| Objective:To study the effects of bile acid nuclear receptor(Farnesoid X Receptor,FXR)on lipopolysaccharide(LPS)-induced macrophage inflammatory response and intestinal barrier injury in mice and to explore the related mechanism.This research includes the following three parts:(1)To observe the survival rate and the injury of various organs of FXR gene knockout mice in the model of LPS-induced endotoxemia,and to clarify the effect of FXR gene on the acute inflammatory response.(2)To investigate the effects of GW4064,a small molecule known to be an agonist of the nuclear receptor FXR,on the secretion and expression of inflammation factors,production of nitric oxide(NO),expression of inducible nitric oxide synthase(iNOS)induced by LPS in RAW264.7 macrophage cells.(3)To observe the effect of FXR agonist obeticholic acid(OCA)on LPS-induced intestinal injury in mice and to explore the related mechanisms.Method:(1)A:Male FXR knockout(FXR-/-)and wild type(WT)C57BL/6 mice were injected intraperitoneally with LPS(30 mg/kg)and the survival time of mice were observed.B:Male FXR-/-and WT C57BL/6 mice were injected intraperitoneally with LPS(10 mg/kg),liver and kidney function were tested by blood biochemistry,secretion of serum inflammatory factors were detected by ELISA,and HE was used to observe histopathological change of the intestines and lungs.(2)The anti-inflammatory effects of GW4064 were investigated through evaluating the production of cytokines,NO and evaluating the protein expression of iNOS,NF-?B in LPS-induced RAW264.7 macrophages.(3)A total of 32 male wild-type C57BL/6 mice were randomly divided into control group(Control,n=8),endotoxemia group(LPS,n=8),and OCA group(OCA,n=8),OCA+endotoxemia group(OCA+LPS,n=8),and orally administered with OCA(10mg/kg)at 48h,24h,1h before LPS(10mg/kg)injected.Animals were sacrificed at 24h after LPS injected,the proximal jejunum and distal ileum specimens were collected for histopathological analysis.The expression of RNA and protein were detected by Quantitative Real-time PCR(qPCR);Immunohistochemistry,Western blot and Immunofluorescence were used to explore the level of protein.Results:(1)Compared with WT mice,the mortality of mice with FXR deficiency was significantly increased in LPS-induced sepsis(P<0.05).Serum AST,ALT and BUN in FXR-/-group were significantly higher than those in WT group(P<0.05),and FXR-/-group were also with more severe injury of lung and intestine.Serum proinflammatory cytokine IL-10 levels were significantly higher than that in WT mice,while anti-inflammatory factor IL-10 concentrations were significantly lower than in WT group(P<0.05).(2)Compared with LPS group,pretreated with GW4064 significantly inhibited the mRNA expression of proinflammatory factors including IL-1?,TNF-a and iNOS(P<0.05);The secretion of TNF-a and MCP-1 in cell supernatant were also reduced(P<0.05)when pretreated with GW4064.In addition,GW4064 significantly suppressed the production of nitric oxide(NO)induced by LPS(P<0.05);Simultaneously,the protein expression of iNOS and activity of nuclear factor NF-?B was also inhibited(P<0.01).(3)Compared with the LPS group,OCA can improve the clinical symptom score(P<0.05)and pathological score(P<0.05);Accordingly,leakage of fluorescein isothiocyanate-dextran was significantly reduced in OCA+LPS group(P<0.01).OCA partially up-regulated the reduction of ZO-1(P<0.05)and Occludin protein(P<0.01)in ileum tissues induced by LPS.Further studies showed that OCA inhibited the expression of TLR4 protein and TLR4,MyD88 mRNA(P<0.05),and suppressed activation of NF-?B pathway(P<0.05)in the ileum tissues.Conclusion:(1)FXR agonist GW4064 has a certain anti-inflammatory effect on macrophages,inhibiting the expression of iNOS and down-regulation of NF-?B may be one of the mechanisms of its anti-inflammatory effect.(2)FXR knockout can significantly aggravate sepsis caused by LPS in mice,while FXR agonist OCA can inhibit LPS-induced intestinal barrier damage through TLR4/NF-?B pathway. |
PDF Full Text Request