TXNDC5 Involved In Rheumatoid Arthritis By Insulin Signaling Pathways

Background:Rheumatoid arthritis(rheumatoid arthritis,RA)prevalence was 0.35%,is a chronic systemic autoimmune disease,and the clinical manifestations are joint damage and synovial inflammation.The main histopathological characteristics of RA are articular synovial cell proliferation,apoptosis,cell infiltration ability enhancement,pannus formation,and a large number of inflammatory cells infiltrations.Patients with RA significantly restricted in joint activities and even cause a physical disability,which threat to human health.But the pathogenic mechanism of RA is not fully clear,and anti-inflammatory drugs are main treatment.Therefore,to carry out the related research about cellular and molecular mechanism of RA is very important for the diagnosis and treatment of RA.Thioredoxin domain containing protein 5(TXNDC5)which encode by TXNDC5 genes belongs to the family of key protein disulfide isomerase,catalytic disulfide bond rearrangement,antioxidant,promoting angiogenesis,participate in a variety of biological functions such as cell inflammation.Our previous studies have found that TXNDC5 were increased in the synovial tissue and blood of patients with RA,and its coding gene is genetic susceptibility genes of RA.Our experimental research proves that are easier to be collagen Ⅱ TXNDC5 transgenic mice induced arthritis.So we suggest that TXNDC5 play an important role in the pathogenesis process of RA,although the specific pathogenic mechanism is not clear.The purpose of this research is to explore TXNDC5 how to participate in the process of RA.In recent years,genomics and other molecular biology found that,TXNDC5 is diabetes susceptibility genes,and play an important role in the process of diabetes.Diabetes is a kind of the insulin secretion defect or insulin disorder characterized by hyperglycemia due to the action of metabolic disease,often accompanied by insulin resistance or abnormal insulin signaling pathway.Others TXNDC5 can not only show that the catalytic reduction of insulin and reduce insulin synthesis,but also can reduce insulin and its receptor binding activity,aggravate diabetes.Epidemiological investigation also showed that RA patients increased risk of type 2 diabetes,the body’s resistance to insulin in patients with increased.In addition,a large number of studies suggest that inflammatory activity of RA can affect the metabolism of sugar,insulin signaling pathways and inflammatory mediators of insulin resistance related pathways,thus increasing the risk of RA patients suffering from diabetes.Given the relationship between RA and diabetes,and the important function of TXNDC5 in the two diseases,suggests TXNDC5 may be involved in development of RA through the insulin signaling pathway.Therefore,this topic plans to explore whether TXNDC5 through insulin signaling pathways involved in RA disease process,analysis the relationship between TXNDC5 and insulin resistance,insulin signaling pathway associated genes.This topic also plans to understand the inner relationship between RA and diabetes at the molecular level.Objective:Rheumatoid arthritis synovial cells(RASFs)is an important component in RA synovial joints,have significantly enhanced proliferation ability,can secrete a variety of inflammatory cytokines and growth factors.promote RA synovial inflammation and joint tissue destruction process.In order to study the relationship between TXNDC5 and pathogenic mechanism of RA,we inhibited TXNDC5 in RASFs with small molecular RNA,and then observe changes in cell proliferation,migration and apoptosis.At the same time,use the PCR array screening TXNDC5 related genes involved in insulin resistance or insulin signaling pathway in RASFs,and then use the Real-time PCR,ELISA and Western blot to confirm PCR array results.PCR array is a technology to rapidly screen genes in signaling pathways associated with disease.The technique used by a signaling pathways or a disease related gene loaded on a chip.It can quickly screen the purpose gene through the Real-time PCR technology.Methods:adopt the method of transient transfection to treat RASFs with anti-TXNDC5 small interfering RNA(siRNA),inhibit TXNDC5 expression.and then the Real-time PCR and Western blot technique to detect TXNDC5 mRNA and protein expression level;Using CCK8 proliferation experiment,cell migration and flow cytometry respectively to detect resistance TXNDC5 siRNA treatment