The Effect Of Electroacupuncture At ST36 On Chronic Colitis And The Related Mechanisms

Objective: Electroacupuncture(EA)at ST36 has been shown to improve acute colonic inflammation induced by dextran sulfate sodium(DSS).However,the role of EA in chronic colitis has not been studied yet.This part aims to evaluate the role of EA in DSS-induced chronic colitis,to explore whether EA can improve chronic colonic inflammation by protecting the intestinal barrier.Methods: Sixty male C57BL/6 mice were randomly divided into Control group,DSS group,DSS with sham EA(DSS + SEA)group,DSS with low-frequency EA(DSS + LEA)group,and DSS with high-frequency EA(DSS + HEA)group,12mice/group.All mice were given 3 cycles of 2% DSS drinking water to induce chronic colonic inflammation,both the EA and SEA treatments were administered throughout the duration of the experiment,30min/day.The disease activity index(DAI)was calculated.When the model is built,all mice were killed and their serum,colons were harvested.Haematoxylin eosin(HE)staining was used to assess the injury of colon and histological score.Western Blot and RT-PCR was used to detect the expression level of inflammatory cytokines in colon tissue,the serum inflammatory cytokines were measured by a cytometric bead array(CBA)kit,the expression of Zona occludens 1(ZO-1),Occludin,adhesion proteins(E-Cadherin)and Mucin2(MUC2)were detected with Western Blot,RT-PCR or immunofluorescence staining.In vivo intestinal permeability was determined using fluorescein isothiocyanate conjugated dextran(FITC-dextran),TUNEL assay was used to detect the apoptosis of intestinal epithelial cells(IECs),the proliferation colonic mucosal of IECs were evaluated by Ki67 immunofluorescence staining,the Immunofluorescence and fluorescence in situ hybridization(FISH)analysis was performed to identify the translocation and infiltration of bacteria in colonic mucosal.Results:(1)Compared with the DSS group,the DAI and histological scores of both the DSS+LEA and DSS+HEA groups were reduced,and only the HEA treatment prevented intestinal shortening in colitis mice.(2)The expression levels of TNF?,IL1?,IL6 and inducible nitric oxide synthase(i NOS)were decreased in both the LEA-treated and HEA-treated mice.Additionally,compared to the DSS group,both LEA and HEA treatments promoted the protein expression of the anti-inflammatory factor IL10.(3)Compared with the DSS group,both the LEA and HEA treatments promoted the expressions of intestinal barrier related proteins ZO-1,Occludin,E-Cadherin and MUC2,maintained the balance of apoptosis and proliferation of IECs,and decreased intestinal permeability.Conclusion: EA can maintain the integrity of the intestinal barrier,thereby to relieve chronic experimental colitis.Objective: The results of the first part confirmed that the EA treatment can preserve the intestinal barrier of colitis mice,but the specific mechanism has not been explained.Previous studies have reported that EA can regulate the gut microbiota in acute colitis mice,and the gut microbiota is closely related to intestinal barrier.Nevertheless,whether EA can preserve the intestinal barrier by modulating gut microbiota and the specific mechanism remains unclear.This part we will further explore effects of EA on gut microbiota of chronic colitis mice,and further build a fecal microbiota transplantation(FMT)model,to evaluate the influence of EA-altered microbiota on intestinal barrier of colitis mice,then further explore whether EA can maintain the integrity of the intestinal barrier by regulating gut microbiota and the potential molecular mechanisms.Methods: Stool samples of the first part mice were used to analyze the alterations in the gut microbiota by 16 S r RNA gene sequencing,and activation of the mitogen activated protein kinase(MAPK)and the signal transducer and activator of transcription 3(STAT3)signal pathway in EA-treated mice was evaluated.Then to further construct a FMT model,the mice were randomly divided into the Control FMT group,DSS FMT group,SEA FMT group,LEA FMT group,and HEA FMT group.All mice were given antibiotic water(1g/L of neomycin,streptomycin,ampicillin,metronidazole;0.5g/L of vancomycin)for 4 weeks to clear gut microbiota and prepare sterile mice.Similarly,chronic colitis was induced in all sterile mice with three cycles of 2% DSS drinking water and used as recipients of FMT.The DAI and histological scores of FMT mice were recorded,the colonic inflammation,intestinal barrier integrity and activation of the MAPK and STAT3 signaling pathway in sterile chronic colitis mice were detected.Results:(1)The diversity of gut microbiota in EA-treated mice was increased,the structure of gut microbiota was similar to that in control group,and the content of beneficial bacteria was increased while the harmful bacteria was decreased.(2)Both the LEA FMT and HEA FMT reduced the DAI and histological scores,increased the expression of ZO-1,Occludin,E-Cadherin and MUC2.(3)The EA treatment and microbiota from EA-treated mice could promoted activation of the MAPK signaling pathway.Conclusion: The EA treatment may promote the MAPK signaling pathway by modulating the gut microbiota,thereby preserving intestinal barrier integrity in mice with chronic colitis.