1130-45.
[13] Ilatovskaya D V, Levchenko V, Lowing A, et al. Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels[J]. Sci Rep, 2015, 5: 17637.
[14] Langer S, Kreutz R, Eisenreich A. Metformin modulates apoptosis and cell signaling of human podocytes under high glucose conditions[J]. J Nephrol, 2016, 29(6): 765-73.
[2] Xu Y, Wang L, He J, et al. Prevalence and control of diabetes in Chinese adults[J]. JAMA, 2013, 310(9): 948-59.
[3] Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues[J]. Cell, 2011,147(4): 728-41.
[4] Fang L, Zhou Y, Cao H, et al. Autophagy attenuates diabetic glomerular damage through protection of hyperglycemia-induced podocyte injury[J]. PLoS One, 2013, 8(4): e60546.
[5] 展照双, 齐丽娟, 吕翠霞,等. 肾气丸与右归丸对肾虚大鼠肾脏细胞凋亡及肾组织内Bcl-2、Fas表达的影响[J]. 山东中医杂志, 2011, 30(6): 412-4.[5] Zhan Z S, Qi L J, Lyu C X, et al. Effects of Shenqi Pill and Yougui Pill on apoptosis of renal cells and expression of Bcl-2, Fas in renal tissue of kidney deficiency rats[J]. Shandong J Tradit Chin Med, 2011, 30(6): 412-4.
[6] 李青, 韩宇博. 金匮肾气丸联合美卡素治疗Ⅲ期糖尿病肾病的临床研究[J]. 光明中医, 2014, 29(3): 576-7.[6] Li Q, Han Y B. Clinical study of Jinguishenqi Pill combined with mekacin in the treatment of stage III diabetic nephropathy[J]. Guangming J Chin Med, 2014, 29(3): 576-7.
[7] 刘忠文. 金匮肾气丸治疗糖尿病肾病的疗效评价[J].中国中医基础医学杂志,2014, 20(6):118-9, 128.[7] Liu Z W. Evaluation of curative effect of Jinkui Shenqi Pill on diabetic nephropathy[J]. Chin J Basic Med Tradit Chin Med, 2014, 20(6):118-9, 128.
[8] 金智生,陈雪,李甜. 金匮肾气丸对实验性2型糖尿病肾病大鼠肾脏组织NO、NOS的影响[J]. 中医药学报,2012,40(1): 60-3.[8] Jin Z S, Chen X, Li T. Effect of Jinguishenqi Pill on NO, NOS in renal tissue of experimental type 2 diabetic nephropathy rats[J]. Acta Chin Med Pharmacol, 2012,40(1): 60-3.
[9] Weil E J, Lemley K V, Mason C C, et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy[J]. Kidney Int, 2012, 82(9): 1010-7.
[10] Shankland S J. The podocyte′s response to injury: role in proteinuria and glomerulosclerosis[J]. Kidney Int, 2006, 69(12): 2131-47.
[11] 丁海华,邱原野,王盈盈, 等. 小檗碱对糖尿病肾病大鼠nephrin、podocin和α3β1整合素表达的影响[J]. 中国药理学通报,2015,31(10): 1414-20.[11] Ding H H, Qiu Y Y, Wang Y Y, et al. Effect of berberine on the expression of nephrin, podocin and intergrin α3β1 in diabetic nephropathy rats[J]. Chin Pharmacol Bull,2015,31(10):1414-20.
[12] Lenoir O, Jasiek M, Henique C, et al. Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis[J]. Autophagy, 2015, 11(7): 1130-45.
[13] Ilatovskaya D V, Levchenko V, Lowing A, et al. Podocyte injury in diabetic nephropathy: implications of angiotensin II-dependent activation of TRPC channels[J]. Sci Rep, 2015, 5: 17637.
[14] Langer S, Kreutz R, Eisenreich A. Metformin modulates apoptosis and cell signaling of human podocytes under high glucose conditions[J]. J Nephrol, 2016, 29(6): 765-73.