Inhibitory Effect Of N-3PUFAs On Acute Ulcerative Colitis In Mice And The Underlying Mechanism

Objective: Male C57BL/6J mice were chosen to induce acute ulcerative colitis(UC)using dextran sulphate sodium(DSS)in order to compare the effects of DHA,EPA as well as DHA+EPA intervention.The underlying mechanism was explored from multiple aspects including colonic mucosal barrier,transepithelial water transport,and intestinal flora to provide a scientific basis for developing effective intervention measures for acute UC.Methods: Sixty 8-week-old mice were randomly divided into six groups according to body weight: control group,DSS group,mesalazine group,DHA group,EPA group and DHA + EPA intervention(i.e.fish oil)group,each group had 10 mice.DHA,EPA and Fish Oil were solved in10% gum arabic solution.DHA group,EPA group and Fish Oil group were given 400 mg/kg·bw of corresponding fatty acids by oral gavage every other day for 30 days to form fatty acids store in colon tissue;the remaining groups were given 10% gum arabic solution.Then,the protocol of DSS induced acute UC was initiated.During this period,mesalazine group received oral administration of 25 mg/kg · bw mesalazine,and the other groups were treated as before.Moreover,oral intervention was conducted daily until the end of the experiment.The control group received normal drinking water.The remaining five groups drank water containing 2% DSS for 7 days and then exposed to normal drinking water.At day 10 of DSS exposure the experiment ended.The body weight,diarrhea and bleeding stool of the mice were recorded daily.The length and weight of the colon were measured to evaluate the shortening and edema of colon,and the levels of serum inflammatory factors INF-γ,IL-6 and IL-1β were tested by ELISA kit.Distal colon tissues were taken for H&E staining,mucus staining and scanning electron microscopy analysis;fatty acid profiles in colon were analyzed by gas chromatography.The m RNA levels of mucin2,glycosylation-modifying key enzymes and ion transporter protein were detected by quantitative rtq PCR.The expression of proteins related tight junction and neutrophil markers MPO were observed by Western Blot.Broadly,the effects of the TLR4/PI3K/AKT pathway,TNF-α/NF-κB pathway and GPR120/c AMP/CREB pathway on ion transporter protein expression were analyzed to reveal the intrinsic potential mechanism for the different effects of n-3 PUFAs on acute UC.Results: Fish oil intervention(i.e.DHA+EPA combined intervention)significantly inhibited acute enteritis in mice.DHA intervention alone also showed inhibitory effect,but EPA intervention had no effect.Fish oil intervention significantly inhibited the weight loss of mice,decreased the DAI score,alleviated colonic edema and colonic shortening,and inhibited the inflammatory injury of colon tissue.The fatty acid spectrum of intestinal tissue of mice showed that the level of arachidonic acid(AA),a pro-inflammatory fatty acid,in colon tissue was significantly increased in DSS group,and the level of AA in EPA intervened group was also higher,while AA level and n-3PUFAs/AA,DHA/AA in fish oil and DHA treated group were markedly decreased and increased respectively.It was suggested that the protective effects of fish oil intervention and DHA intervention on acute enteritis may be related to altered fatty acid composition in colon.Further analysis showed that fish oil intervention significantly decreased the level of MCP-1 in colon tissue.DHA and fish oil intervention also decreased the level of neutrophil marker MPO,inhibited neutrophil infiltration and decreased the content of serum inflammatory factors INF-γ,IL-6 and IL-1β.The results of AB/PAS staining and scanning electron microscope showed that fish oil and DHA increased the mucus secretion of goblet cells,which may be related to the enhancement of MUC2 and glycosylation-modifying key enzymes expression.Fish oil and DHA enhanced the expression of tight junction proteins Claudin-1 and Occludin.Fish oil and DHA also downregulated TLR4/PI3K/AKT pathway thereby promoted the expression of ion transporter protein NHE3,up-regulated GPR120/PKA/CREB pathway thereby enhancing the expression of aquaporin AQP3,as a result,promoted intestinal water absorption and improved diarrhea symptoms.Fish oil and DHA intervention maintained the intestinal anaerobic environment of mice,promoted the diversity of flora by up-regulating the abundance of anaerobic bacteria producing short-chain fatty acids,and inhibiting the abundance of aerobic and facultative anaerobes.Conclusion: This study found that the combined treatment of DHA and EPA can significantly inhibit acute colitis in murine UC model,DHA exhibits inhibitory effect to some extent,while EPA treatment has no protective effect.The potential mechanism for inhibitory effect of combined DHA and EPA treatment in ulcerative colitis may involve the following aspects:(1)Promote the synthesis and glycosylation of MUC2 and enhance the mucous barrier of the colonic mucosa.(2)Up-regulate the expression of tight junction proteins Claudin2 and Occludin to maintain the integrity of the mechanical barrier.(3)Inhibit the infiltration of inflammatory cells and the production of pro-inflammatory factors,reduce the inflammatory damage of the colon.(4)Inhibit the activation of TLR4/PI3K/AKT,TNF-α/NF-κB pathway,and promote the activation of GPR120/PKA/CREB pathway,promote the expression of ion transporter protein and aquaporin 3,promote trans-epithelial water absorption,and alleviate diarrhea.(5)Improve the diversity of intestinal flora,up-regulate the abundance of anaerobic bacteria that produce short-chain fatty acids,inhibit the abundance of aerobic and facultative anaerobic bacteria,which is helpful to maintain the intestinal microbial homeostasis.