Effects Of Macamides On DSS-induced Colitis In Mice

Macamine is a kind of characteristic active ingredient in Maca,and Maca extracts rich in macamide had many biological activities such as antioxidation,enhancing immunity and improving reproductive health.Nbenzyl docosahexaenoic acid amide(NB-DHA)is a new amide compound with the highest degree of unsaturation in macamides,and its structure is similar to Docosahexaenoic acid(DHA).As a new DHA derivative with high unsaturation,NB-DHA has potential and considerable research value.In recent years,the incidence of ulcerative colitis has gradually increased,and it has become a common disease threatening human health.Its pathogenesis is related to many factors,and the current treatment methods cannot obtain stable curative effect,so it is necessary to develop more safe and effective treatment strategies to ensure the therapeutic effect.Objective:In order to seek the best therapeutic effect and drug treatment potential of this compound,and to provide a new idea for treating ulcerative colitis,this study independently synthesized NB-DHA and another macamide in maca with the highest content,N-benzyl hexadecanamide(NBH)based on the synthesis and purification method optimized by previous experiments of our research group,and applied dextran sodium sulfate(DSS)-induced mouse colitis model to observe the influence of NB-DHA on enteritis mice and the change of relative number of Th17/Treg cells in vivo,and compared with NBH to explore the difference of efficacy between the two typical macamides.Methods:1.Synthesis and purity identification of NB-DHA and NBHTwo kinds of macamides with different saturation were synthesized and purified independently by using microalgae oil and palmitic acid as raw material according to saponification reaction principle,and the purity of the compounds was identified by high-pressure liquid chromatography(HPLC)based on the previous experimental results of the research group,and the structure of NB-DHA was preliminarily identified by LC-MS2.Effects of NB-DHA and NBH on DSS-induced colitis in miceThe enteritis of C57BL/6 mice was induced by 2.5% DSS aqueous solution for 7 days,and was given orally every day 3 days before the establishment of the model.The concentration of NB-DHA and NBH was 100 mg/kg,and the concentration of sulfasalazine(SASP)was 50 mg/kg.After the model was established,the animals were killed.During the modeling period,the body weight,fecal occult blood,diarrhea and mental state of each mouse were monitored daily,and the disease activity index was calculated.The spleen was weighed and the spleen index was calculated.HE sections of mouse colon tissue and spleen were made to observe the pathological injury of colon and immune activation of spleen.ELISA was used to detect the concentrations of TNF-α,IL-1β,IL-6,IL-10 and MPO in mouse intestinal tissue.3.Effects of NB-DHA and NBH on intestinal barrier protein and relative quantity of Th17/Treg cells in DSS-induced colitis miceThe expression of tight junction proteins ZO-1 and occludin in colon tissues of mice was detected by IHC method,and analyzed the difference of gray value.The number ratio of Th17 and Treg cells in mesenteric lymph nodes of mice was detected by flow cytometry,and the mRNA expression levels of ZO-1,occludin,ROR-γ,Foxp3,IL-10 and IL-17 A in colon tissues were detected by q PCR technique.Results:1.Synthesis and purity identification of NB-DHA and NBHBy saponification reaction and high-performance liquid chromatography,NB-DHA and NBH monomer compounds were synthesized with DHA fish oil/palmitic acid and benzylamine as raw materials.The purity of NB-DHA monomer compounds was 95%,and that of NBH monomer compounds was 98% by high performance liquid chromatography.According to LC-MS analysis,NB-DHA had benzene ring and amide bond structure,and its total molecular weight was 417.00.2.Preventive and alleviating effects of NB-DHA and NBH on mice with colitisAfter administration,NB-DHA and NBH can alleviate the weight loss caused by colitis.According to the calculation of disease activity index(DAI),NB-DHA and SASP significantly reduced the scores.NB-DHA and NBH both can alleviate the shortening of colon length caused by colitis.The results of colon HE staining showed that NB-DHA and NBH both reduced inflammatory cell infiltration of colitis,and the score also decreased significantly,and the effect of NB-DHA was better than NBH.ELISA was used to determine the concentrations of TNF-α,IL-1β,IL-6,IL-10 and MPO in the supernatant of colon homogenate,and the results showed that NB-DHA and NBH decreased the concentrations of TNF-α,IL-1β and IL-6,increased the concentrations of IL-10,and decreased the content of MPO.3.Protective effects of NB-DHA and NBH on intestinal barrier in mice with colitisImmunohistochemical results showed that NB-DHA and NBH could reduce the destruction of intestinal mucosal tight junction protein ZO-1 and occludin.The results of q PCR showed that NB-DHA and NBH both upregulated the mRNA expression of ZO-1 and occludin,and protected the intestinal tight junction protein ZO-1 and occludin to some extent.4.Effects of NB-DHA and NBH on the relative quantity of Treg/Th17 cells in mice with colitisHE staining results of spleen showed that NB-DHA could inhibit immune overactivation in inflammatory mice,and the spleen index was lower than that in model control group.The results of flow cytometry showed that NB-DHA and NBH could increase the relative quantity of Treg cells and the relative quantity of Th17 cells was decreased.The q PCR results showed that NB-DHA and NBH could significantly increase the mRNA expression levels of Foxp3 and IL-10,and decrease the mRNA expression levels of IL-17 A and ROR-γ,but the effect of NB-DHA was better than that of NBH,which promoted the inflammation to subside.Conclusion:In this study,two kinds of macamides were synthesized according to the independent synthesis method of our research group,one of which is N-benzyl hexadecanamide(NBH),which is the most abundant in maca,and the other is N-benzyl docosahexaenoic acid amide(NB-DHA),which has the highest unsaturation in maca,with the purity of both over 95%.Furthermore,the relieving effects of the two compounds on ulcerative colitis mice were discussed.It was concluded from the aspects of body weight,colon injury,intestinal barrier tight junction protein and immune cell regulation that NB-DHA and NBH administered three days in advance could effectively relieve the colon inflammation of mice,and the effect of NB-DHA was similar to that of positive drug SASP,but better than NBH.