The Role Of Ceramide-associated Synthetase In The Regulation Of Interleukin-6 Production In Fibroblast-like Synoviocytes From Patients With Rheumatoid Arthritis

Background:Rheumatoid Arthritis(RA)is a systemic inflammatory autoimmune disease characterized by chronic destructive joint disease.The main pathological features of RA include synovial hyperplasia,inflammatory cell infiltration and pannus formation.Complex cytokine and chemokine networks localized in peripheral blood and synovium of RA patients perpetuate the inflammatory environment.Fibroblast-like synoviocytes(FLS)are not only the effector cells of RA joint inflammation,but also involved in the destruction of joints.In the joint target stage,FLS,which interact with innate immune cells and adaptive immune cells,produce a large number of inflammatory cytokines,then construct the proinflammatory microenvironment of RA joints.FLS and relative cytokines play a pivotal role in the occurrence of RA.As a key pro-inflammatory factor,IL-6 plays a variety of roles in the pathogenesis of RA,which interacts with FLS and other immune cells,promotes tissue damage,acute phase response and autoimmune response.IL-6 receptor blockers have been proved efficaciously in clinical application.And the regulation of IL-6 signal transduction pathway is regarded as an important target for the treatment of RA.Sphingolipids are important components of cell membranes,which associated with cellular functions.Sphingolipids are involved in the regulation of production of IL-6,while pro-inflammatory cytokines such as TNF-α and IL-1 β can affect the metabolism of sphingolipids.The metabolism between sphingolipids is carried out with the participation of a large number of enzymes.Ceramide is generally considered as the backbone of sphingolipids,which is involved in multiple diseases and immune regulation,by reprogramming given signals.The ceramide-associated synthetase ASM and GBA1 by which ceramide generates,are associated with IL-6 production in tumor cells.ASM/ceramide plays an independent positive role in the stability of IL-6 m RNA expression through the p38 pathway,resulting in increased IL-6 production.Moreover,ASM participates in the invasive biological behavior of tumor cells by regulating the production of IL-6.GBA1 is negatively correlated with IL-6 production in breast cancer cells by the GBA1-ceramide-p38δ pathway.Therefore,in this study,we speculated that ASM and GBA1,which related to ceramide synthesis,regulated the generation of IL-6 in RAFLS through p38 pathway,and affected the abnormal proliferation,migration,invasion and anti-apoptotic ability of RAFLS.In this study,we focused on peripheral blood of RA patients and RAFLS to further explore the regulatory effect of ceramide-associated synthetase ASM and GBA1 on IL-6 in RA and its related mechanism.Part Ⅰ: Expression of ceramide-associated synthetase ASM and GBA1 in peripheral blood of patients with rheumatoid arthritisObjective: To study the expression of ASM and GBA1 in peripheral blood of patients with RA.To analyze the correlation between ASM,GBA1 and levels of TNF-α,IL-6,and other clinical data.Methods: A total of 42 patients with RA were collected,who were admitted to the department of rheumatology and immunology of the first affiliated hospital of China medical university and met the 2010 Rheumatoid Arthritis Classification Criteria,as well as 15 healthy controls with the matched age and gender were also collected.The clinical data of patients,including age,gender,duration,combined organ involvement,medication history,number of swollen joints of 28 counted(SJC),number of tender joints of 28 counted(TJC),VAS score,erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),rheumatoid factor(RF),anti-cyclic citrullinated peptide(anti-CCP)and routine clinical laboratory test results,the disease activity index(DAS28 ESR,DAS28 CRP,CDAI,SDAI)and joint bone erosion classification,were all collected.The levels of ASM,GBA1,TNF-α,IL-6 in peripheral blood of RA patients were detected by ELISA.Comparisons between RA patients and healthy controls were made using either a independent sample t test or a mann-whitney rank sum test.Spearman correlation analysis(non-parametric correlation analysis)was used to analyze the correlation between ASM and GBA in serum and TNF-α,IL-6 and clinically index.Results: Among the 42 patients with RA,there were 4 patients(10%)with remission and low disease activity(DAS28-CRP ≤3.2),13 patients(41%)with moderate disease activity(3.2 < DAS28-CRP < 5.1),and 25 patients(59%)with severe disease activity(DAS28-CRP ≥5.1).Patients with RA were mostly with moderate and severe disease activity.The expression levels of ASM and GBA1 in the serum of patients with RA were significantly higher than those in the healthy control group(p=0.0419,p<0.0001).Spearman correlation analysis showed that serum GBA1 level was negatively correlated with IL-6(r=-0.4957,p=0.0008),and was positively correlated with PLT(r=0.4625,p=0.002)and CRP(r=0.3946,p=0.0097).There was no significant correlation between serum ASM and GBA1,IL-6,TNF-α,PLT,ESR,CRP,RF,and anti-CCP.Conclusion: The expression levels of ASM and GBA1 related to ceramide synthesis in peripheral blood of RA patients were increased,and GBA1 and IL-6 levels were negatively correlated.ASM and GBA1 may play different roles in the pathogenesis of RA by regulating inflammation.PartⅡ: Role of ceramide-associated synthetase ASM and GBA1 