Mechanism Underlying The Alleviation Of Ulcerative Colitis In An Mouse Model By Pterostilbene Treatment

Ulcerative colitis(UC)is a chronic inflammatory disease of the intestine characterized by recurrent or prolonged inflammation of the intestinal mucosa.The lesion is located in the colon and rectum,and the incidence rate is increasing worldwide.The main symptoms of UC are bloody stool and diarrhea,which seriously affects the quality of life of UC patients.The pathogenesis of UC is still unclear,and its disease progression mainly includes intestinal barrier function damage,intestinal mucosal inflammation and gut microbiota dysbiosis.Although the use of biological drugs has increased the clinical remission rate of UC in the past decade,the long-term cure rate has no significant increase,and the expensiveness of biological drugs has introduced a heavy economic burden to social and public healthcare system.Therefore,the development of effective drugs for UC with much less economic impact is of great significance to UC patients and national medical care.Natural plant products are abundant in nature and produce a large number of secondary metabolites with rich structural diversity,including a large number of small molecular compounds with anti-inflammatory activity,which is one of the main sources of research and development of UC drugs.Pterostilbene(PTE)is a kind of polyphenol compound that widely exists in grape trees and blueberries.It has high bioavailability and metabolic stability.PTE has anti-inflammatory functions and antioxidant biological activities in various inflammatory animal models.However,the mechanism underlying the alleviation of UC by using PTE is till large unknown,and it has been less investigated in combination with our current understanding of US pathophysiology.This study elucidates the molecular cell biological mechanism underlying PTE therapeutic functions for UC in vivo and in vitro and provides a scientific basis for further clinical trials and finally its application for UC clincal treatment in the future.The main results of my Ph D project are as follows:The first part was to establish a UC mouse model and evaluate the curative effectiveness of PTE for UC therapy.The UC mouse model was induced by dextran sulfate sodium(DSS),and the success of the model was assessed by the observation of mouse body weight changes,appraisal of disease activity index(DAI),colonic histopathological score,and colonic inflammatory factor levels during the PTE treatment.After PTE administration,the symptoms of UC mice were significantly relieved,and the reduction of DAI score and colon histopathological score was pronounced.ELISA analysis showed that PTE treatment significantly reduced the levels of TNF-α,IL-6,IL-1β and ROS in the colonic tissue of UC mice and CRP level in plasma.PTE also increased colonic tissue SOD and GSH-px levels in the colonic tissue.Western blotting analysis showed that PTE treatment introduced higher expression levels of tight junction proteins(TJs,e.g.,ZO1 and Occludin)and adherent junction proteins(AJs,e.g.,E-cadherin,β-catenin)in the colonic tissue,decrease plasma FITC-dextran levels in DSS-induced mice.Finally,Western blotting analysis showed that PTE treatment activated the nuclear factor E2-related factor 2(Nrf2)pathway,increased downstream heme oxygenase 1(heme oxygenase-1,HO-1)and quinone NADH dehydrogenase 1(NQO1)expression,and inhibited the nuclear factor kappa B(nuclear factor kappa B,NF-κB)pathway.These results indicated that PTE can significantly alleviate the symptoms of UC mice,reduce colonic inflammatory response and oxidative stress while maintaining intestinal barrier function,and these effects may be produced through the activation of Nrf2 pathway.The second part of my thesis studied the mechanism by which PTE inhibits colonic inflammation in DSS-induced mice.The macrophage(MΦ)inflammation model,including mouse MΦ RAW264.7 line and human monocyte THP1 line,was generated by lipopolysaccharide(LPS)treatment to further investigate and reveal the mechanism underlying the attenuation or inhibition of MΦ inflammatory response post PTE treatment.First,the optimization of working concentration of PTE was carried out by the detection of CCK-8 toxicity and ELISA assays for the quantification of inflammatory cytokine expression.Western blotting analysis showed that PTE significantly increased the level of Nrf2 in the nuclei of RAW264.7 cells and THP1 cells while increasing the expression of downstream HO-1 and NQO1.PTE treatment decreased LPS-induced ROS levels in RAW264.7 cells and THP1 cells,and increased the levels of SOD and GSH-px in cells,and these effects were significantly reduced by transfection of Nrf2 si RNA.ELISA analysis showed that PTE treatment also introduced the reduced expression levels of TNFα,IL-6 and IL-1β in LPS induced RAW264.7 cells and THP1 cells,which were reversed by transfection of Nrf2 si RNA.Western blotting analysis showed that PTE could inhibit the