基于内质网应激-自噬通路研究茯苓多糖对2型糖尿病小鼠肠道屏障功能损伤和炎症反应的影响

Poria cocos polysaccharide improves intestinal barrier function and at⁃tenuates inflammatory response in type 2 diabetic mice through endo⁃plasmic reticulum stress-autophagy pathway

目的:探讨茯苓多糖(PCP)对2型糖尿病(T2DM)小鼠肠道屏障功能损伤和炎症反应的影响及潜在机制。方法:取C57BLKS/JNju小鼠为对照(control)组,将db/db小鼠分为模型(model)组、低剂量(6 mg/kg)PCP(PCP-L)组、高剂量(12 mg/kg)PCP组(PCP-H)组、内质网应激(ERS)诱导剂衣霉素(Tm;1 mg/kg)组、PCP(12 mg/kg)+Tm(1 mg/kg)组,每组12只。进行口服葡萄糖耐量试验和胰岛素耐量试验;检测小鼠空腹血糖(FBG)及空腹血清胰岛素(FINS)水平,计算胰岛素抵抗指数(HOMA-IR);检测肠道通透性,以及血清二胺氧化酶(DAO)、D-乳酸(D-LA)、内毒素(ET)、白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)水平;观察小鼠肠组织病理学变化和肠上皮细胞自噬情况;检测肠组织闭合蛋白(occludin)、闭锁小带蛋白1(ZO-1)、蛋白激酶R样内质网激酶(PERK)、p-PERK、真核生物翻译起始因子2α(eIF2α)、p-eIF2α、转录激活因子4(ATF4)、C/EBP同源蛋白(CHOP)、微管相关蛋白1轻链3A/B(LC3A/B)、beclin-1和p62蛋白水平。结果:与control组相比,model组小鼠各时点血糖和曲线下面积,FBG、FINS和HOMA-IR,荧光素异硫氰酸酯-葡聚糖浓度,肠组织损伤评分,血清DAO、D-LA、ET、IL-6、TNF-α和IL-1β水平,自噬小体数量,肠组织p-PERK/PERK比值、p-eIF2α/eIF2α比值、LC3-II/LC3-I比值,以及ATF4、CHOP和beclin-1蛋白水平显著升高,肠组织occludin、ZO-1和p62蛋白水平显著降低(P<0.05);与model组相比,PCP-L组和PCP-H组小鼠上述指标显著改善(P<0.05);Tm可显著激活自噬,减弱PCP对T2DM小鼠肠道屏障功能的保护作用(P<0.05)。结论:PCP可减轻T2DM小鼠肠黏膜损伤和炎症反应,改善肠道屏障功能,其作用机制可能与抑制ERS引起的自噬激活有关。

AIM:To investigate the effects of Poria cocos polysaccharide(PCP)on intestinal barrier function and inflammatory response in type 2 diabetes mellitus(T2DM)mice and its underlying mechanism.METHODS:C57BLKS/JNju mice were chosen as control group,and db/db mice were divided into model group,low-dose(6 mg/kg)PCP(PCP-L)group,high-dose(12 mg/kg)PCP(PCP-H)group,endoplasmic reticulum stress(ERS)inducer tunicamycin(Tm;1 mg/kg)group,and PCP(12 mg/kg)+Tm(1 mg/kg)group,with 12 rats in each group.Oral glucose tolerance test and insulin tolerance test were performed.Fasting blood glucose(FBG)and fasting serum insulin(FINS)levels were detected,and insulin resistance index(HOMA-IR)was calculated.The intestinal permeability,and the serum levels of diamine oxidase(DAO),D-lactic acid(D-LA),endotoxin(ET),interleukin 6(IL-6),IL-1βand tumor necrosis factorα(TNF-α)were detected.The pathological changes of mouse intestinal tissue and the autophagy of intestinal epithelial cells were observed.The protein levels of occludin,zonula occludens-1(ZO-1),protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,eukaryotic translation initiation factor 2α(eIF2α),p-eIF2α,activating transcription factor 4(ATF4),C/EBP homologous protein(CHOP),microtubule-associated protein 1 light chain 3A/B(LC3A/B),beclin-1 and p62 were detected.RESULTS:Compared with control group,the blood glucose and its area under curve,FBG,FINS,HOMA-IR,fluorescein isothiocyanate-dextran concentration,intestinal tissue damage score,serum DAO,D-LA,ET,IL-6,TNF-αand IL-1βlevels,number of autophagosomes,p-PERK/PERK ratio,p-eIF2α/eIF2αratio,LC3-II/LC3-I ratio,and ATF4,CHOP and beclin-1 protein levels in model group were significantly increased,while the protein levels of occludin,ZO-1 and p62 were significantly reduced(P<0.05).Compared with model group,the above indicators in PCP-L and PCP-H groups were significantly improved(P<0.05).Treatment with Tm significantly activated autophagy and weakened the protective effect of PCP on the intestinal barrier function of T2DM mice(P<0.05).CONCLUSION:The PCP reduces intestinal mucosal damage and inflammation in T2DM mice through the inhibition of autophagy caused by ERS.