CAJ | 학술논문

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.
목적 관찰당뇨병신병대서골격기중자서수평적변화급저단백연합α-동산음식대기적간예작용,탐토저단백연합α-동산개선당뇨병신병대서골격기위축적궤제.방법 24주령Goto-Kakizaki대서분위3조:정상단백조(22％락단백음식,간칭NPD조)、저단백조(6％락단백음식,간칭LPD조)급저단백연합α-동산조(6％락단백음식,간칭Keto조),매조15지;15지Wistar대서급여정상단백음식,작위대조조(간칭CTL조).48주후검측각조대서비목어기중자서-용매체표지물미관상관단백1경련3B(LC3B)、Bcl2/선병독E1B결합단백3(Bnip3)、조직단백매L(Cathepsin L)적mRNA급단백함량,전경진일보험증자서-용매체도경적변화.결과 여CTL조대서상비,NPD조LC3B、Bnip3급Cathepsin L적mRNA균교고(균P＜0.05).차NPD조LC3B-Ⅰ、LC3B-Ⅱ、Bnip3급Cathepsin L적단백균교CTL조고(0.82±0.33비0.25±0.07,0.76±0.38비0.20±0.12,1.25±0.30비0.56±0.19,1.29 ±0.40비0.69±0.20).여NPD상비,LPD조LC3B、Bnip3급Cathepsin L mRNA함량초저,차LC3B-Ⅰ、LC3B-Ⅱ、Bnip3급Cathepsin L적단백함량초저,단차이균무통계학의의(균P＞0.05).여NPD조급LPD조상비,Keto조상술자서표지물적mRNA명현저,차기단백함량명현저.전경시NPD조급LPD존재다량적자서소체혹자서성용매체,이CTL조급Keto조검사음성.결론 당뇨병신병대서골격기존재착자서-용매체도경활화적현상.저단백연합α-동산음식가조단골격기중자서-용매체도경적활화,진이완해골격기위축.
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.