| The intestinal mucosal barrier is an important barrier to protect the human body from exogenous pathogens,mainly composed of epithelial cells connected by tight junction proteins.Tight junction proteins(TJs)are the basis of the epithelial barrier,which are mainly responsible for solute transport of cells and play an important role in regulating cell differentiation and permeability.Dysfunction of tight junction proteins will cause changes in the structure and function of intestinal mucosal barriers,and the development of various diseases such as inflammatory bowel disease,autoimmune disease,hyperlipidemia and other related diseases.Therefore,it is of great significance to explore the protection mechanism of the intestinal mucosal barrier and to develop related protective products.Tangerine peel,which is not only a traditional gastrointestinal protective drug but also a homologous food substance in China,has various biological activities such as anti-oxidation,anti-inflammatory and anti-tumor.Previous studies in our laboratory have confirmed that the most abundant representative polymethoxylated flavonoid,Nobiletin(NOB),has good anti-inflammatory effects.In this study,we focused on the feasibility and mechanism of maintaining intestinal mucosal stability by the tangerine peel.In this study,we first constructed a mouse intestinal epithelial monolayer to mimic the intestinal mucosal barrier in vitro and verified whether the tangerine peel and NOB have a protective effect on it,and the transepithelial electrical resistance(TEER)was used to detect the integrity of mouse epithelial monolayer.We found that tangerine peel and NOB can prevent the inflammatory stimulation from impairing the permeability of intestinal epithelial monolayer in mice,suggesting that tangerine peel and NOB may have protective effects on the intestinal mucosal barrier.Therefore,we used a model of mouse inflammatory bowel disease induced by Dextran sulfate sodium salt(DSS)for in vivo experiments and randomly divided mice into blank control group,DSS treatment group,0.01% NOB group,10% tangerine peel group,with 12 mice in each group.The blank control group was fed with normal diet.The other three groups consumed normal feed,0.01% NOB and 10% dried tangerine peel,while drinking 3% DSS water.The results showed that tangerine peel and NOB could significantly inhibit DSS-induced weight loss,the colon shortened and the stool shapeless even a bloody stool in mice.In addition,fluorescein isothiocyanate(FITC)experiments confirmed that both 0.01% NOB and tangerine peel can improve the intestinal mucosal barrier damage caused by DSS,further indicating that tangerine peel and NOB have a good protective effect on the intestinal mucosal barrier in mice.We also performed HE staining histological analysis on the colon of mice and demonstrated that both tangerine peel and NOB could inhibit DSS-induced villus shortening and structural damage in mice.The above results indicate that tangerine peel and NOB have a good protective effect on the intestinal mucosal barrier of mice.In order to investigate the mechanism of tangerine peel and NOB on the intestinal mucosal barrier of mice,we examined the expression of a series of tight junction proteins in intestinal epithelial cells by real-time quantitative(RT-PCR).It was found that DSS treatment in the colon of mice can lead to Claudin family members Claudin-7(CLDN7)be significantly down-regulated,while 0.01% NOB and dried tangerine peel reversed DSS-induced inhibition of claudin-7 expression.In theory,the expression of claudin-7 during intestinal epithelial cell differentiation is usually regulated by the nuclear receptor hepatocyte nuclear factor 4 alpha(HNF4-?).To verify whether the signaling pathway of HNF4-?-claudin-7 is involved the protective mechanism of tangerine peel or NOB,we first detected the expression of HNF4-? in mouse colon by RT-PCR and immunohistochemistry,indicating that tangerine peel and NOB can up-regulate the expression of HNF4-? markedly.To further validate the transcriptional regulation of NOB-induced HNF4-? on claudin-7,we specifically knocked down HNF4-? in HT-29 cells by RNA interference technique and found that HNF4-? knockdown was able to inhibit NOB-induced claudin-7 expression significantly.At last,further luciferase reporter experiments showed that the vector activity of the HNF4-? binding site in the mutant claudin-7 promoter was lower than that of the wild-type construct.Taken together,our results indicate that tangerine peel and its representative polymethoxylated flavonoid NOB can protect the intestinal mucosal barrier by inducing HNF4-? transcriptional regulation of claudin-7. |
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