从“脾与小肠相通”探讨益糖康调控db/db小鼠支链氨基酸代谢修复小肠黏膜屏障损伤的作用机制

Mechanism of Yitangkang regulating the metabolism of branched-chain amino acids in db/db mice to repair the damage of intestinal mucosal barrier from'the spleen communicating with the small intestine'

目的:基于“脾与小肠相通”理论观察从脾论治方药复方益糖康对db/db小鼠支链氨基酸代谢紊乱的调控及修复小肠黏膜屏障损伤的作用机制。方法:选取36只db/db小鼠,按照随机数字法分为模型组、支链氨基酸分解剂组(BT2组)以及益糖康组(YTK组),另选取12只db/m小鼠作为对照组。分别给予不同的干预措施。5周后,检测小鼠体质量及空腹血糖(FBG)水平,UPLC-MS/MS方法检测小鼠血清内支链氨基酸水平,HE染色检测小肠组织病理的变化,Western Blot和RT-qPCR法检测小肠黏膜屏障相关封闭蛋白1(Claudin1)、紧密连接蛋白闭合蛋白(Occludin)、闭锁连接蛋白-1(ZO-1)蛋白及mRNA表达情况。结果:与对照组比较,模型组小鼠的体质量和FBG水平均显著升高(P<0.01),亮氨酸(Leu)、异亮氨酸(Ile)、缬氨酸(Val)水平均显著升高(P<0.01),HE染色结果显示模型组小鼠小肠黏膜结构破坏,可见大量的炎症浸润,Claudin1、Occludin、ZO-1蛋白及mRNA表达均显著降低(P<0.01);治疗后与模型组比较,YTK组和BT2组小鼠体质量、FBG水平均显著降低(P<0.05,P<0.01);Leu、Ile、Val水平均显著降低(P<0.05,P<0.01),HE染色结果显示小肠黏膜结构改善、炎症损伤减少,Claudin1、Occludin、ZO-1蛋白及mRNA表达显著升高(P<0.01),且YTK组和BT2组比较无显著性差异。结论:脾与小肠功能失调为2型糖尿病(T2DM)发生的重要原因,饮食不节引起的支链氨基酸代谢紊乱会引起小肠黏膜屏障损伤,而从脾论治方药益糖康能够改善支链氨基酸代谢紊乱,并可以修复小肠黏膜屏障损伤,发挥治疗T2DM的作用。

Objective:Based on the theory of‘the spleen communicating with the small intestine',to observe the effects of compound Yitangkang on the regulation of branched-chain amino acid metabolism disorder in db/db mice and the mechanism of repairing the damage of intestinal mucosal barrier.Methods:Thirty-six db/db mice were selected and divided into db/db group,BT2 group and YTK group according to random number method,and 12 db/m mice were selected as control group.Different intervention measures were given.After 5 weeks,body weight and fasting blood glucose(FBG)levels were measured,serum branched-chain amino acids were detected by UPLC-MS/MS method,pathological changes of small intestine were detected by HE staining,and proteins and mRNA expressions of mucosal barrier related Claudinl,Occludin and ZO-1 were detected by Western Blot and RT-qPCR.Results:Compared with db/m group,body weight and FBG levels in db/db group were significantly increased(P<0.01),leucine(Leu),isoleucine(Ile)and valine(Val)levels were significantly increased(P<0.01),and the small intestinal mucosal structure of db/db group was damaged by HE staining,and a large number of inflammatory infiltrates could be seen.The proteins and mRNA expression of Claudin1,Occludin and ZO-1 were decreased(P<0.01).After treatment,body weight and FBG levels in YTK group and BT2 group were significantly decreased compared with db/db group(P<0.05,P<0.01).Leu,Ile and Val levels were significantly decreased(P<0.05,P<0.01),HE staining results showed that intestinal mucosal structure improved,inflammatory damage decreased,and protein and mRNA expression of Claudin1,Occludin and ZO-1 significantly increased(P<0.01).There was no significant difference between YTK group and BT2 group.Conclusion:The dysfunction of spleen and small intestine is an important cause of type 2 diabetes mellitus(T2DM).The metabolism disorder of branched-chain amino acids caused by improper diet may lead to the damage of intestinal mucosal barrier.Yitangkang,a prescription based on spleen,can improve the metabolism disorder of branched-chain amino acids,repair the damage of intestinal mucosal barrier,and play a role in T2DM treatment.