levels of phosphorylated p65 and JNK in RAW264.7 cells and THP1 cells after LPS treatment,and cell immunofluorescence(IF)results showed that PTE introduced much less p65 nuclear translocation in RAW264.7 cells and THP1 cells after LPS stimulation.These effects can be inhibited by transfection of Nrf2 si RNA.These results indicated that PTE treatment significantly alleviated MΦ oxidative stress and the inflammatory response through the activation of Nrf2 pathway.The third part of the project studied the mechanism by which PTE maintains intestinal mucosal barrier function physiologically.Human Caco-2 cells were selected to conduct assays for the assessment the integrity of Caco-2 monolayer after TNF-α treatmentas a model.Western blotting analysis showed that PTE significantly increased the level of Nrf2 nuclear reloction in Caco-2 cells and simultaneously increased the expression of downstream HO-1 and NQO1.PTE treatment significantly alleviated the transepithelial electrical resistance(TEER)of the TNF-α-induced Caco-2 monolayer and reduced the permeability to FITC-dextran,but the treatment of Nrf2-specific inhibitor ML385 reversed the forementioned effects.PTE treatment ROS levels in the Caco-2 after TNF-α treatment,and increased the expression levels of SOD and GSH-px,which could be reversed after the cells were treated with ML385.Western blotting analysis showed that PTE inhibited p65 phosphorylation in the Caco-2 cell monolayer after their administration of TNF-α.The IF results showed that PTE inhibited p65 nuclear translocation,and these effects were significantly reversed by ML385 treatment.Western blotting analysis showed that there was no significant difference in the expression of TJs and AJs in Caco-2 monolayers in each group,and PTE significantly reduced the expression myosin light chain kinase(MLCK)and the phosphorylation levels of myosin and ezrin in TNF-α treated Caco-2 monolayers.IF showed that PTE reduced the colocalization of p-Ezrin and p-MLC around ZO-1,and relieved cell shrinkage,and these effects were also significantly reversed by ML385 treatment.These results indicate that PTE can promote healing of the injury of intestinal epithelial cells(IECs),which is made by the alleviation of oxidative stress of IECs to maintain the physiological structure of TJs and AJs under the inflammatory condition through Nrf2 pathway,and ultimately improve intestinal epithelial barrier function.The last part my thesis project is to evaluate the effect of PTE treatment on gut intestinal microbiota homeostasis.After the DNA was extracted from the feces of the mice in each group,the bacterial 16 S r RNA gene V3–V4 region was amplificated using PCR,and then sequencing was performed using the Illumina Mi Seq platform.R was used to conduct bioinformatics analysis on the sequencing results.The results showed that PTE significantly increased the Chao1 index,Shannon index and Simpson index of the gut microbiota in UC mice;principal coordinate analysis(PCo A)showed that after PTE administration,the gut microbiota matrix of mice was closer to that of the control group.Species composition analysis and linear discriminant analysis effect size(LEf Se)showed that PTE increased the abundance of beneficial bacteria such as Lachnospiraceae and Oscillospira in the gut microbiota in UC mice and reduced the abundance of harmful bacteria such as Allobaculum,Bacteroides and Paraprevotella to maintain intestinal microbiota homeostasis.Spearman correlation analysis showed that Roseburia,Prevotella and Rikenella were significantly positively correlated with the expression ZO-1 and Occludin but negatively correlated with colonic permeability.Allobaculum,Bacteroides and Paraprevotella were significantly correlated with the levels of TNF-α,IL-6,IL-1β and ROS in colonic tissue,and significantly negatively correlated with SOD activity and GSHpx activity in colonic tissue.The above results show that PTE administration can improve the diversity of the gut microbiota in UC mice and maintain intestinal microbiota homeostasis by increasing the abundance of probiotic bacteria and reducing the abundance of harmful bacteria.In summary,this study is the first to systematically elucidate the mechanism by which PTE significantly alleviates the symptoms of UC mice.The alleviation of oxidative stress and the inflammatory response through the activation of Nrf2 pathway,the promotion of healing of intestinal mucosal barrier injury by improving the expression of TJs and AJs and recovering the physiological structure of TJs and AJs,and the amelioration of intestinal microbiota homeostasis by increasing the colonization and abundance of probiotics and reducing the colonization and abundance of opportunistically pathogenic bacteria are the major mechanisms underlaying the therapeutic effects of PTE treatment for UC.The above finding reveal the therapeutic potential of PTE for UC and provides a scientific basis for the clinical application of PTE for UC in the future.