Study On The Mechanism Of Polyphenols From Juglans Regia In Improving Ulcerative Colitis In Mice

Ulcerative colitis(UC)is a chronic non-specific inflammatory disease of the colon and rectum,the incidence of which is increasing worldwide year by year.Traditional drug treatment is associated with serious adverse effects,so it is important to explore functional nutritional factors from natural products that have an ameliorative or palliative effect on UC.Walnut(Juglans regia)is a genus of walnut(Juglans)in the family Juglandaceae.The heartwood is the septum of walnut,which has a variety of medicinal activities as a traditional Chinese medicine,but there are problems such as low exploitation and potential value has not been exploited.Walnut heartwood is rich in polyphenols,flavonoids and other active ingredients,and polyphenols are known to have good anti-inflammatory activity,but the anti-colitis effect and mechanism of walnut heartwood polyphenols have not been reported yet.Therefore,in this study,the extraction process of the heartwood polyphenols was optimized using orthogonal tests,and the crude polyphenol composition of the heartwood was identified by AB Sciex QTOFs high resolution mass spectrometry,while the heartwood polyphenols were purified using D101 macroporous resin.Animal tests were conducted using purified centella polyphenols,and the effects and mechanisms of centella polyphenols on the improvement of ulcerative colitis in mice were investigated in terms of basal indexes,intestinal barrier,inflammatory response and intestinal microorganisms.Animal experiments were as follows: 36-week-old male C57BL/6J mice were randomly divided into 3 groups,blank control group(Control group),model group(DSS group)and polyphenol-treated group(WDP),and the mice were fed with 3.0% DSS solution and also intervened with 100 mg/kg of Centella asiatica polyphenol solution for 12 days,during which time their body weight was monitored daily and the DAI score.After 12 days,the colon length and spleen weight of mice were measured;H&E staining was used to observe the histopathological damage of mouse colon;immunohistochemistry was used to observe the infiltration of inflammatory cells and the expression level of intestinal barrier protein;Alisin Blue staining was used to detect the number of colonic cup cells;ELISA standard kit was used to detect the serum inflammatory factor content of mice;RT-q PCR was used to detect the expression level of inflammation and intestinal barrier in mouse colon tissue;16Sr RNA sequencing was used to detect the structure and diversity of mouse intestinal microorganisms.The results showed that the optimum extraction conditions for walnut heartwood polyphenols were 1:90 g/m L,40 min sonication time,45°C sonication temperature and30% ethanol concentration,and the content of heartwood polyphenols was 121.2±1.36mg/g under these extraction conditions;Using liquid chromatography,the six phenolic compounds with high content in the polyphenols of walnut heartwood were identified as:desmethoxycurcumin,protocatechuic aldehyde,protocatechuic acid,vanillin,phenylethanol and ellagic acid.Walnut heartwood polyphenols were purified using D101 to achieve a purity of 80.5%.The results of animal experiments showed that walnut heartwood polyphenols improved the effects of weight loss,increased DAI scores,shortened colon and enlarged spleen in mice with colitis;they also improved histopathological damage,reduced inflammatory cell infiltration in mice with colitis,restored the expression of intestinal barrier proteins,maintained the function of mucus secretion in colonic tissues,reduced MPO activity,serum inflammatory factor content and colonic tissue m RNA levels of inflammatory factors in colonic tissues;And centella asiatica polyphenols reversed m RNA expression levels of intestinal tight junction-related genes in mice with colitis;In addition,walnut phloem polyphenols increased the relative abundance of the beneficial bacteria Firmicutes,Desulfobacterota,Patescibacteria,Deferribacterota,Lachnospiraceae,Oscillospiraceae,Lachnospiraceae＿NK4A136＿group,Lactobacillus and reduced the relative abundance of the harmful bacteria Enterobacteriaceae,Bacteroides,Escherichia-shigella,Faecalibaculum.Correlation analysis showed that harmful bacteria were positively associated with inflammatory genes(TNF-a,IL-1β,IL-6,IFN-γ)and serum inflammatory factors(TNF-a,IL-1β)in colon tissue,and beneficial bacteria were positively associated with intestinal tight junction-related genes(ZO-1,Occludin,Claudin-1,Muc-2).The results showed that walnut heartwood polyphenols improved DSS-induced intestinal flora dysbiosis,increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria,raised the intestinal barrier in mice with colitis,reduced inflammatory cytokine infiltration and decreased the level of inflammation in mice with colitis,thus exerting the effect of improving ulcerative colitis in mice.The results of the study may provide an experimental basis for the development of functional food products for the relief of ulcerative colitis by walnut heartwood polyphenols,and also provide new ideas for the study of the mechanism of polyphenols’ effect on improving colitis.