Effects Of Alpinetin On Intestinal Epithelial Barrier In Ulcerative Colitis

Objective: Inflammatory bowel disease(IBD),including Crohn’s disease and ulcerative colitis,is a group of chronic inflammatory disorders of the gastrointestinal tra ct characterized by intestinal inflammation and mucosal damage.Recent years,the incidence of IBD obviousl y increases.The pathogenesis of IBD is still not clear,studies show that disruption of the intestinal epithelial barrier is an important pathogenic factor in IBD.Immune dysfunction and oxidative stress also play important roles in the pathogenesis of IBD.As the most important component of the intestinal epithelial barrier,a functional tight junction(TJ)contributes to prevent the entry of luminal pathogens,such as bacteria,virus and endotoxins into the mucosa lamina propria and blood circulation.Dysregulation of tight junction in the barrier will lead to an increased gut permeabilit y.Nowadays,achieving and maintaining clinical remission and mu cosal healing is considered as the therapeutic goal in IBD.Recent studies suggest that increased tight junction permeabilit y occurs in earl y disease stage and promotes disease initiation,therefore,the integrit y of tight junction may be a key factor for mucosal healing.Tight junctions are multi-protein complexes composed of transmembrane proteins,peripheral membrane proteins and regulatory molecules that include kinases.The most important transmembrane proteins are claudins and occludin,in charge of r egulating tight junction permeabilit y and maintaining cell polarit y.Peripheral membrane proteins,such as zonula occludens 1(Zo1)and Zo2,are crucial for tight junction assembl y and maintenance,partl y owing to the fact that these proteins own multiple domains for interaction with other proteins,including claudins,occludin and cytoskeleton protein.It has been reported that claudin-2 is elevated in intestinal mucosal biopsies of patients with IBD,and occludin and Zo-1 protein expression decreased.Cur rentl y,IBD treatment mainl y depends on anti-inflammatory drugs,immune s ystem suppressors and antibiotics,which existed many resistances problem as well as major adverse events and poor treatment responses.Therefore,the development of novel therapies f or IBD is imminentl y needed.Alpinetin,a novel plant flavonoid isolated from Alpinia katsumadai Hayata,is a traditional Chinese medicine.It has been reported that alpinetin showed anti-inflammatory,antibacterial,antioxidant,anti-tumor and other important therapeutic activities.In previous studies,the protective effects of alpinetin on acute lung injury and experimental colitis in mice have been confirmed.However,little is known on the protective effect and mechanism of alpinetin on intestinal epit helial barrier function in IBD mice.The aim of the present study is to investigate the protective effects and mechanism of Alpinetin on the expression of tight junction protein in dextran sulfate sodium(DSS)-induced colitis mice.In addition,the relatio nship between tight junction and mucosal healing in human ulcerative colitis will also be studied.Methods: 1.The correlation between intestinal mucosal healing and the expression of tight junction proteins in human ulcerative colitis.Subjects selection: colonic biopsies and clinical data were collected from UC patients and controls in Shengjing hospital of China medical universit y.IBD was diagnosed according to the Consensus of Inflammatory bowel disease diagnosis and treatment(Guangzhou,2012).Contro l tissues were the normal margins from patients without IBD who underwent resection for large adenoma or carcinoma.Expression levels of tight junction proteins(occludin,ZO-1 and claudin-2)were assessed by RT-PCR and immunohistochemical.And then,the co rrelation between intestinal mucosal healing and tight junction proteins expression in ulcerative colitis was anal yzed.2.The effect of different concentrations of alpinetin on UC mice.A total of 42 C57BL/6 male mice were randoml y divided into seven grou ps(n=6)including normal group,Alpinetin(100 mg/kg)group,3% DSS group,3%DSS+Alpinetin(25mg/kg)group,3%DSS+Alpinetin(50mg/kg)group and 3% DSS+Alpinetin(100 mg/kg)group,3%DSS+5-ASA(200mg/kg)group with the experiment time for 7 days.The diet,act ivit y,weight loss,loose stools,bloody stools of UC mice were observed and recorded everyday.On the eighth day of experiment,all mice were anesthetized using chloral hydrate and sacrificed.The protective effects of alpinetin on UC mice were evaluated by comparing each group’s disease activity index(DAI),body weight,length of bowel,histologic changes and histologic lesion scores.Intestinal epithelial tight junction changes were assessed by transmission electron microscopic.3.The protective effect and mechanism of alpinetin on intestinal epithelial barrier function in mice with UC.A total of 30 C57BL/6 male mice were randoml y divided into five groups(n=6)including