Potential Therapeutic Activity Of Berberine In Thyroid-associated Ophthalmopathy

PurposeThyroid-associated ophthalmopathy(TAO)is an autoimmune disease with multiple organ involvement,mild cases can cause changes in the appearance of the eye,serious cases will lead to serious dysfunction.The incidence of this disease tops the list of adult orbital disease.There are several pathophysiological processes in the orbit of TAO patients,characterized by expansion of adipose tissue,orbital inflammation,enlargement and fibrosis of extraocular muscles.Orbital fibroblasts(OFs)act as the core of forementioned pathologic changes.Undergoing the infiltration of autoimmune cells and activation of autoantigen,OFs could develop various degrees of adipognesis and fibrosis,along with hyaluronan(HA)production and overwhelming release of pro-inflammatory cytokines.The inflammatory microenvironment in orbit would deteriorate due to this inflammatory cascade.Nowadays,the pharmacotherapy of TAO is still a worldwide conundrum.Glucocorticoid has been applied as a classical management in active TAO cases with its well-known limitations and side-effects.And the emerging targeted drugs and biological agents,like teprotumumab and tocilizumab,are not widely available yet and may impose a heavy burden economically on TAO patients.Thus,continuous exploration for TAO drugs possesses high scientific and social value.“Huang Lian”,as a classical Chinese herbal medicine,is extensively used in traditional Chinese medicine.The main active ingredient extracted from Coptis,berberine(BBR),is mainly used to treat diarrhea caused by gastrointestinal infection.Recently,many studies found that BBR has satisfactory therapeutic effects in models related to metabolic diseases,lipid abnormalities and fibrosis,while the exact effects of BBR on TAO remain unclear.In this study,we intend to choose the primary cultured OFs obtained from TAO patients and control subjects as the research objects.Several cell models were established to explore the therapeutic effects of BBR on TAO comprehensively and study the underlying mechanisms.The experimental results could help us discuss whether BBR is worth considering as a potential candidate for TAO treatment and lay a foundation for following animal experiments and clinical trials.Methods1.OFs were cultured from TAO patients and control subjects.To determine the safe range of BBR concentration,the cytotoxicity of BBR was detected by CCK8 assay based on the certain concentration gradient.2.The adipogenesis of OFs was induced by commercial adipogenic differentiation medium(DM)and BBR was added according to the concentration gradient.After induction,the lipid droplets formed in OFs were displayed by Oil Red O staining,which was subsequently extracted by using analytical-grade isopropanol for the quantification.3.At predefined time points during adipogenic process,the expression of adipogenic indicators(PPARγ,adiponectin,c/EBPα,FABP4,Perilipin-1)at gene and protein level were detected by RT-q PCR and Western Blot assays.4.TAO-OFs were divided into PM group,DM group(adipogenic induction)and BBR group(adipogenic induction + BBR co-treatment).RNA sequencing(RNA-seq)was conducting after cell lysis using Trizol reagent.Differentially expressed genes(DEGs)were screened and used to conduct pathway-related classification and pathway-riched analysis.5.The signaling pathways which is riched by DEGs and associated with lipid metabolism were selected and verified by Western Blot.6.OFs were co-treated with IL-1β and BBR according to the concentration gradient.The m RNA expression levels of intracellular inflammatory factors(IL-6,PTX3,COX-2,IL-1β)were detected by RT-q PCR,and the protein concentrations of the above inflammatory factors were detected by ELISA7.OFs were co-treated with IL-1β and BBR.The vital proteins in NF-κB signaling pathway were determined by Western Blot.8.OFs were co-treated with TGF-β1 and BBR according to the concentration gradient.The culture medium was collected and