Therapeutic Effect And Mechanism Of Berberine On Dextran Sodium Sulfate-induced Intestinal Inflammation In Mice

Early weaning piglets’ gut inflammation often leads to diarrhea and eventually leads to the decline of growth performance and even death of piglets,which has a huge economic loss to the breeding industry.Berberine(BBR)is a kind of anti-inflammatory drug which is non-prescription medicine in human.Because of its good effect,safety,and low price,it has potential value for the treatment of piglet’s intestinal inflammation.The previous research of our research group showed that berberine has a good therapeutic effect on weaned piglet diarrhea.This experiment uses transcriptomics combined with in vitro cell models and in vivo animal models to study the effect and mechanism of berberine in the treatment of intestinal inflammation,and provide reference for the application of berberine in pig production.The test is divided into four parts:1.The effect of berberine on dextran sodium sulfate(DSS)-induced enteritis in mice and its effect on lysozyme secretion.Kunming mice(8 weeks old,female)with similar body weight(about 25g)were selected for the experiment.120 Kunming female mice were randomly divided into 6groups(control group,DSS group,DSS+NS group,DSS+25mg/kg BBR,DSS+50mg/kg BBR and DSS+100mg/kg BBR).There are 5 replicates for each treatment,and the experiment is 15 days in total.After adapting to the environment for the first 3 days,starting from the 4th day,except for the blank control group,all five groups were given DSS(3%)free drinking water for 7 days,and then except for the DSS group,The remaining four groups were treated with normal saline(NS),25mg/kg BBR,50mg/kg BBR,and 100mg/kg BBR for 5 days.During the experiment,the weight,disease activity index(DAI)and expression of intestinal lysozyme of the mice were counted respectively.The results showed that the body weight of DSS-induced mice decreased significantly from the 4th to the 10 th day,but the body weight began to increase after the BBR treatment(from the 11 th to the 15 th day),and the weight gain effect increased with the increase of the berberine dose increase.Disease activity index(DAI)showed that BBR can reduce DSS-induced ulcerative colitis in a concentration-dependent manner.H-E histological sections showed that DSS had serious damage to the colon of mice.After 5days of treatment in different groups,the 50mg/kg BBR group(P<0.05)and 100mg/kg group(P<0.01)significantly reduced the DSS-induced inflammatory damage.The results show that berberine can alleviate DSS-induced intestinal inflammation in mice.In addition,immunofluorescence staining of ileum sections of mice in different groups showed that compared with the control group,the content of lysozyme in the ileum of mice was significantly increased after DSS treatment for 7 days(P<0.01).After BBR treatment,the content of lysozyme in the ileum was significantly reduced(P<0.05),and the effect became more significant as the concentration of BBR increased.The results show that berberine can alleviate the DSS-induced intestinal inflammatory injury in mice and inhibit the secretion of lysozyme.2.Transcriptome analysis of IEC-18 inflammatory cell model induced by LPS treated with berberine.In this experiment,four experimental groups were set up(control group,LPS group,LPS + BBR group and LPS + DMSO group).The cells in control group were cultured in normal medium for 12 h;cells in LPS group were cultured in LPS(10 μg/ml)medium for12 h;cells in LPS + BBR group were cultured in LPS(10 μg/ml)medium for 12 hours,and then in BBR(100 μM)medium for 24 h.Finally,cells in LPS + DMSO group were cultured in LPS(10 μg/ml)medium for 12 h,and then in DMSO(0.2 %)medium for 24 h.Each group had three repeats,the total RNA of different groups was extracted for sequencing and analysis of transcription.There were 11258(90.4 % of the total number of coding genes)genes expressed among four groups.On the other hand,117,89,114 and118 genes were expressed in the control group,LPS group,LPS + BBR group and LPS +DMSO group respectively.Principal component analysis(PCA)results showed that LPS-induced cell inflammation after BBR treatment,the expression of cell gene transcription levels changed significantly.The GO and KEGG functional enrichment analysis of differentially expressed genes(DEGs)between the LPS group and LPS+BBR group found that the "DNA replication initiation" enrichment degree was the highest in the GO enrichment analysis,followed by the "centromeric organization ".Subsequently,KEGG enrichment analysis was performed.The results show that the top 20 KEGG pathways enriched by DEGs are "steroid biosynthesis","DNA replication","TNF signaling pathway" and "cytokine-cytokine receptor interaction".The results of WGCNA showed that most of the key annotation genes were rich in "endocytosis","TNF signal pathway","chemokine signaling pathway","toll-like receptor