N-3 PUFAs Alleviate DSS Induced Colitis By Inhibition Of MTORC1

Background and aimsThe inflammatory bowel diseases(IBD)comprise two types of chronic intestinal disorders:Crohn's disease(CD)and ulcerative colitis(UC),the occurrence of these two diseases and the pathological mechanisms of them have not been clearly definited.The occurrence of these diseases were so long and repeated that cutting down the quality of patients' life and adding the load of public health enterprise.Thus,it's important to discover the pathological mechanisms and the cause of IBD.Nucleotide oligomerization domain 2(NOD2)and components of the interleukin-23-type 17 helper T-cell(Th17)pathway had shown their role in inducing IBD.Some researches pointed that lots of inflammatory cytokines involved in intestinal inflammation such as IL-1??TNF-? and IFN-?,the interaction of these cytokines with intestinal epithelial cells links to IBD.Changes in diet and antibiotic use have probably contributed to the increased prevalence of inflammatory bowel diseases.The anti-inflammatory ability of polyunsaturated fatty acids(PUFAs)may be mediated by a lower production of the most powerful arachidonic acid metabolite,leukotriene B4,as well as by a reduction in another arachidonic acid metabolite,thromboxane A2,which is an early abnormality of special pathogenic significance.In addition,n-3 PUFAs can inhibit IL-1? and TNFs production.Giving the rich DHA and EPA diet can effectively alleviate the symptoms of colitis in rats which induced colitis using DSS.But the mechanism of n-3 PUFAs on the occurrence of IBD is still not clear.Mammalian target of rapamycin(mTOR)is a serine-threonine kinase plays animportant role in many cellular functions,such as cell growth,proliferation,autophagy,proteins translation regulating and cell aging.The activity of mTOR links to the occurrence and retention of immflamation.A large part of the immune response depends on the proliferation and clonal expansion of antigen-specific T cells,which depends on mTOR activation.It also said that mTOR is at the fulcrum of the immunoregulatory balance.Studies have pointed out that short-term treatment with rapamycin reduces the severity of the pathologic damage observed in mice with chronic colitis.In addition,some of the members of our group have discovered that there have close relation between the activity of mTOR and n-3PUFAs.We conclude that the activation of mTOR should affect the occurrence or pathological mechanisms of colitis,it is interesting to discover how it influences IBD.Our preliminary experiment results showed that DHA riched fish oil gavage will ease the development of colitis induced by DSS in C57 mice.The bodyweight lost caused by colitis,the degree of colon shortening and rectal bleeding symptoms were improved.At the same time,immunohistochemistry showed that the biopsies of mice colon tissue biopsies were conducted;DSS induced colitis increased intestinal epithelial cell mTORCl activity in mice.But in DHA gavaged mice,the activation was inhibited.Then,we also extracted the mouse colon epithelial cells for Western blotting.We found that the expression of phosphorylated ribosomal protein S6(pS6)expression increased in mice that induced colitis by DSS.In DHA gavage mice,expression of pS6 in colon epithelial cells was inhibited.So we think,n-3 PUFAs may affect the occurrence and development of colitis by regulate mTOR signal pathway.The purpose of this study is to investigate if mTOR will participate in the process that n-3 PUFAs relieve DSS induced colitis.We hope we can influence on the occurrence and development of colitis through the study of n-3 PUFAs and mTOR,so as to search for new mechanisms to enrich the IBD treatment.Methods1.First,we conducted experiments in mice reproduction,fat-1 mice and C57 mice bred mice with fat-1 positive,set fat-1 negative littermates as control.In addition,we use the Cre-loxP system to reproduction CAC-Tsc1-/-mice,and Tsc1-/-littermates as controls;fat-1 mice and CAC-Tsc1-/-mice were mated to breed fat1-CAC;Tsc1-/-mice,set CAC Tsc1-/-mice littermates as controls.Then we use DSS for induce colitis in mice.2.Then,changes in body weight lost and occult blood test results during the colitis model would be detected,to calculate the disease activity score.After modeling,the mice colon length measurement and histological slices would done.H&E staining was used to observe the colon tissue structure changes.Then we calculate the pathological score according to the scoring standard.Severity of colitis in mice were analyzed and decided by integrating of these indicators.3,Next,we also use immunohistochemical and immunofluorescence to find out the expression of pS6 in mice colon epithelial,in order to determine the activation level of mTORC1.In addition,we use the EDTA to extract the mouse colon epithelial