| ObjectiveDioscin is a kind of natural steroidal saponin with antibacterial,anti-inflammatory,hypolipidemic,anti-tumor and other pharmacological activities,which is used for the treatment of stenocardia and coronary heart disease.The study aims to investigate the protective effect of dioscin on ulcerative colitis(UC)induced by dextran sodium sulfate(DSS)in mice,and explore the potential mechanisms.Methods1.Effect of dioscin on DSS-induced UC in mice.Male Balb/C mice were randomly divided into normal group,model group,dioscin groups and sulfasalazine(SASP)group.Mice in the normal group drank distilled water freely,while mice in the other groups drank 3%DSS solution freely.Simultaneously,mice in the dioscin and SASP groups were intragastricly administrated with dioscin(40,80,160mg/kg)and SASP(200mg/kg)for 7 days respectively,while the remaining groups of mice were given distilled water of the same volume.During the experiment,bodyweight,fecal status and blood in the stool of mice in each group were recorded every day.On the 8th day of the experiment,all mice were sacrificed and the colons were isolated which length was measured subsequently.Pathological changes of colon tissues were observed by H&E staining.The activity of myeloperoxidase(MPO)in colonic tissue was detected.The expression levels of TNF-α,IL-6,IFN-γ and IL-10 in colon tissues were detected by Elisa.The expression and distribution of activated molecules F4/80,M1-type macrophage(M1 Mφ)surface marker CD86 and M2-type macrophage(M2 Mφ)marker CD206 were detected by immunofluorescence assay.Moreover,western blot was used to detect the protein expression of CD86 and CD206 in colon tissues.2.Mechanism of dioscin Regulating Mφ Polarization.2.1 Effect of dioscin on M1 Mφ Polarization.RAW264.7 cells were stimulated with 250ng/mL LPS and 100ng/mL IFN-γ for 24 hours to establish model of M1 Mφ polarization.Meanwhile,dioscin(0.625,1.25,2.5μ M)was given to the drug administration groups for 24 hours.The polarization of M1 Mφ(F4/80 and CD86)was analyzed by flow cytometry.The gene expression of Ml Mφ(iNOS,TNF-α,IL-6)was detected by real-time fluorescence quantitative PCR(qRT-PCR).The expression of TNF-α,IL-6,IL-1β in the cell supernatant was detected by Elisa,and biochemical kit was used to detect the secretion of NO and lactic acid and glucose uptake.The protein expressions of Raptor,HIF-1α,HK-2,PKM2,LDHA and CD86 were detected by western blot.2.2 Effect of dioscin on M2 Mφ Polarization.RAW264.7 cells were treated with dioscin(0.625,1.25,2.5 μM)for 48 hours,and cells in the positive group were treated with 50ng/mL IL-4 for 48 hours.The polarization of M2 Mφ(F4/80 and CD206)was analyzed by flow cytometry.The gene expression of M2 Mφ(Arg-1,IL-10,Yml)was detected by qRT-PCR.The secretion of IL-10 and the free fatty acid uptake were detected by Elisa.The protein expressions of Rictor,PPAR-γ,CPT1A,CPT2,ACSL1 and CD206 were detected by western blot.2.3 Effect of dioscin on M2 Mφ Polarization while inhibiting fatty acid oxidation.Dioscin(2.5 μM)or IL-4 combined with Etomoxir(100 μM),a fatty acid oxidation inhibitor,stimulated cells for 48 hours.Gene expression of Arg-1,IL-10 and Yml was detected by qRT-PCR.IL-10 secretion and free fatty acids uptake were detected by Elisa.And protein expression of CPT1A,CPT2,ACSL1 and CD206 were detected by western blot.2.4 Effect of dioscin on M1 Mφ Polarization while activating mTORC1.The M1 Mφ polarization model was established with the stimulation of 250ng/mL LPS+100ng/mL IFN-γ for 24 hours,meanwhile the co-intervention of 2.5μM dioscin and 10mM mTORCl agonist L-Leucine was conducted for 24 hours.The polarization of M1 Mφ(F4/80 and CD86)was analyzed by flow cytometry.The gene expression of iNOS,TNF-α,IL-6 was detected by qRT-PCR.The expression of TNF-α,IL-6 in the cell supernatant was detected by Elisa,and biochemical kit was used to detect the secretion of NO and lactic acid and glucose uptake.The protein expressions of Raptor,HIF-1α,HK-2,PKM2,LDHA and CD86 were detected by western blot.2.5 Effect of dioscin on M2 Mφ Polarization while suppressing mTORC2.Cells were stimulated by dioscin or IL-4 combined with 1μM mTORC2 inhibitor JR-AB2-011 for 48 hours.The polarization of M2 Mφ(F4/80 and CD206)was analyzed by flow cytometry.Gene expression of Arg-1,IL-10 and Ym1 was detected by qRT-PCR.Expression of IL-10 in cell supernatant and free fatty acids uptake were detected by Elisa,and protein expression of Rictor,PPAR-γ,CPT1A,CPT2,ACSL1 and CD206 were detected by western blot.3.Effect of dioscin on DSS-induced UC in mice while activating mTORC1 and suppressing