group(anti-TXNDC5 siRNA group)RASFs proliferation,migration and apoptosis.Conrtrol group including the blank control group,negative control group(Allstar siRNA treatment group);Using insulin resistance RT2 Profiler PCR Array and insulin signaling pathway RT2 Profiler PCR Array respectively analysis anti-TXNDC5 siRNA transfection RASFs and Allstar siRNA transfection RASFs,found TXNDC5 downstream regulation genes by comparing the difference between the related gene expression;The Real-time PCR and ELISA method validation PCR array rsesults;The Real-time PCR and Western blot detection s IGFBP1(IGF binding protein 1)and IGFBPI(IGF binding protein 3)expression level in RA and Osteoarthritis(Osteoarthritis,OA)synovial tissue.Results:the experimental results showed that inhibiting TXNDC5 expression.RASFs cell proliferation(P=0.003)and migration ability significantly decrease(P=0.002),apoptosis increased significantly(P=0.006);Between the anti-TXNDC5 siRNA group and Allstar siRNA group,insulin resistance RT2 Profiler PCR Array detected IGF-1(insulin like growth factor 1),PCKI(phosphoenolpyruvate carboxykinase 1),SLC2A4(solute carrier family member 2 and 4)and IL1R1(interleukin 1 receptor type I)gene,insulin signaling pathway RT2 Profiler PCR Array detected IGFBPI(insulin like gro,wth factor binding protein 1)gene.Furthermore,Real-time PCR confirmed IGFBPI increased significantly(P=0.005)in anti-TXNDC5 siRNA group,and IGF-1,PCK1,SLC2A4 and IL1R1 expression has no statistical significance difference between the two groups;ELISA confirm IGFBP1 protein levels increased significantly(P<0.001)in cell culture of anti-TXNDC5 siRNA group.There were studies reported that IGFBP3 expressed(insulin like growth factor binding protein 3)in RASFs,and play an important role in IGF signal pathway.IGFBP3 and IGFBP1 are all important IGF-1 binding protein,and their function is very similar.So we detected IGFBP3 expression with Real-time PCR.According to the results,IGFBP3 expression has no difference between the two groups.Real-time PCR analysis IGFBP1 expression in the organization of RA and OA,the results show that RA synovial tissues IGFBPI mRNA expression is significantly lower than OA synovial tissue(P=0.011).Similarly,Western blot results show that the RA organization IGFBP1 protein expression is significantly decreased in OA group(P=0.014).The experimental results show that the inhibition TXNDC5 in RASFs,IGFBP1 expression quantity increased,while the expression of IGF-1 and IGFBP3 did not change.Western blot results show that compared with OA synovial tissue,IGFBP1 expression is reduced in RA synovial tissues.Conclusion:these results indicate that.inhibiting TXNDC5 in RASFs,IGFBP1 expression increased.Our preliminary work has proved TX:NDC5 were highly expressed in RA synovial tissue.Therefore.this research results suggested that high RASFs express TXNDC5 may inhibit IGFBP1 expression.Because IGFBP combined with IGF-1 can inhibit the activity of IGF.Thus.our results also suggest that RA synovial increased TXNDC5 by inhibiting IGFBP 1 expression of higher the ratio of IGF-1/IGFBP1,and inhibit the activity of IGF-1.Has been reported,IGF-1 have the function of the proinflammatory and inhibiting cell apoptosis,and it also can promote a variety of cell proliferation,differentiation and migration.After this study found that inhibit TXNDC5 expression,RASFs cell proliferation,migration ability is reduced,apoptosis rate increased.This may be due to higher IGFBP1 inhibits the activity of IGF-1,so cells behavior have changed.In a word,combined with the previous work and the results of this study,we recommend TXNDC5 involved in RA disease process is as follows:increased TXNDC5 in RA synovial cells stimulate the IGF-1 to stimulate RASFs proliferation,infiltrating ability enhancement,and inhibiting apoptosis,the results promote the progression of RA by inhibiting IGFBP1 instead of IGFBP3 expression activity.This study found IGFBP1 lower expression in RA synovial tissue also proved the proposal.In addition,some people also found that people with diabetes blood IGFBP1 expression level apparently unusual,IGF-1 activity changes obviously.The results of this study also suggests TXNDC5 and IGFBP1 expression of regulation may be inner link between RA and diabetes one of the important molecular mechanism.TXNDC5 negative control IGFBP1 expression and found that affect RASFs cell function which offers a new way for studying the pathogenesis of RA.