signaling pathway" and "MAPK signal pathway".The DEGs of LPS group and LPS +BBR group were constructed into a new gene set,and then cluster analysis of differentially expressed genes was carried out.The results showed that KEGG enrichment analysis showed that DEGs were enriched in autophagy,lysosome,nod like receptor signaling pathway and AMPK signaling pathway.In addition,we also screened the different genes according to anti-inflammatory performance.Compared with LPS and LPS+BBR group,the functions and pathways rich in DEGs were DNA replication,cell cycle,apoptosis,leukocyte migration and NF-κB and AP-1 pathway.The above results show that BBR can inhibit the inflammatory signal pathway mediated by NF-κB and AP-1,down-regulate the expression of chemokines,and prevent the migration of leukocytes;BBR regulates autophagy through AMPK pathway.3.The regulation of berberine on autophagy.Transcriptomics analysis results show that BBR regulates autophagy-related genes.In order to further study the effect of BBR on IEC-18 cell autophagy,we used monodansylcadaverine(MDC)staining to detect the number of autophagosomes in six groups of IEC-18 cells(control group,LPS group,LPS+DMSO group,LPS+25 μM BBR group,LPS+50 μM BBR group,LPS+100 μM BBR group),three replicates in each group,and the expression levels of several autophagy-related proteins were detected by western blotting.After IEC-18 cells were treated with LPS,the number of MDC green fluorescent bodies was significantly reduced(P<0.05),while after BBR treatment,the number of MDC green fluorescent bodies increased significantly(P<0.05),and the effect of BBR was dependent on the concentration(P<0.05).The gene expression was checked by RNA sequencing and analyzed in the KEGG database,and it was found that BBR can increase the autophagy level of IEC-18 cells through the AMPK/MTOR/ULK1 pathway.We also tested the protein expression levels of these related genes,and the results showed that BBR significantly increased the expression of p-AMPK(P<0.05),p-ULK1(P<0.05)and LC3B(P<0.05)protein,and inhibited p-The expression level of MTOR protein(P<0.05).The results showed that berberine increased the autophagy level of IEC-18 cells through AMPK/MTOR/ULK1 pathway.4.Effects of berberine and autophagy promoters/inhibitors on DSS-induced enteritis and Paneth cell lysozyme secretion in mice.120 Kunming female mice were randomly divided into 6 groups(control group,DSS group,DSS+NS group,DSS+BBR group,DSS+Rapamycin(RAPA)group,DSS+BBR+3-Methyladenine(3-MA)group),each with 5 replicates,the mice in the same group were randomly divided into 5 cages,with 4 mice in each cage.The experiment lasted 15 days.After 3 days of environmental adaptation,from the 4th day onwards,except for the control group,the remaining five groups were given DSS(3%)free drinking water for 7days.Subsequently,the remaining four groups except the DSS group were treated with normal saline(NS),100mg/kg BBR,2mg/kg RAPA,100mg/kg BBR+15mg/kg 3-MA for 5 days.The results showed that after 5 days of treatment in different groups,histological scores,villus length/capsular depth(V/C),and DAI index showed that the NS group could not significantly reduce the inflammation damage(P>0.05),but the BBR and RAPA groups could significantly reduce the inflammation damage(P<0.01).However,compared with the BBR-only treatment group,the 3-MA combination therapy reduced the efficacy of BBR(P<0.01).The results indicate that the promotion of autophagy may be one of the mechanisms by which BBR relieves DSS-induced enteritis in mice.Subsequently,q-PCR was used to detect the transcription levels of ileum-related genes in different groups of mice,and the results showed that autophagy can affect the expression of antimicrobial peptide secretion-related genes(Lyz1,Camp,Defa1,Defa5)in Paneth cells.The ileum of different groups was collected,and the content of Paneth cell lysozyme and autophagy were detected by immunofluorescence using lysozyme(Lyz1)and LC3 b antibody.In each crypt,Lyz1(red)was distributed in the bottom of the crypt.DSS significantly increased the number of Lyz-positive crypts and the number of Lyz-positive Paneth cells in each crypt.Compared with the DSS group,the BBR and RAPA groups significantly reduced the number of Lyz-positive crypts and the number of Lyz-positive Paneth cells in each crypt,while 3-MA significantly reduced the effect of BBR.The results showed that berberine inhibited the secretion of lysozyme by promoting autophagy.Conclusion: 1.Berberine has a good therapeutic effect on DSS-induced intestinal inflammation in mice.2.Berberine can inhibit the secretion of intestinal Paneth cells lysozyme,and its effect is related to the promotion of autophagy.3.Transcriptome analysis results show that berberine can inhibit the inflammatory pathway mediated by NF-κB and AP-1,reduce the expression of chemokines,and prevent the migration of leukocytes to exert anti-inflammatory effects.