Mechanistic Studies On Anti-ulcerative Colitis Effect Of Tiliroside In View Of HIF-1α/glycolysis And Macrophage Balance

Objective:Ulcerative colitis(UC)is a chronic and non-specific inflammatory disease.In recent years,the prevalence and incidence of UC have been increasing in the Chinese population.At present,the clinical therapeutic drugs include 5-aminosalicylic acid,corticosteroids,immunosuppressants and biological agents.However,these therapeutic agents have obvious side effects,and some patients are still insensitive to them,resulting in further deterioration of the disease and eventually having to undergo surgical resection.In order to improve the life quality of UC patients,there is an urgent need to develop safer and more effective therapeutic drugs.The Tibetan medicine Lvluohua is the dried flower bud of Edgeworthia gardneri of the Araceae family.It is commonly used in treating diabetes,coronary heart disease,hypertension,and vascular inflammation diseases.The flower contains many components such as dicoumarins,flavonoids,fatty acids and triterpenes.Among them,tiliroside,the flavonoid with the highest content,has been proven to have anti-inflammatory,antioxidant and wound healing effects.We established colitis model in mice to study the inhibitory effect and mechanism of tiliroside on colitis.Methods:1.The female C57BL/6 mice and male BALB/c mice aged 6-8 weeks were selected to construct dextran sulfate sodium(DSS)-and TNBS-induced colitis models.5-aminosalicylic acid(5-ASA)or different dosage of tiliroside(12.5,25.0,50.0mg/kg)were orally administered to the mice daily from the day of modeling.In addition,the DSS-induced colitis model was reproduced in the mice treated with clodronate liposome and control liposome,which has been demonstrated to remove macrophages.The survival rate of mice in each group was recorded,the disease activity index(DAI)scores of body weight loss,blood in the stool and diarrhea were calculated,the colon length was measured,the myeloperoxidase(MPO)activity in the colons was detected,and the pathological changes of colons were detected by H&E staining.2.The polarization of macrophage in colons was detected by immunofluorescence assay;the mRNA expressions of M1 macrophage markers(Tnf-α,Il-1β,Inos)and M2macrophage markers(Arg1,Chil3,Cd206)were detected by using qPCR assay;the proportion of CD4~+,CD8~+T cells and CD19~+B cells in the colonic mucosal lamina propria was dected by using flow cytometry assay.3.The effect of tiliroside on the viability of BMDMs was detected by using MTT assay.LPS and IFNγwere used to induce M1 macrophages,while IL-4 was used to induce M2 macrophages.The effects of tiliroside on mRNA expressions of macrophage polarization markers(Tnf-α,Il-1β,Inos,Arg1,Chil3,Cd206)and LPS,IFN receptors(Tlr4,Cd14 and Ifngr)was detected by using qPCR assay.4.The effect of tiliroside on glucose uptake was detected by using the glucose analogue 2-NBDG;the effect of tiliroside on the mRNA expressions of glycolytic enzymes in M1 macrophages was detected by using qPCR assay;the production of lactate was detected by using the kits;the extracellular acidification rate(ECAR)was detecte by using the glycolytic rate assay kit.5.The expression of hypoxia inducible factor 1α(HIF-1α)in M1 macrophages was detected by using the qPCR and western blotting assays.The effect of tiliroside on HIF-1αubiquitination was detected by using the immunoprecipitation assay.Results:1.Tiliroside significantly alleviated the body weight loss of DSS-induced colitis mice,reduce the DAI scores,inhibit the shortening of the colon length,down-regulated the colonic MPO activity,and improves the colonic pathological damage.Similarly,tilirosidesignificantly reduces the mortality of TNBS-induced colitis mice,inhibited the shortening of the colon,down-regulated the colonic MPO activity and improved the colonic pathological damage.Results of the two colitis models collectively indicate that tiliroside possesses a good anti-colitis effect.2.The immunofluorescence results indicated that tiliroside(25.0,50.0 mg/kg)significantly decreased the proportion of M1(CD68~+iNOS~+)and increased M2(CD68~+CD206~+)macrophage phenotype in the colonic lamina propria of colitis mice.The qPCR results showed that tiliroside significantly downregulated mRNA expressions of M1(Il-1βand Inos)and upregulated mRNA expressions of M2(Arg1,Chil3,and Cd206)in colon.Tiliroside(12.5,25.0,50.0 mg/kg)exerted little effect on the mRNA expressions of Cdh1,Cldn7 and Ocln and infiltration of CD4~+T cells,CD8~+T cell,CD19~+B cells in the colonic lamina propria of DSS-induced colitis mice,indicating that the effect of tiliroside on UC was independent of epithelial barreier function,T and B cells.In addition,the DSS-induced colitis mice treated with control liposomes presented several colitis symptoms,evidenced by increased DAI scores,shorter colon length,increased MPO activity,serious pathological damage in colons,and tiliroside alleviated the development of colitis.More importantly,the protective effect of tiliroside on colitis was not stronger in the combination with clodronate liposomes,suggesting that tiliroside inhibited colitis through a macrophage-dependent mechanism.3.The MTT results showed that tiliroside(5,10,20,40,80μM)exerted no effect on cell viability of BMDMs.By establishing the polarization model in mouse-derived and human-derived macrophages,it was shown that tiliroside(10,20,40μM)significantly inhibited the expression of M1 type macrophage markers(Tnf-α,Il-1β,Inos),but had no effect on M2 macrophage markers(Arg1,Chil3,Cd206).4.In M1 type macrophages,tilirosde(10,20,40μM)significantly inhibited glucose uptake,mRNA levels of glycolysis-related enzymes(Glut1,Eno1,Pkm,Pdk1,Aldolase,Ldha,Pgam,Pfk,Gapdh),lactic acid production and extracellular acidification rate(ECAR).5.The effect of tiliroside on hypoxia inducible factor 1α(HIF-1α)was investigated by using western blotting and qPCR assays.The results showed that tiliroside(10,20,40μM)remarkably reduced the protein level of HIF-1α.Conversely,tiliroside exerted little effect on the mRNA expression of HIF-1αat the concentration of 10,20 and 40μM.The further pulse-chase experiment indicated that tiliroside facilitated the turnover of HIF-1αprotein in the presence of cycloheximide(CHX,15μg/ml).Thus,it is reasonable to believe that the decrease of the HIF-1αprotein level in tiliroside-treated BMDMs is due to increased degradation rather than decreased synthesis.Subsequently,proteasome inhibitor MG-132 and lysosome inhibitor Baf A1 were employed to probe the potential pathways of tiliroside-mediated HIF-1αdegradation.MG132 treatment significantly inhibited the tiliroside-induced reduction of HIF-1αprotein levels,while Baf A1 treatment did not block the effect of tiliroside.Finally,co-immunoprecipitaton was used to detect the ubiquitination of HIF-1αprotein by tiliroside.The results showed that tiliroside promoted the ubiquitination accumulation of HIF-1αprotein.Conclusion:1.Tiliroside ameliorates UC by restoring the balance of M1/M2macrophages in colonic mucosal lamina propria.2.Tiliroside can promote HIF-1αdegradation by enhancing ubiquitin-proteasome pathway,thereby inhibites the glycolysis process and prevents macrophages M1 polarization.