detected by ELISA for the levels of HA and the m RNA levels of HA synthase(HAS1,HAS2,HAS3).9.OFs were co-treated with TGF-β1 and BBR according to the concentration gradient.The m RNA and protein levels of fibrotic markers(ACTA2,FN1,COL1A1,COL3A1)were detected by RT-q PCR and Western Blot.Results1.When the concentration of BBR was less than 20μM,the cell viabilities of TAO-OFs and CON-OFs were stable.BBR was noncytotoxic until a concentration of >20 μM,the downtrends of cell viability were consistent among 24 h,48h and 72 h treatment duration.2.Under the adipogenic differentiation medium,the lipid droplets were substantially accumulated in TAO-OFs,but not CON-OFs.BBR dose-dependently suppressed this effect and the difference detected by absorbance quantification had statistical significance.3.The expression levels of adipogenic markers(PPARγ,adiponectin,c/EBPα,FABP4,perilipin-1)were decreased by BBR treatment at m RNA and protein levels.The descent degree of m RNA expression was higher with long treatment duration.4.According to the result of RNA sequencing,there were 8989 DEGs and 10276 differential transcripts in PM-versus-DM,5227 DEGs and 5297 differential transcripts in DM-versus-BBR with statistical significances.Adjusting the threshold |log2FC| >1,there were 2466 DEGs and 5051 differential transcripts in PM-versus-DM,663 DEGs and 2081 differential transcripts in DM-versus-BBR.Among them,853 DEGs and 2140 differential transcripts were upregulated in PM-versus-DM,while 374 DEGs and 1103 transcripts were downregulated in DM-versus-BBR.At last,99 DEGs and 254 differential transcripts were screened after the intersection of two datasets and selected to obtain kegg＿pathway classification and enrichment.5.Signal transduction,lipid metabolism and endocrine system topped the list of kegg＿pathway classification.PPARγ,adipocytokine and AMPK signaling pathway had the most enrichment of DEGs.The downsteam proteins(SREBP1,SCD1)of AMPK pathway were upregulated during adipogenesis and this elevation was blocked by BBR treatment.6.After IL-1β stimulation of OFs in vitro,m RNA expression of inflammatory factors(IL-6,PTX3,COX-2,IL-1β)in OFs increased,and the content of these inflammatory factors in culture supernatant increased significantly.BBR treatment inhibited the induction effect concentration-dependently.There was no significant difference in the inhibition of BBR between TAO-OFs and CON-OFs,but the secretion and m RNA expression of various inflammatory factors were significantly different between the two cell groups.7.After IL-1β stimulation of TAO-OFs in vitro,the protein expressions of MYD88,IKKα and NF-κB p65 increased significantly.BBR treatment inhibited NF-κB p65,but the changes of MYD88 and IKKα were not obvious.8.After TGF-β1 stimulation of OFs in vitro,the content of HA in culture supernatant was significantly increased,and the m RNA expression of HA synthetase genes(HAS1,HAS2,HAS3)in OFs was raised,which was inhibited by BBR treatment.There was no significant difference in HA content between TAO-OFs and CON-OFs groups.The expression of HAS1 m RNA in TAO-OFs group was significantly higher than that in CONOFs group,while the expression of HAS2 and HAS3 m RNA was lower.9.After TGF-β1 stimulation of OFs in vitro,the m RNA and protein expressions of fibrotic markers(ACTA2,FN1,COL1A1,COL3A1)were significantly increased,and the upregulation was concentration-dependently inhibited by BBR.No significant difference exists in the inhibitory effect between TAO-OFs and CON-OFs.Conclusions1.BBR exhibited no cytotoxicity to OFs when the concentration was less than 20μM.The possible influence of BBR on subsequent experiments could be excluded.2.TAO-OFs had potent adipogenic differentiation ability,while CON-OFs were delicate.BBR substantially inhibited the adiponesis of TAO-OFs concentrationdependently,possibly by blocking PPARγ and partial AMPK signaling pathways.3.BBR can significantly inhibit IL-1β-mediated OFs inflammation and TGF-β1-mediated HA secretion and fibrosis.