| Background:Rheumatoid arthritis(RA)is a chronic autoimmune disease characterized by inflammatory cell infiltration,extensive synovial hyperplasia with pannus formation,and destruction of articular cartilage and bone matrix.It is characterized by symmetrical,destructive progression that eventually leads to joint deformity and loss of function.So far,there is no effective treatment method,so it is of great clinical significance to study the pathological mechanism of RA.The main constituent cells in RA synovium are rheumatoid arthfitissynovial fibroblasts(RASF).The main pathogenic mechanism of RA is the abnormal proliferation of synovial cells and extensive infiltration of inflammatory cells.Deeply exploring the immune imbalance,inflammatory response,and synovial cell proliferation caused by RASF and its metabolic changes during the pathogenesis of rheumatoid arthritis is a meaningful direction for the treatment of RA.1,25-Dihydroxyvitamin D3(1,25(OH)2D3)is the main active form of vitamin D and can regulate calcium and phosphorus metabolism in the human body.The hormonal form of vitamin D3 has been recognized as an immunomodulatory hormone for 30 years.Experimental studies have shown that 1,25(OH)2D3 is involved in a variety of immune diseases,regulating the production of immune cells and cytokines.Vitamin D(VD)levels are closely related to RA.The level of serum 1,25(OH)2D3 in patients with moderate to severe RA was significantly lower than that in patients with mild RA.Clinically,the immunomodulatory capacity of RA patients was significantly associated with adjuvant therapy with 1,25(OH)2D3.1,25(OH)2D3 supplementation reduces pain in RA patients,serum VD levels are inversely correlated with RA disease activity,VD supplementation is a beneficial drug for RA treatment,and 1,25(OH)2D3 intake increases Afterwards,the incidence of RA can be reduced to a certain extent.In conclusion,the disease activity of RA is closely related to the level of serum 1,25(OH)2D3,however,the mechanism of action of 1,25(OH)2D3 in the treatment of RA is still unclear.Vitamin D Binding Protein(VDBP)is a transporter that transports 1,25(OH)2D3 between blood and cell membranes.VDBP is widely divided into articular chondrocytes and synovial cells at the site of RA injury.Our previous study found that VDBP in synovial tissue was significantly lower in RA patients compared with osteoarthritis patients.However,whether VDBP can inhibit the viability of RASF has not been clearly reported.Here,we aimed to investigate the synergistic effect of VDBP and 1,25(OH)2D3 on synovial cell proliferation and apoptosis in RA patients and whether VDBP is involved in the therapeutic effect of 1,25(OH)2D3 on CIA rats.Our findings may provide new strategies and directions for the treatment of RA.ObjectsTo investigate the therapeutic effect of VDBP and 1,25(OH)2D3 on rheumatoid arthritisMethodsPart Ⅰ:The expression of VDBP is decreased in the synovium,synovial fluid and synovial cell RASF of RA patients.We collected the synovial membrane and synovial fluid of the knee joints of RA patients and OA patients,and firstly analyzed the expression of VDBP in the synovium of RA patients(n=3)and OA patients(n=3)by immunohistochemistry.VDBP staining was stronger in OA synovium than in RA synovium.Statistical analysis showed that the expression level of VDBP was higher in OA synovium(P<0.05).Secondly,we took the joint fluid of RA patients(n=16)and OA patients(n=12)respectively,and detected the VDBP level in the joint fluid of RA and OA patients by ELISA.The results showed that the level of VDBP in the synovium of OA patients was significantly higher than that of RA patients.Again,we isolated and cultured synovial fibroblasts(RASF and OASF)from 3 RA patients and 3 OA patients.Western Blot results showed that the expression level of VDBP protein in OASF was significantly higher than that in RASF.Part Ⅱ:VDBP can enhance the effect of 1,25(OH)2D3 on RASF viability and apoptosis.RASFs were first treated with different concentrations of human recombinant VDBP(0,1,5,10,50,100 ng/ml)for 72 hours.Then,CCK-8 assay was performed to detect cell viability.The results showed that VDBP in the experimental groups(1,5,10,50 and 100 ng/ml)significantly inhibited the viability of RASF compared with the control group(0 ng/ml VDBP)(P<0.05).The VDBP concentration of 5 ng/ml had stronger inhibitory effect on cell viability than 1 ng/ml.However,at other concentrations(10,50 and 100 ng/ml),the inhibitory effect on RASF activity was not significantly enhanced with increasing VDBP concentration(P>0.05).Second,RASFs were treated with different concentrations of 1,25(OH)2D3(0,1,5,10,50 and 100 nmol/L)for 72 h,and cell viability was detected by CCK-8 assay.The results showed that 1,25(OH)2D3 in the experimental group(1,5,10,50 and 100 nmol/L)significantly inhibited RASF compared with the blank control group(0 nmol/L 1,25(OH)2D3)viability(P<0.05).The cell viability at 10 nmol/L was significantly lower than that at 5 nmol/L(P<0.05).But at 50 and 100 nmol/L,the inhibitory effect on cell viability had no further significant change compared with 10 nmol/L(P>0.05).Again,to analyze the combined effects of 1,25(OH)2D3 and VDBP on cell viability,RASF was treated with 10 nmol/L 1,25(OH)2D3 and different concentrations of human recombinant