cells,then using Western blotting to determine the expression level of the protein that correlated with mTORC1 activity.Using Trizol reagent extract the total mRNA of colonic epithelium to determin the changes in transcript levels of inflammatory factors IL-1 ?and IL-6 in mice.Results1?n-3 PUFAs can alleviate DSS induced colitis in C57 mice.We found that in the last two days of modeling the weight of control group significantly decreased compared with the experimental group(p<0.05).The intake of n-3 PUFAs can prevent weight decline caused by the colitis.In addition,the colon of experimental group is longer than the control group;As to the ratio of weight and colonic length,the experimental group is closer to the blank control group than the control group.In the last two days of modeling,rectal bleeding score and disease activity score(DAI)of experimental group was significantly lower than the control group(p<0.05).From the histopathological point of view,pathological scores of colon tissue of the experimental group also lower than the control group(p<0.05).2?Fat-1 mice relieve DSS induced colitis.We insert the fat-1 gene into the mouse called fat-1 mouse and use DSS to induce colitis.We found that fat-1 mouse can alleviate weight decrease caused by colitis,particularly in the last two days of modeling,the difference compared with the control group is significant(p<0.05).At the same time the colon length of fat-1 mice were also longer than the control group.We can in view of the ratio of weight and colonic length,fat-1 mice is closer to the blank control group than the control group.Finally,the pathological scores of the fat-1 mouse's colon tissue were lower than that of the control group(p<0.05).That indicate the less damage of the colon.3?The n-3 PUFAs can reduce mTORCl activity of colonic epithelial cells in DSS-induced colitis,no matter it is ectogenic or endogenic.We used Immuohistochemical stainin to detect pS6(s235/236)in mouse colon tissue,the results show that pS6(s235/236)expression increased in colonic epithelial cells of colitis mouse.However,the pS6(s235/236)expression is suppressed in colonic epithelial cells which from fat-1 mouse and the mouse intake n-3 PUFAs.In addition,we extracted the proteins from colonic epithelial cells,the western blotting indicate pS6(s235/236)were increased during colitis,but pS6(s235/236)expression in fat-1 mice and the DHA-taken mice had recovered to normal levels.4?The mice which knocked out Tscl specificity in colon epithelial cells shows more seriours in DSS induced colitis.We used Cre-loxp system to obtained Tscl knockout mice specific in colonic epithelial cells,known as CAC;Tsc1-/-mice.We fed knockout mice and its' littermate DSS to induce colitis,we found,the weight drop of CAC;Tsc1-/-mice was heavier than the control group(p<0.05).Meanwhile,CAC;Tsc1-/-mice also had a shorter colon,higher disease activity score and colonic tissue pathology score than the control group.Using colonic epithelial cells' RNA to do the fluorescence quantitative PCR,which showed that the transcription of pro-inflammatory cytokines IL-1? and IL-6 were significantly increased compared with the control group.5.The fat1-CAC;Tsc1-/-mice can relieve colitis induced by DSS.We transferred fat-1 gene into the CAC;Tsc1-/-mice,then get fat1-CAC;Tsc1-/-mice and its'DSS-induced colitis model.During the modeling of fat1-CAC;Tsc1-/-mice,the decrease of weight was smaller than the CAC;Tsc1-/-mice(p<0.05),while the length of the colon showed the crosscurrent.Compared with CAC;Tscd1-/-mice,the ratio of weight and colonic length of fat1-CAC;Tsc1-/-mice had significantly improved(p<0.05).DAI score also showed fat-1 gene can ease colitis progress in CAC;Tsc1-/-mice and the pathology score of colon tissue of fat1-CAC;Tsc1-/-mice was lower than the control group.RT-PCR indicated fat1-CAC;Tsc1-/-mRNA transcription of IL-1? and IL-6 were reduced.6?Fpr2 expression in colonic epithelial cells of CAC;Tsc1-/-mice have been upregulated during colitis,while fat-1 gene can inhibit this phenomenon and activity of mTORC1.Immunohistochemistry of fat1-CAC;Tsc1-/-mouse colon tissue,compared with the control group we found the expression of pS6(s235/236)had decreased.Western blotting showed the same result.Fpr2 mRNA levels of colonic epithelial cells of CAC;Tsc1-/-mouse rose while fat1-CAC;Tsc1-/-mouse decreased.Conclusion1.Dss induced colitis and mTORC1 activating can be relieved by external or internal n-3 PUFAs.2.Knocking out Tsc1 specificity in colon epithelial cells causes enhancing expression of mTORClactivity and aggravate the colitis induced by DSS.3.Fat-1 reverse the aggravates of DSS induced colitis in the mice whichknocked out Tscl specificity in colon epithelial cells.4.The reduction of mTORC1 activity may bring the relieve effect in fat1-CAC;Tsc1-/-mice in colitis induced by DSS.