in the regulation of interleukin-6 in RAFLSObjective: To isolate,culture and identify RAFLS.To investigate the effects of ASM and GBA1 on IL-6 production,proliferation,apoptosis,migration and invasion of RAFLS.Methods: Synovial tissue samples were collected during knee arthroplasty surgery from10 patients with RA according to 2010 Rheumatoid Arthritis Classification Criteria.The tissue was digested by collagenase to isolate primary FLS.Expression of Vimentin in FLS was detected by immunofluorescence.The expression IL-6 levels of cellular supernatant and IL-6 m RNA in RAFLS was detected by ELISA and real-time PCR.Cell proliferation was detected by CCK8 assay,apoptosis was detected by flow cytometry(Annexin V/PI),and cell migration and invasion were detected by scratch assay and Transwell invasion assay.Results: Primary synovial cells were isolated and cultured in vitro,cells passage 3-4 with high expression of Vimentin,which identified them as RAFLS.Different concentrations of IL-1β were used to stimulate RAFLS for different periods of time.In each stimulus group,IL-1β 2.5ng/ml and IL-1β 10ng/ml groups showed significant increasing in concentration of IL-6 in cell supernatant compared with the control group(p<0.05,p<0.0001).Induction of 2.5ng/ml for 24 hours was selected as the condition for stimulating RAFLS.Tocilizumab(IL-6 receptor blockers),Desipramine(ASM inhibitor),Tocilizumab combined with Desipramine and Tocilizumab combined with conduritol β epoxide(GBA inhibitor)significantly down-regulated IL-6 protein and m RNA expression in RAFLS stimulated by IL-1β(p<0.05,p<0.001 or p<0.0001).Conduritolβ epoxide significantly upregulated IL-1 β to stimulate IL-6 m RNA expression in RAFLS(p<0.0001).The expression levels of IL-6 protein and m RNA in Tocilizumab combined with desipramine were significantly lower than those in Tocilizumab alone(p<0.05,p<0.0001).Tocilizumab,desipramine and Tocilizumab combined with desipramine significantly inhibited IL-1 β induced RAFLS proliferation(p<0.01,p<0.05,p<0.001).However,conduritol β epoxide significantly increased the proliferation ability of IL-1β induced RAFLS(p<0.05),and each group of drugs had no significant effect on apoptosis.Tocilizumab,desipramine,and Tocilizumab combined with desipramine significantly inhibited IL-1 β-induced RAFLS migration and invasion(p<0.05).Conduritol β epoxide significantly increased the RAFLS migration and invasion ability induced by IL-1 β and treated with Tocilizumab(p<0.05,p<0.0001).Conclusion: Functional ASM inhibitors can down-regulate the secretion of IL-6 by RAFLS and inhibit the proliferation,migration and invasion of RAFLS cells.The combination of functional ASM inhibitors and IL-6 receptor blockers can synergistically inhibit the proliferation,migration and invasion of RAFLS cells by RAFLS.Therefore,functional ASM inhibitors such as tricyclic antidepressants may have a potential therapeutic effect on synovial inflammation of RA,and can be used in combination with IL-6 inhibitor for synergistic treatment.GBA1 inhibitor can up-regulate the secretion of IL-6 in RAFLS and enhance the proliferation,migration and invasion of RAFLS cells.GBA1 may be involved in the disease process of RA.Part Ⅲ: Ceramide-associated synthetase ASM and GBA1 regulate the production of IL-6 in RAFLS through the p38 pathwayObjective: To study the molecular mechanism by which ASM and GBA1 regulate the production of IL-6 in RAFLS.Methods: RAFLS of P3-6 were isolated and cultured.The total and phosphorylated of MAPK signaling pathway p38α,p38δ,ERK,JNK in the RAFLS were detected by Western Blot.GBA and ASM of RAFLS were knocked down by lentivirus transfection.The expressions of GBA m RNA,ASM m RNA and GBA and ASM protein after lentivirus transfection were detected by Real-time PCR and Western Blot.The expression of IL-6 levels of cellular supernatant and IL-6 m RNA in RAFLS was detected by ELISA and real-time PCR.Results: The phosphorylation of p38α,p38δ,ERK and JNK induced by IL-1β was significantly inhibited by desipramine,Tocilizumab and Tocilizumab combined with desipramine(p<0.0001).The phosphorylation of p38α and p38δ was down-regulated by desipramine + Tocilizumab compared with Tocilizumab group(p<0.01,p<0.05).Conduritol β epoxide significantly up-regulated the phosphorylation of p38α,p38δ,ERK and JNK induced by IL-1β(p<0.05,p<0.01,p<0.0001).RAFLS was transfected by lentivirus plasmid to knockdown GBA or ASM.Compared with the negative control group,the expressions of m RNA and protein in the transfection group of LV-GBA and LV-ASM were significantly decreased.Silencing GBA of RAFLS significantly increased IL-1β-induced expression of IL-6 protein and m RNA(p<0.001),meanwhile up-regulate the phosphorylation of p38 α,p38 δ,ERK and JNK(p<0.0001,p<0.01,p<0.05,p<0.01).Silencing ASM of RAFLS significantly down-regulated IL-6 protein and m RNA levels induced by IL-1β(p<0.0001),and reduced phosphorylation of p38α,p38δ,ERK,and JNK(p<0.0001).SB202190,a p38 MAPK inhibitor,can significantly down-regulate IL-6 protein and m RNA levels induced by IL-1β in RAFLS after GBA and ASM lentivirus transfection(p<0.0001).Conclusion: Both functional ASM inhibitors and LV-ASM can down-regulate IL-6secretion of RAFLS by inhibiting p38 activation.The combination of functional ASM inhibitors and IL-6 inhibitors can synergistically inhibit p38 activation.GBA inhibitors and LV-GBA can promote the secretion of IL-6 by activating the p38 pathway.