mTORC2.Male Balb/C mice were randomly divided into normal group,model group,dioscin(160mg/kg)group,L-Leucine(100mg/kg)group,dioscin+L-Leucine group,JR-AB2-011(4mg/kg)group,and dioscin+JR-AB2-011 group.Mice in the normal group drank distilled water freely,while remaining mice drank 3%DSS solution freely for 7 days to establish UC model.At the same time,mice in dioscin group,dioscin+L-Leucine group and the dioscin+JR-AB2-01l group were given dioscin for 7 days,while other mice were given distilled water of the same volume by gavage.Dioscin+L-Leucine group,L-Leucine group,dioscin+JR-AB2-011 group and JR-AB2-011 group were intraperitoneally injected with L-Leucine or JR-AB2-011 for 7 days.During the experiment,the weight,fecal status and blood in the stool of mice were recorded every day.On the 8th day,the colons were isolated and its length was measured.Pathological changes of colon were observed by H&E staining.The expression of TNF-α,IL-6 and IL-10 in colon tissues was detected by Elisa.Expression and distribution of F4/80,CD86 and CD206 in colon tissues were detected by immunofluorescence.Besides,the protein expression of Raptor,Rictor,HIF-1α,PPAR-γ,CD86 and CD206 in colon tissues was detected by western blot.Results1.The results in vivo showed that DSS stimulation caused the mice to be depressed,bad appetite,weight loss,loose stools and blood in the feces,etc.In addition,MPO activity in colon tissues increased after mice induced by DSS.Also,the levels of TNF-α,IL-6 and IFN-γ increased,and the levels of IL-10 decreased.H&E staining showed obvious inflammatory infiltration of colon and severe loss of colonic glands after DSS stimulation.The results of immunofluorescence and western blot showed that the expression of CD86 was increased and that of CD206 was decreased in the colon of UC mice.Dioscin significantly ameliorated the pathological symptoms of UC,reduced the inflammatory infiltration of colon tissues,the activity of MPO and the expression level of TNF-α,IL-6 and IFN-γ in colon tissues,meanwhile increased the expression level of IL-10.The expression of CD86 was decreased and the expression of CD206 was increased.2.On the investigation to M1 Mφ polarization,the results showed that RAW264.7 cells were induced by LPS+IFN-γ,which significantly increased the mRNA expression of iNOS,TNF-α and IL-6,the secretion of TNF-α,IL-6,IL-1β,NO and lactic acid,the uptake of glucose,and the protein expression HK-2,PKM2,LDHA,Raptor,HIF-1α and CD86.After the intervention of dioscin(0.625,1.25,2.5μM),the expression of the above indicators was down-regulated.3.In the study of M2 Mφ polarization,the results indicated that RAW264.7 cells were stimulated by dioscin(0.625,1.25,2.5 μM)or IL-4(50 ng/mL),which markedly increased the mRNA expression of Arg-1,Ym1 and IL-10,the secretion of IL-10,the intake of free fatty acids,as well as the protein expression of Rictor,PPAR-γ,CD206,CPT1A,CPT2 and ACSL1.4.In the study of M2 Mφ polarization,when Etomoxir was used to inhibit fatty acid oxidation,the mRNA expression of Arg-1,Ym1 and IL-10 intervened by dioscin was weakened,the uptake of free fatty acids and the secretion of IL-10 were decreased,and the protein expression of CPT1A,CPT2,ACSL1 and CD206 was also attenuated.5.In the investigation of M1 Mφ polarization,while using L-Leucine activated mTORCl,dioscin remained the effect on reducing the level of Raptor,HIF-1α,HK-2,PKM2,LDHA and CD86,and reducing the mRNA expression of iNOS,TNF-α,IL-6,also reducing the secretion of NO,lactic acid,TNF-α and IL-6 and glucose uptake.6.In the study of M2 Mφ polarization,when mTORC2 was suppressed by JR-AB2-011,dioscin increasing the protein expression of Rictor,PPAR-γ,CPT1A,CPT2,ACSL1 and CD206 was neutralized,increasing the mRNA expression of Arg-1 IL-10 and Ym1,and the secretion of IL-10 and free fatty acid uptake were also attenuated.7.The results in vivo revealed that under the intervention of L-Leucine,dioscin still improved the DSS-induced UC in mice,reduced the secretion of TNF-α and IL-6 as well as reduced the expression of Raptor,HIF-1α and CD86 in colon tissues.With the intervention of JR-AB2-011,the effect of dioscin on ameliorating UC in mice was weaken,and the effect on increasing the secretion of IL-10 and the expression of Rictor,PPAR-γ,CD206 in colon tissues was also neutralized.ConclusionDioscin ameliorates DSS-induced UC in mice,which may be related to the inhibition of mTORCl/HIF-1α-glycolysis-M1 Mφ polarization pathway,and the promotion of mTORC2/PPAR-γ-fatty acid oxidation-M2 Mφ polarization pathway. |
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