| Purpose: In this study,we simulated the process of T2 DM injury through a db/db mouse model and observed the ameliorative effects of the Chinese herbal compound on T2DM-induced small intestinal inflammation,oxidative stress and apoptosis,in order to further elucidate the mechanism of T2 DM on intestinal protection and provide some theoretical basis for the clinical application of the Chinese herbal compound Yixiangkang.Material and method:Twenty-four SPF-grade male,9-week-old C57BL/6J db/db(hereafter referred to as db/db)mice were randomly divided into three groups: the model group(db/db group),the YTK group(YTK group),and the western drug(liraglutide)control group(LIRA group),and 8 heterozygous db/m mice in the same cage were selected as the normal control group(db/m).group).The Chinese medicine compound Yixiang Kang consisted of 12 herbs including Huang Lian,Huang Bai,Huang Qi,Huang Jing,Bai Zhu and San Qi.After one week of acclimatization feeding,mice in the YTK group were given decoction by gavage at 30 g-kg-1-d-1.liraglutide was injected subcutaneously at 200μg-kg-1-d-1 daily.db/m and db/db groups were given distilled water by gavage for 5 weeks in total.After 5 weeks of drug treatment,animals were sampled and tissues were isolated after12 h fasting of mice.Changes in small intestinal pathology were detected by HE staining;fasting blood glucose(FBG)of harvested mice was detected by blood glucose test paper;fasting serum insulin(FINS)and serum IL-6,IL-1β and TNF-a expression were detected by ELISA assay.Their serum levels were also measured by kits for SOD,MDA and CAT;expression of small intestinal IL-6 and IL-1β by immunohistochemistry;expression of small intestinal Occludin,Claudin,Claudin5 protein and SOD2,as well as apoptosis signaling molecules BCL-2,Bax,caspase3 by Western blot.Small intestinal apoptosis was detected by TUNEL assay in each group of mice.Results:1.General condition of mice: the mice in the blank control group were in good mental condition,with shiny hair,agile activity,normal eating and drinking;the mice in the model group were in poor mental condition,with rough hair,reduced activity,slow reflection,bowed back and curled body,and increased eating and urine volume;after treatment,compared with the model group,the mice in the YTK and Liraglutide groups were more responsive,with better shiny hair,and improved eating,drinking and urine volume.2.Glucose and insulin levels: compared with db/m,blood glucose and insulin were significantly higher in the db/db group(P<0.05);compared with the db/db group,both the YTK and LIRA groups significantly reduced blood glucose and insulin(P<0.05).3.Serum levels of IL-6,IL-1β and TNF-a:Compared with the db/m group,serum inflammatory factors IL-6,IL-1β and TNF-a were significantly increased in the db/db group of mice(P<0.05);compared with the db/db group,IL-6,IL-1β and TNF-a were significantly decreased in the YTK and LIRA groups(P<0.05).4.HE staining results: db/m mice had intact small intestinal tissue structure,neat villi,regular mucosal morphology,visible intact mucosal base and abundant small blood vessels,and no infiltration of inflammatory cells;db/db mice had structural destruction of small intestine,partial defect,fracture,necrosis and loss of small intestinal villi,and visible infiltration of large number of inflammatory cells;after YTK and LIRA treatment,the inflammatory damage of small intestinal mucosa was reduced and villi were improved in mice.5.Expression of tight junction proteins Occludin,Claudin1 and Claudin5: Western blot results showed that the expression of Occludin,Claudin1 and Claudin5 proteins in the small intestine of mice in the db/db group was significantly decreased compared with mice in the db/m group(P<0.05);while compared with mice in the db/db group,the expression of the above proteins was significantly upregulated in the small intestine of mice in the YTK and LIRA groups(P<0.05).6.Expression of small intestinal inflammatory factors IL-6 and IL-1β: immunohistochemical results showed that the expression of IL-6 and IL-1β in the mucosa and submucosa of db/db mice was significantly enhanced compared with the db/m group(P<0.05);while YTK and LIRA groups showed moderate expression compared with the db/db group,which was significantly attenuated compared with the db/db group(P<0.05).7.Levels of serum SOD,MDA and CAT: compared with the db/m group,serum SOD and CAT were significantly decreased and MDA was significantly increased in the db/db mice(P<0.05);compared with the db/db group,SOD and CAT were significantly increased and MDA was significantly decreased in the YTK and LIRA groups(P<0.05).8.Small intestinal SOD2 expression: Western blot results showed that the expression of small intestinal SOD was significantly decreased in the db/db group compared with the db/m group(P<0.05);the expression of SOD2 was significantly increased in the YTK and LIRA groups compared with the mice in the db/db group(P<0.05).9.Small intestinal apoptosis:Western blot results showed that the number of small intestinal apoptotic vesicles was significantly higher in mice in the db/db group compared with mice in the db/m group,and the ratio of Bax/Bcl-2 and Cleaved/Pro-caspase3 were significantly higher in the small intestine(P<0.05);compared with mice in the db/db group,the expression of SOD2 was significantly higher in mice treated with YTK and LIRA group,the ratio of Bax/Bcl-2 and Cleaved/Pro-caspase3 in the small intestine were significantly decreased after treatment(P<0.05).Conclusion:1.The herbal compound Yiangkang can improve the structural damage of small intestine in db/db mice.2.Chinese herbal compound can inhibit the inflammatory response in serum as well as small intestine of db/db mice.3.The Chinese herbal formula can attenuate the oxidative stress response in the serum and small intestine of db/db mice.4.The Chinese herbal formula can improve apoptosis in small intestine of db/db mice. |
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