VDBP(0,1,5,10,50,100 ng/ml)for 72 hours.Compared with the control group and the 1,25(OH)2D3 group(10 nmol/L),the 1,25(OH)2D3+VDBP group had a statistically significant inhibitory effect on the viability of RASF(P<0.05).Among them,the groups treated with 10 nmol/L 1,25(OH)2D3+5ng/ml VDBP and 10 nmol/L 1,25(OH)2D3+10ng/ml VDBP had similar levels in inhibiting the activity of RASF(P>0.05).Finally,10 nmol/L 1,25(OH)2D3,5ng/ml VDBP and l0nmol/L 1,25(OH)2D3+5ng/ml VDBP were added to RASF cells and co-cultured for 72 hours.The CCK-8 assay showed that 1,25(OH)2D3 and VDBP had a significant inhibitory effect on the viability of RASF cells compared to the control group.The combined application of 1,25(OH)2D3+VDBP had the strongest inhibitory effect on the viability of RASF cells.Therefore,we can think that VDBP can enhance the inhibitory effect of 1,25(OH)2D3 on RASF activity.Apoptosis experiment:RASF cells were co-cultured with 10 nmol/L 1,25(OH)2D3,5ng/ml VDBP and 10nmol/L 1,25(OH)2D3+5ng/ml VDBP for 72 hours.The apoptosis of RASF cells was observed by flow cytometry.The results showed that both the 1,25(OH)2D3 group and the VDBP group could promote the pro-apoptosis of RASF cells,while the combined use of 1,25(OH)2D3+VDBP could enhance the pro-apoptotic effect(P<0.05).Compared with 1,25(OH)2D3 group,the pro-apoptotic effect of 1,25(OH)2D3+VDBP group was stronger,indicating that VDBP could enhance the effect of 1,25(OH)2D3 on RASF cell apoptosis(P<0.05).Cell cycle experiments:RASFs were treated with 10 nmol/L 1,25(OH)2D3,5ng/ml VDBP and 10nmol/L 1,25(OH)2D3+5ng/ml VDBP for 72 hours,respectively.RASF cell cycle was observed by flow cytometry.However,the cell cycle results showed that 1,25(OH)2D3,VDBP and 1,25(OH)2D3+VDBP had no significant effect on the number of RASFs in G0/1,S or G2 phases(P>0.05),indicating that VDBP and 1,25(OH)2D3 had no significant effect on the cell cycle of RASF(P>0.05).Different concentrations of 1,25(OH)2D3(0,1,10,100 nM)were added to RASF cells and co-cultured for 72 hours.WB results showed that compared with the control group,adding 1,25(OH)2D3 to RASF cells could promote the expression of VDBP(P<0.05);The expression of VDBP did not increase significantly(P>0.05).It shows that 1,25(OH)2D3 can stimulate RASF cells to produce VDBP,but the expression of VDBP does not increase with the increase of 1,25(OH)2D3 concentration,which is not a concentration-dependent linear relationship.Part III:VDBP may be involved in the treatment of CIA rats with 1,25(OH)2D3.A rat CIA model of rheumatoid arthritis was constructed,treated with 1,25(OH)2D3,and divided into NC group,CIA group and 1,25(OH)2D3 group.CIA rats were treated with an equal volume of normal saline or 1,25(OH)2D3,and the inflammatory curve was drawn to detect inflammatory factors to evaluate the therapeutic effect of 1,25(OH)2D3 on CIA.Through experiments,we observed that,compared with the normal control group,the CIA model group had swollen ankles,stiffness,decreased activity,lethargy,and weight loss.After 1,25(OH)2D3 treatment,the above symptoms were relieved.The inflammation curve showed that,compared with the normal control group,the inflammation in the CIA group reached a peak around 11 days after treatment(P<0.05).At 17 days of treatment,the 1,25(OH)2D3-treated group showed no statistically significant changes in inflammation compared with the normal control group.Compared with the CIA group,the inflammation was relieved in the 1,25(OH)2D3 treatment group at 17 days of treatment,and the difference was statistically significant.After 18 days of 1,25(OH)2D3 treatment,the serum of rats was collected to detect the changes of cytokines.Compared with the normal control group,the results showed that the serum levels of IL-10,IFN-y,TNF-α,GM-The concentrations of CSF and IL-6 were significantly increased,and the changes of other cytokines had no statistical difference.Compared with the CIA group,the concentration of IL-6 in the 1,25(OH)2D3 treatment group was decreased,and the changes of other cytokines were not statistically different.Rat serum and joint lavage fluid were collected,and the expression of VDBP was detected by ELISA.The expression results of VDBP in the joint lavage fluid showed that:compared with the control group,there was no statistical difference in the concentration of VDBP in the joint lavage fluid of the CIA group;There was no statistical difference in the concentration of VDBP in the liquid.The results of the expression of VDBP in the blood showed that compared with the control group,the concentration of VDBP in the serum of the CIA group was significantly lower(p=0.0015).Compared with the CIA group,there was no significant difference in the concentration of VDBP in the serum of the 1,25(OH)2D3 treatment group.ConclusionsThis study found that the expression of VDBP in synovial tissue,synovial fluid and RASF cells of RA patients was decreased,and VDBP could promote the apoptosis of RASF cells.1,25(OH)2D3 can promote the expression of VDBP in RASF cells,but in a concentrationindependent manner.In addition,VDBP could enhance the inhibition of RASF viability and the effect of apoptosis by 1,25(OH)2D3.This study provides a new theoretical basis for the treatment of RA with 1,25(OH)2D3 and VDBP. |
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