Experimental Study Of Betulinic Acid Inhibiting The Adipogenesis,Fibrosis And Inflammation Of Orbibital Fibroblasts

Purpose Thyroid-associated ophthalmopathy(TAO)is a common thyroid-specific autoimmune disease.It is the most common extrathyroidal change in diffuse toxic goiter.The common ocular symptoms are exophthalmos,eyelid retraction,diplopia,and so on.Orbital fibroblasts(OFs)are the main effector cells involved in the pathogenesis of thyroid associated ophthalmopathy.There are no studies on the role of betulinic acid(BA)in TAO.The aim of this study is to investigate the role and mechanism of betulinic acid in adipose differentiation,fibrosis and inflammation of TAO by using orbital fibroblasts from TAO patients.MethodThe orbital adipose-derived fibroblasts of TAO patients were extracted by tissue block attachment method for subculture.Cell immunofluorescence was used to identify fibroblasts.Cell Counting Kit-8(CCK-8)and flow cytometry were used to explore the effects of BA at different concentrations on the proliferative activity,cell cycle and apoptosis of fibroblasts,and the safe concentration was determined.Fibroblast differentiation into adipocytes was induced by lipogenic induction medium,and BA,mTOR inhibitor MHY1485,mTOR agonist Rapamycin or SREBP inhibitor Fatomycin were added for intervention treatment.The m RNA levels of FABP4,PPARγ,CEBPA were detected by real-time quantitative PCR(RT-q PCR).The production of lipid droplets were detected by oil red O staining.The expression levels of FABP4,PPARγ,CEBPA,SREBP,p-mTOR/mTOR were detected by Western Blot.Fibroblasts were co-cultured with BA and TGF-β to induce their fibrosis.The m RNA levels of ACTA2,COL1A1,COL1A2,and FIBRONECTIN were detected by RT-q PCR.The protein levels of ACTA2,COL1A1,p-ERK/ERK were detected by Western Blot.The level of hyaluronic acid in cell supernatant was detected by Enzyme Linked Immunosorbent Assay(ELISA).Fibroblasts were co-cultured with BA.IL-1β to induce an inflammatory response.The m RNA levels of IL-6,COX-2,ICAM-1,and TNF-α were detected by RT-q PCR,and the secretion levels of IL-6 and TNF-α in cell supernatant were detected by ELISA.ResultsOrbital adipose-derived fibroblasts from TAO patients could be successfully obtained by tissue adherent method,and could be successfully cultured in vitro.BA at 0.5,1 and 2 μM had no significant effect on the viability,cell cycle and apoptosis of OFs.BA inhibited the production of lipid droplets,and downregulated the m RNA and protein expression of FABP4,PPARγ and CEBPA,and inhibited the activation of mTOR/SREBP signaling pathway in OFs.The inhibitory effects of BA on lipid droplet formation,expression of adipogenesis-related proteins FABP4 and PPARγ,and activation of mTOR-SREBP pathway could be neutralized by mTOR agonists.The expression of mTOR-SREBP pathway and adipogenesis-related protein down-regulated by Rapamycin could be further inhibited by BA.The expression of SREBP and the expression of adipogenic-related protein down-regulated by Fatostatin could be further inhibited by BA.BA significantly inhibited the TGF-β-induced expressions of ACTA2 and COL1A1 and the secretion of hyaluronic acid,and reduced the phosphorylation level of ERK in OFs.BA significantly inhibited IL-1β-induced expression of IL-6,TNF-α,COX-2,ICAM-1 and secretion of IL-6 and TNF-α in OFs.ConclusionBetulinic acid inhibits adipogenic differentiation of TAO-OFs by inhibiting the mTOR-SREBP signaling pathway.Betulinic acid inhibits TGF-β-induced fibrosis of TAO-OFs,and the mechanism is related to the phosphorylation level of ERK.Betulinic acid also inhibits IL-1β-induced production of inflammatory cytokines by TAO-OFs.Above conclusions indicate that BA has potential therapeutic role in TAO management.