normal group,3%DSS group,3%DSS+Alpinetin(25mg/kg)group,3%DSS+Alpinetin(50mg/kg)group and 3%DSS+Alpinetin(100mg/kg)group with the experiment time for 7 days.Expression levels of tight junction proteins(occludin,ZO-1 and claudin-2)were assessed b y immunohistochemical staining,western blot and RT-PCR.The levels of Nrf2,HO-1,STAT3,IL-6,were determined by western blot and RT-PCR.4.The mechanism of alpinetin on intestinal epithelial barrier function in UC mice.The expression levels of Nrf2,HO-1,STAT3,IL-6 and tight junction proteins(occludin,ZO-1 and claudin-2)were studied by western blot and RT-PCR before and after giving ZNPP,a competitive inhibitor of HO-1.Results: 1.Among the 48 individuals recruited,there were 40 UC and 8 controls.MCS and MES showed increasing trend in clinical remission group,mild active gro up,moderate active group and severe active group,and the differences were statisticall y significant in each group(P<0.01).Compared with active UC patients,the Geboes index was obviousl y decreased in control group and in remission(P<0.01).The m RNA ex pression of ZO-1 and occludin was significantl y decreased in UC patients compared with controls.The m RNA expression of The decrease was more pronounced in UC patients with non musosal healing in alleviate group.The m RNA expression of occludin was signifi cantl y decreased in UC compared with controls without any significant difference in alleviate group.The expression of occludin and ZO-1 deceased,while Claudin-2 showed increased expression by immunohistochemical detection in UC in comparison to the contr ol group.In clinical alleviate group,the express of ZO-1 in mucosal healing group was significantl y different with non-mucosal healing.2.Compared with control group and only Alpinetin group,DSS groups and 4 treatment groups appeared to show higher wei ght loss,DAI scores,histological scores and MPO activity.Compared with DSS group,weight loss,DAI scores,histological scores and MPO activity appeared to be obviously reduced in 4 treatment groups to various extent,there was no significant difference when compared Alpinetin(100mg/kg)treatment group with 5-ASA group and compared control group with only Alpinetin group(P> 0.05).Intestinal epithelium ultrastructure was observed using transmission electron microscopy,in the control and only Aplinetin group,the tight junction appeared as an electron-dense belt at the apex of the intestinal epithelial cells,indicating an intact intestinal mucosal barrie;In DSS group,the intercellular space was widened,the tight junction was indistinct,and the density was reduced;In Aplinetin-treated and 5-ASA groups,the density of the tight junctions was increased compared with that in the DSS group.3.The expression of tight junction proteins(occludin,claudin and ZO-1)was anal yzed by immunohistochemistry,we stern blot and RT-PCR.Compared with control group,the expression of occludin and ZO-1 obviousl y reduced in DSS group,while the expression of claudin-2 increased.Compared with DSS group,expression of occludin and ZO-1 upregulated in Aplinetin treated g roup,while the expression of claudin-2 decreased.Compared with control group,MDA content increased and SOD activit y decreased in DSS group,but in Aplinetin treated group,MDA content reduced and SOD activit y increased,which compared to DSS group.4.The expression of p STAT3 and IL-6 was anal yzed by western blot.The expression of p STAT3 and IL-6 obviousl y increased in DSS group compared with control group.Compared with DSS group,p STAT3 and IL-6 reduced,while Nrf2 and HO-1 increased in Aplinetin trea ted group,the differences were statisticall y significant when compared to Alpinetin(100mg/kg)treated group(P<0.05).Compared with Alpinetin(100mg/kg)treated group,in ZNPP group,the expression of HO-1 obviously reduced,but the nuclear Nrf2 protein had no significant change,while the protein expression of p STAT3 and IL-6 reduced(P<0.05),the expression of ZO-1 significantl y reduced(P<0.05),however,there was no significant change in occludin and claudin-2 protein expressions.Conclusion: 1.The expression of tight junction proteins(Zo-1 and occludin)declined,while the expression of claudin-2 increased in UC patients.The expression of ZO-1 was related with mucosal healing.Tight junction proteins may be used as a predictor of mucosal healing an d the effect of treatment in UC patients.2.Alpinetin significantl y attenuated the intestinal inflammation in UC mice by oral administration in a dose-dependent manner.And no toxic side effects were shown.3.Alpinetin exerted beneficial eff ects on intestinal barrier function by regulating the expression of tight junction proteins(ZO-1,occludin and claudin-2).Alpinetin regulated the tight junction proteins may be through activating Nrf2/HO-1 and inhibiting IL-6/STAT3 signal pathway.4.Alpinetin regulated the tight junction proteins may be partl y through Nrf2/HO-1 regulating IL-6/STAT3 signal pathway.