糖尿病肾病大鼠骨骼肌自噬-溶酶体途径水平变化及低蛋白联合α-酮酸饮食对其作用

Autophagy-lysosome pathway in skeletal muscle of diabetic nephropathy rats and the effect of low-protein diet plus α-keto acids on it

目的 观察糖尿病肾病大鼠骨骼肌中自噬水平的变化及低蛋白联合α-酮酸饮食对其的干预作用,探讨低蛋白联合α-酮酸改善糖尿病肾病大鼠骨骼肌萎缩的机制.方法 24周龄Goto-Kakizaki大鼠分为3组：正常蛋白组（22％酪蛋白饮食,简称NPD组）、低蛋白组（6％酪蛋白饮食,简称LPD组）及低蛋白联合α-酮酸组（6％酪蛋白饮食,简称Keto组）,每组15只;15只Wistar大鼠给予正常蛋白饮食,作为对照组（简称CTL组）.48周后检测各组大鼠比目鱼肌中自噬-溶酶体标志物微管相关蛋白1轻链3B（LC3B）、Bcl2/腺病毒E1B结合蛋白3（Bnip3）、组织蛋白酶L（Cathepsin L）的mRNA及蛋白含量,电镜进一步验证自噬-溶酶体途径的变化.结果 与CTL组大鼠相比,NPD组LC3B、Bnip3及Cathepsin L的mRNA均较高（均P＜0.05）.且NPD组LC3B-Ⅰ、LC3B-Ⅱ、Bnip3及Cathepsin L的蛋白均较CTL组高（0.82±0.33比0.25±0.07,0.76±0.38比0.20±0.12,1.25±0.30比0.56±0.19,1.29 ±0.40比0.69±0.20）.与NPD相比,LPD组LC3B、Bnip3及Cathepsin L mRNA含量稍低,且LC3B-Ⅰ、LC3B-Ⅱ、Bnip3及Cathepsin L的蛋白含量稍低,但差异均无统计学意义（均P＞0.05）.与NPD组及LPD组相比,Keto组上述自噬标志物的mRNA明显低,且其蛋白含量明显低.电镜示NPD组及LPD存在多量的自噬小体或自噬性溶酶体,而CTL组及Keto组检查阴性.结论 糖尿病肾病大鼠骨骼肌存在着自噬-溶酶体途径活化的现象.低蛋白联合α-酮酸饮食可阻断骨骼肌中自噬-溶酶体途径的活化,进而缓解骨骼肌萎缩.

Objective To explore the regulation of autophagy-lysosome pathway （ALP） in skeletal muscle of diabetic nephropathy and examine the effect of low protein diet plus α-keto acid on ALP.Methods A total of 45 24-week-old Goto-Kakizaki rats were randomized to receive normal protein （22％） diet （NPD）,low-protein （6％） diet （LPD） or low-protein （5 ％） plus α-keto acids （1％） diet （Keto） （n =15 each）.Wistar control rats had a normal protein diet.The mRNA and protein levels of ALP markers LC3B,Bnip3,Cathepsin L in soleus muscle were evaluated at 48 weeks.Electron microscopy was used to confirm the changes of autophagy.Results Compared with CTL group,the mRNA levels of LC3B,Bnip3,Cathepsin L in soleus muscle of rats on NPD were higher,and protein levels of LC3B-Ⅰ,LC3B-Ⅱ,Bnip3,Cathepsin L in soleus muscle of rats on NPD also higher than CTL group （0.82 ± 0.33 vs 0.25 ± 0.07,0.76±0.38vs0.20±0.12,1.25±0.30 vs0.56±0.19,1.29±0.40 vs0.69±0.20）.The mRNA levels of LC3B,Bnip3 and Cathepsin L in LPD group were slightly lower,compared with NPD group.However there was no statistical significance.Similarly the protein levels of LC3B-Ⅰ,LC3B-Ⅱ,Bnip3 and Cathepsin L in LPD group were slightly lower with no statistical significance.In contrast,the mRNA levels of LC3B,Bnip3 and Cathepsin L were greatly lower in Keto group in comparison with NPD and LPD.And protein levels of LC3B-Ⅰ,LC3B-Ⅱ,Bnip3 and Cathepsin L were also greatly lower in Keto group in comparison with NPD and LPD.Additionally,autophagosome or autolvsosome was found in NPD and LPD groups by electron microscopy.Conclusions ALP is activated in skeletal muscle of diabetic nephropathy rats.And low protein plus α-keto acid decrease the activation of ALP and improve muscle wasting.