Effects And Mechanisms Of 5-ASA And High-fat Diet In Ulcerative Coliti

Part Ⅰ The correlation between serum lipids level and 5-ASA efficacy in patients with ulcerative colitisObjective:To investigate the relationship between serum lipid levels and disease activity and prognosis of Ulcerative colitis(UC)and the effect of 5-Aminosalicylic acid(5-ASA).Methods:Retrospective data from 497 patients with UC who had detailed lipid reports from January 2003 to September 2020 were collected consecutively and divided into short-term lipid cohort and long-term lipid cohort,univariate,multivariate,and survival analyses were performed to assess the association of serum lipid levels with disease activity,long-term outcomes,and 5-ASA efficacy.At the same time,the correlation between blood lipids and disease activity and prognosis was evaluated by subgroup stratified analysis.All patients were followed up until 30 September of the 2021.Results:1.Patients with dyslipidemia showed more severe disease activity compared with UC patients with normal lipids.The level of high-density lipoprotein cholesterol(HDL-C,p<0.05)was negatively correlated with disease activity index(DAI)(p<0.05).2.Compared with patients with transient dyslipidemia and normal lipid levels,patients with persistent dyslipidemia had a higher risk of UCrelated surgery(Hazard ratio[HR]:3.27,95%confidence interval[CI]:1.86-5.75,p<0.001)and tumorigenesis(HR:7.92,95%CI:3.9715.78,p<0.001),who had shorter surgery-and tumor-free survival(both p<0.001).The levels of HDL-C(both p<0.001)and Apolipoprotein A1(ApoA1,both p<0.05)levels were negatively associated with risks of surgery and tumorigenesis.3.In the long-term lipid cohort of UC receiving 5-ASA,the proportion of drug escalation was higher in the low HDL-C group compared with the high HDL-C group(100.0%vs.74.4%,p<0.001),whereas the proportion of mucosal healing was lower(23.2%vs.41.8%,p=0.014).Low HDL-C levels were independent determinants of drug escalation(HR:1.63,95%CI:1.242.18,p<0.01)and mucosal healing(HR:0.56,95%CI:0.33-0.96,p<0.05)in 5-ASA-treated patients with UC.Patients in the low HDL-C group had earlier medicine upgrade(HR:1.72,95%CI:1.31-2.25,p<0.001)and achieved later mucosal healing(HR:0.57,95%CI:0.34-0.97,p<0.05)than those in the high HDL-C group.Conclusion:1.In patients with UC,persistent dyslipidemia is associated with a higher risk of severe disease activity and worse prognosis.Lipid patterns should be evaluated to improve the management of UC patients with high-risk factors.2.The UC patients treated with 5-ASA in low HDL-C group exhibited worse drug efficacy and longer-term outcomes than those in high HDL-C group.Blood lipid patterns,especially the level of HDL-C,should be considered in medication strategy and the time of drug ladder therapy.Part II The effect and immunity mechanism of 5-ASA and highfat diet on acute colitis in miceBackgroud and Objective:The level of serum lipid is correlated with disease activity and prognosis in patients with UC,and 5Aminosalicylic acid(5-ASA)has a poor anti-inflammatory effect in patients with dyslipidemia.The aim of this study was to explore the effect of 5-ASA and high fat diet(HFD)on acute colitis and its immunity mechanism.Methods:1.Dextran sulfate sodium(DSS)animal models of colitis were established in C57BL/6 mice and divided into six groups:control diet normal control(CD-NC)group,CD-DSS model group,CD-5ASA treatment group,HFD-NC Group,HFD-DSS model group and HFD-5ASA treatment group.Colitis severity was assessed by weight loss,disease activity index(DAI),colonoscopic colitis score,colon length,and colonic histopathology score.Meanwhile,serum samples were collected to detect the level of blood lipids,and colon samples were collected to detect the expression of cytokine messenger RNA(mRNA)by quantitive real-time polymerase chain reaction(qRT-PCR).2.The intestinal lamina propria immune cells were isolated and extracted from each experimental group.The proportion and type of intestinal immune cells were detected by flow cytometry method,multiple immunofluorescences were used to assess the intestinal macrophage infiltration.3.The mucosal expression profiles of UC patients and normal controls were obtained from Gene Expression Omnibus(GEO)database,and the Immune infiltration analysis were operated by