2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione Protects Palmitic Acid Induced Min6 Cell Dysfunction And Its Mechanim

Objective: To investigate the protection of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione(DMDD)on palmitic acid(PA)induced MIN6 cell dysfunction and the regulation of DMDD on TLR4/MyD88/NF-κB signaling pathway.Methods: MIN6 cellsweredividedinto6groups:normal controlgroup(NC group),model group(PA group),positivecontrolgrou p(TAK-242group),and andlow-,medium-andhigh-dosages of DMDD-treatedgroups(DMDDL,DMDDM,and DMDDH groups).1 Effect of DMDD on insulin levels,inflammatory cytokines levels and apoptosis in MIN6 cell:(1)Effect of DMDD on insulin levels in MIN6 cell: Cell activity wasdetectedbyCCK-8.The levels of insulin weredetectedby using enzyme-linked immuno sorbent assay(ELISA)commerciallyavailable kits.(2)Effect of DMDD on inflammatory cytokines levels in MIN6 cell: The levels of insulin,interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and monocyte chemotactic protein-1(MCP-1)weredetectedby ELISA.The expressions of IL-6,TNF-α,MCP-1 mRNA were analyzedby quantitative real-time PCR.(3)Effect of DMDD on MIN6 cell apoptosis: The rates of apoptosis weredetectedby using Annexin-V-FITC/Pl,Hoechst33342/PI kits.The ultrastructure of apoptosis was observed by transmission electronmicroscopy.The expressions of Caspase-3,Bax,Bcl-2 mRNA were analyzedby quantitative real-time PCR.The expressions of Caspase-3,Bax,Bcl-2 were analyzedby Western blot.2 Effect of DMDD on TLR4/MyD88/NF-κB signaling pathway in MIN6 cell: The expressions of TLR4,MyD88,NF-κB p65 mRNA were analyzedby quantitative real-time PCR.The expressions of TLR4,MyD88,phospho-NF-κB p65(p-NF-κB p65),cytoplasm phospho-IκBα(p-IκBα)were analyzedby Western blot.Results:1 Effect of DMDD on insulin levels,inflammatory cytokines levels and apoptosis in MIN6 cell:(1)Effect of DMDD on insulin levels in MIN6 cell: Compared with the PA group,the extracellular insulin content,intracellular insulin content,and total insulin content were increased in DMDDM,DMDDH and TAK-242 groups(p<0.05 or p<0.01).(2)Effect of DMDD on inflammatory cytokines levels in MIN6 cell: Compared with the PA group,the expressions of IL-6,TNF-α,MCP-1 mRNA and proteins were decreased in DMDDL,DMDDM,DMDDH and TAK-242 groups(p<0.05 or p<0.01 or p<0.001).(3)Effect of DMDD on MIN6 cell apoptosis: Annexin-V-FITC/Pl and Hoechst33342/PI analysis Show that,compared with the PA group,the total rates of apoptosis were decreased in DMDDL,DMDDM,DMDDH and TAK-242 groups(p<0.05 or p<0.01 or p<0.001).Transmission electronmicroscopy analysis Show that,compared with PA group,the ultrastructure of apoptosis was decreased in DMDDL,DMDDM,DMDDH and TAK-242 groups.Quantitative real-time PCR and western blot analysis Show that,compared with the PA group,the expressions of Caspase-3,Bax mRNA were decreased,Bcl-2 mRNA and protein were increased and Bcl-2/Bax ratio was increased in DMDDM,DMDDH and TAK-242 groups(p<0.05 or p<0.01 or p<0.001).Compared with the PA group,the expressions of Caspase-3,Bax proteins were decreased in DMDDH and TAK-242 groups(p<0.05 or p<0.01).2 Effect of DMDD on TLR4/MyD88/NF-κB signaling pathway in MIN6 cell: Compared with the PA group,the expressions of TLR4 mRNA and protein,MyD88 mRNA and protein,NF-κB p65 mRNA and p-NF-κB p65 nuclear protein and cytoplasm p-IκBα protein were upregulated in LPS+PA group,but were downregulated in TAK-242+PA group,DMDDH+PA group(p<0.05).Compared with the LPS+PA group,the expressions of TLR4 mRNA and protein,MyD88 mRNA and protein,NF-κB p65 mRNA and p-NF-κB p65 nuclear protein and cytoplasm p-IκBα protein were downregulated in LPS+DMDDH+PA group(p<0.05).Compared with the TAK-242+PA group,the expressions of TLR4 mRNA and protein,MyD88 mRNA and protein,NF-κB p65 mRNA and p-NF-κB p65 nuclear protein and cytoplasm p-IκBα protein were downregulated in TAK-242+ DMDDH+PA group(p<0.05).Conclusions: DMDD can increase the decreaction of insulin levels in PA induced MIN6 cell,attenuate the inflammatory response and apoptosis in PA induced MIN6 cells.The protection mechanism of DMDD on PA induced MIN6 cell dysfunction may be partial associated with inhibiting the TLR4/MyD88/NF-κB signaling pathway.