二氢杨梅素通过SIRT3调节2型糖尿病小鼠肠上皮屏障功能作用

Dihydromyricetin regulates intestinal epithelial barrier function of type 2 diabetes mellitus mice through SIRT3

目的研究二氢杨梅素(dihydromyricetin,DHM)对高脂饮食(high-fat diet,HFD)诱导2型糖尿病小鼠肠上皮屏障功能的作用,并揭示SIRT3在介导DHM改善肠上皮屏障中的作用机制。方法选用7周龄的SPF级雄性野生型(WT)小鼠18只和SIRT3敲除(SIRT3 KO)小鼠30只,分别按随机数字表法分为对照组(NC)、高脂组(HFD)、高脂加二氢杨梅素组(HFD+DHM),定期监测动物体质量、摄食量。干预12周,检测小鼠肠道通透性。小鼠处死后,检测血清生化指标和LPS水平及肠道组织病理学。利用分离的小鼠小肠上皮细胞和Caco-2细胞系,通过Western blot检测紧密连接蛋白(ZO-1、Occludin)和qRT-PCR检测炎症因子(TNF-α、IL-1β、IL-6)表达。进一步利用慢病毒(LV-SIRT3)感染Caco-2细胞敲降SIRT3表达,DHM和LPS干预后检测相关炎症因子和紧密连接蛋白表达。结果HFD诱导WT和SIRT3 KO小鼠体质量明显增加、糖脂代谢异常、肠道通透性增加、肠上皮结构改变(小肠隐窝深度和上皮绒毛长度减少)、肠上皮细胞紧密连接蛋白表达降低和炎症因子表达增加,且与WT小鼠相比,HFD诱导的SIRT3 KO小鼠相关指标改变具有统计学意义(P<0.05)。DHM干预后WT小鼠上述检测指标改善具有统计学意义(P<0.05),但DHM的作用在SIRT3 KO小鼠中不明显,差异无统计学意义(P>0.05)。体外实验发现,在LV-SIRT3感染的Caco-2细胞中,DHM对LPS诱导的炎症因子表达增加和紧密连接蛋白表达降低作用不明显,且差异无统计学意义(P>0.05)。结论SIRT3在DHM增加小肠上皮紧密连接蛋白表达、降低炎症因子表达、维持肠上皮屏障完整性和减少通透性、改善糖脂代谢和延缓T2DM发生中发挥重要作用。

Objective To investigate the effect of dihydromyricetin(DHM)on high-fat diet(HFD)-induced intestinal epithelial barrier function in mouse model of type 2 diabetes mellitus(T2DM),and to reveal the mechanism involved with SIRT3 in mediating DHM in the process.Methods Eighteen 7-week-old SPF male wild-type(WT)mice and 30 SIRT3 KO mice were randomly divided into the control group(NC),HFD group and HFD+DHM group,respectively.The intervention time was 12 weeks.The body weight and food intake of the animals were monitored regularly.At week 12 of intervention,intestinal permeability were tested.After the mice were sacrificed,serum biochemical indexes and LPS levels as well as the intestinal histopathology were detected and observed.The expression of tight junction proteins(ZO-1,Occludin)and inflammatory factors(TNF-α,IL-1βand IL-6)in the isolated small intestinal epithelial cells and Caco-2 cells were detected by qRT-PCR and Western blotting.Then the Caco-2 cells were further infected with lentivirus(LV-SIRT3)to knock down the SIRT3 expression,and the expression levels of above molecules were detected after DHM and LPS treatment.Results In the WT and SIRT3 KO mice,HFD induced a significant increased body weight,abnormal glucose level and lipid metabolism,increased intestinal permeability and obvious changes in intestinal epithelial structure(reduced small intestinal epithelium crypt depth and villi length),as well as decreased levels of tight junction proteins and elevated levels of inflammatory factors.Furthermore,the alterations induced by HFD in SIRT3 KO mice were more significant than those in WT mice(P<0.05).The administration of DHM notably ameliorated the associated changes induced by HFD in the WT mice(P<0.05)instead of the SIRT3 KO mice(P>0.05).In the LV-SIRT3-infected Caco-2 cells,DHM failed to inhibit LPS-induced enhanced expression of the inflammatory cytokines and decreased expression of the tight junction proteins(P>0.05).Conclusion SIRT3 plays an important role in DHM-induced increased expression of small intestinal epithelial tight junction proteins,reduced expression of the inflammatory factors,maintenance of the intestinal epithelial barrier integrity and permeability and improvement of glycolipid metabolism,and thus is beneficial to delay the development of T2DM.