CIBERSORT algorithm.Colon tissue samples from UC patients were divided into three groups:normal lipid group(n=3),high TC group(n=3)and low HDL-C group(n=3).The intestinal macrophages were detected by immunohistochemistry before and after 5-ASA treatment.Four.4.The inflammatory macrophage model of THP-1 was constructed in vitro.The differentially expressed genes(DEGs)were screened by transcriptome sequencing after intervention with 5-ASA and lipid mixture(LM)respectively,gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were performed.5.The inflammatory macrophage model was established by using THP-1 and peritoneal macrophages of mice in vitro.The toxicity of 5-ASA,LM and HDL-C was determined by CCK8 method after single or combined treatment with 5-ASA,LM and HDL-C,the expression of inflammatory factors was detected by qPCR.Results:1.Compared with the CD-DSS group,the DAI,degree of colon shortening,colonoscopic colitis score and colonic histopathology score in the CD5ASA group were decreased on the 10th day in the CD-5ASA group,but the difference was not significant(all p>0.05).Compared with HFD-DSS group,decrease of HDL-C level was reversed in HFD-5ASA group(p<0.05).At the same time,DAI,degree of colon shortening,colonoscopic colitis score and colonic histopathology score were significantly decreased in HFD-5ASA group compared to HFD-DSS group(all p<0.05).Compared to the DSS model group,the relative expression levels of interleukin-1 Beta(IL-1β),interleukin-6(IL-6)and tumor necrosis factor alpha(TNFα)mRNA in colon mucosa were significantly decreased(all p<0.05),and relative expression of interleukin-10(IL-10)mRNA was increased(p<0.001)in HFD-5ASA group,but there were no significant changes in CD-5ASA group.Flow cytometry and multiple immunofluorescences showed that the proportions of intestinal monocytes,macrophages and the M1 macrophages in HFD-5ASA group were significantly lower than that in HFD-DSS group(n=6,all p<0.05),when the proportion of M2 macrophages was increased(p<0.05).2.In immune infiltration analysis of GEO database,the proportions of intestinal M0 and M1 macrophages in active UC patients were higher than that in healthy subjects(all p<0.05),and the proportion of M2 macrophages was lower(p<0.0001).CD68 immunohistochemistry results from human colon tissue specimens showed that the colonic lesion sites of UC patients with normal blood lipids were less infiltrated by macrophages after 5-ASA treatment than before treatment(n=3,p<0.05).However,no significant changes were found in the groups with high total cholesterol(TC)and low HDL-C between before and after treatment(n=3 each group,all p>0.05).3.In the RNA sequencing results of the in vitro inflammatory macrophage model,GO enrichment analysis showed a significant correlation between 5-ASA treatment and inflammatory response and macrophage differentiation,while lipid mixture(LM)intervention was significantly correlated with inflammatory response and drug response pathways.KEGG pathway enrichment analysis showed that DEGs were enriched in inflammatory bowel disease(IBD)pathways,cytokine signals pathways,and immune cell related pathways after 5-ASA treatment.DEGs were mainly enriched in inflammatory factor signaling pathways and IBD pathways after LM treatment.Compared with the blank group,the control group upregulated the expression of various inflammatory factors,inflammatory factor receptors,and chemokine related genes.Both 5-ASA and LM treatments normalized the altered gene expression levels in the control group.(All p<0.05)4.Results in both THP-1 and PM inflammatory macrophage models in vitro demonstrated that single agent 5-ASA significantly up-regulated the expression of anti-inflammatory factors and single agent LM or HDL-C significantly down-regulated the expression of pro-inflammatory factors.5-ASA combined with LM or HDL-C down-regulated proinflammatory factors and up-regulated anti-inflammatory at the same time.Conclusion:1.5-ASA combined with high-fat diet could increase the level of HDL-C,meanwhile inhibit the infiltration of intestinal macrophages,and decrease the proportion of M1 macrophages in DSS induced colitis in mice.Addictinally,the expression of proinflammatory factors was inhibited,and the expression of antiinflammatory factors was enhanced of intestinal mucosa in HFD-5ASA group.2.5-ASA combined with lipid treatment has a direct antiinflammatory effect by down-regulating the levels of pro-inflammatory factors and up-regulating the levels of anti-inflammatory factors.Part III Effects of 5-ASA and high-fat diet on gut microbiota in acute colitis in miceBackground and Objective:Diet and gut microbiota are core factors in the pathogenesis of ulcerative colitis(UC),and the combination of 5ASA and high fat diet(HFD)significantly alleviated colitis in mice.This study aims to explore the relationship between the antiinflammatory effects of 5-ASA and HFD on acute colitis and gut microbiota.Methods:We used C57BL/6 mice to build the dextran sulfate sodium(DSS)colitis models.The experimental animals were divided into six groups of CD-NC,CD-DSS,CD-5ASA,HFD-NC,HFD-DSS,and HFD-5ASA.Collect fresh fecal samples from mice and divide them into three groups based on the collection time:baseline group included CDO(before CD treatment)and HFD0(before HFD treatment);after dietary pretreatment group included CD1(after CD pretreatment)and HFD1(after HFD pretreatment);after 5-ASA treatment group included CD-NC,CD-DSS,CD-5ASA,HFD-NC,HFD-DSS,and HFD-5ASA.Based on the V3-V4 variable region,16S rDNA amplicon sequencing technology was used to analyze the feces of mice in each group.α and β diversity analysis were conducted to compare the diversity and structural differences of gut microbiota among different groups,as well as the abundance differences of microbiota at the phylum,family,and genus levels.LEfSe analysis was operated to identify characteristic bacterial taxa in each group,and Spearman correlation analysis was used to explore the correlation between various clinical indicators,levels of inflammatory factors and blood lipids,and abundance of fecal bacteria on genus level.Results:1.Among the four groups(CD0,HFD0,CD1,and HFD1)before and after dietary pretreatment,there were 962 common amplicon sequence variants(ASVs)in a total of 68 samples.There was no significant difference inα diversity analysis(p>0.05),but β diversity analysis showed significant difference in microbial structure between the HFD-treated and CD-treated group(p<0.001).T-test analysis was conducted on the bacteria with the highest abundance changes on phylum,family,genus,and species levels.The results showed that HFD treatment caused structural changes in the gut microbiota of normal mice at all levels(all p<0.05).2.In the six experimental groups(CD-NC,CD-DSS,CD-5ASA,HFD-NC,HFDDSS,and HFD-5ASA groups),there were a total of 36 samples with 209 common ASVs.Compared with the CD-NC group,there was no significant difference in the abundance of gut microbiota in the HFD-NC group(Tukey test,p>0.05),but biodiversity increased(Wilcox test,p<0.05).DSS treatment significantly reduced the abundance and diversity of fecal microbiota,and decreases were only inhibited in the HFD-5ASA group(all p<0.05).DSS treatment downregulated the abundance of various beneficial and intermediate bacteria and upregulated the proportion of harmful bacteria at the phylum,family,and genus levels,but changes were only reversed in the HFD-5ASA group(all p<0.05).The LDA effect size(LEfSe)analysis showed that the number of specific taxa in the groups of CD-NC,CD-DSS,CD-5ASA,HFD-NC,HFD-DSS,and HFD5ASA were 5,5,3,11,1,and 6,respectively.Spearman correlation analysis determined the abundance of the Lachnospiraceae_NK4A136_group at the genus level and clinical indicators including DAI score,endoscopic score,histopathology score and intestinal macrophage infiltration were negatively correlated,but positively correlated with colon length.At the same time,the abundance of Lachnospiraceae_NK4A136_group was negatively correlated with the expression level of pro-inflammatory factors and positively correlated with the expression level of IL-10(all p<0.01).In addiction,the abundance of Lachnospiraceae_N K4A136_group was positively correlated with the level of serum HDL-C.Conclusion:HFD treatment can regulate the structure of intestinal flora in normal mice,and 5-ASA conbined with HFD can improve the imbalance of intestinal flora in the DSS induced mice.Lachnospiraceae_NK4A136_group shows a potentially anti-inflammatory effect in HFD-5ASA group,suggest that would be a beneficial bacterium associated with serum HDL-C level.