| Objective: Rheumatoid arthritis(RA)is a complex systemic autoimmunity which is characterized by erosive arthritis.The treatments we know still cannot cure or permanently get rid of RA symptoms.Even the systemic regular treatment still could not help some patients get out of irreversible joint deformities and loss of function.The etiology of autoimmune rheumatic diseases is not fully understood,and which may involve genetic and environmental factors.Vitamin D may inhibit humoral immune response,enhance immune tolerance,then prevent or reduce autoimmune diseases.More and more studies have found that vitamin D(VD)deficiency is related to RA.Studies on the potential role of VD supplementation in RA treatment have been inconsistent.In order to determine whether RA patients benefit from the supplement of vitamin D,we designed clinical trials,animal experiments and in vitro experiments to explore the related effects and mechanisms of vitamin D on RA.This study was conducted through the following three aspects:1.Correlation of 25-hydroxyvitamin D level with rheumatoid arthritis-related indicators and comorbid diseases:We compared the difference of 25-hydroxyvitamin D levels between RA patients and healthy people and analyzed its correlation with RA activity.Case-control studies were conducted to analyze whether vitamin D deficiency was one of the risk factors for RA with rheumatoid vasculitis(RV)or coronary heart disease.2.The effect of vitamin D on collagen-induced arthritis rat model:The collagen-induced arthritis(CIA)model of rats was used to verify whether CIA modeling can mimic the decrease of serum 25(OH)VD levels in RA patients,and whether supplementation with vitamin D could cure the vitimin D deficiency in the model rats,and affect the blood calcium,blood lipids,and histopathological manifestations of joints and synovium in rats,as well as inflammatory factor levels and lymphocyte subsets.3.The mechanism of vitamin D on collagen-induced arthritis rat model:We investigated whether the low 25(OH)VD level of CIA model rats is related to the high expression of Vitamin D receptor(VDR).And we study the effect of 25(OH)VD level on nuclear factor-?B(NF-?b)signaling pathway.We investigated the effects of CIA modeling and 25(OH)VD levels on vascular function related factors in CIA rats.We studied the effect of 25(OH)VD on cytokines secreted by human synovial tissue.Methods: 1.We recruited newly diagnosed RA patients and healthy controls,and recorded the course of disease,morning stiffness,the number of tender joints,the number of swelling joints,C-reactive protein(CRP),Erythrocyte sedimentation rate(ESR),Rheumatoid factor(RF),Anti-Cyclic citrullinated peptide(ACCP)antibody,X-rays of both hands,global health(GH)parameters of patients,calculate 28 joints Disease Activity Score(DAS28)in the RA group.We detected the serum 25-hydroxyvitamin D levels of the two groups of subjects at the time of enrollment.For patients and healthy controls with vitamin D deficiency,a one-time vitamin D3 supplement was given.VD3 oral solution was 300,000 units,and the serum 25-hydroxyvitamin D levels of the two groups were re-measured at intervals of 4 weeks.The t test was used to analyze the difference of the 25-hydroxyvitamin D level between the two groups of subjects before and after the vitamin D3 intervention.Spearman's correlation coefficient for ranked data7 was used to evaluate the correlation between the 25-hydroxyvitamin D level and the changes of 25-hydroxyvitamin D level before and after VD3 intervention with DAS28,CRP,RF,ACCP,GH,number of tender joints,and number of swelling joints.RA patients with RV and RA patients with coronary heart disease were recruited respectively as experimental groups.Match 1: 2 ratio by age and disease course,RA patients without RV and RA patients without coronary heart disease were recruited respectively as controls.CHOL,TG,HDL,LDL,serum albumin(Alb),serum calcium,ESR,CRP,RF,and immunoglobulins M,G,A,complement C3,C4,ACCP antibodies,TNF-?,L-6,ANA,ANCA,25-hydroxyvitamin D and D-dimer were tested in all patients.Multivariate conditional logistic regression analysis was used to screen for risk factors for RV or coronary heart disease in RA patients.Cox model was used to explore factors related to RV recurrence.2.We selected 30 female 4 weeks age Wistar rats with a body weight of about 100 grams.Rats are grouped by weight after adaptive feeding.Blank control group 6 rats,blank administration group 6 rats,and the remaining 18 rats were subjected to CIA modeling.Two weeks later,the successful model was selected,model control group 6 rats and model administration group 6 rats.Two weeks after the model was made,we administered VD3 with an intragastric needle.The rats in the blank administration group and the model control group were given 30,000 u / kg of vitamin D3.The blank and model control groups were given the same volume of double-distilled water.Rat weight and arthritis scores were recorded weekly.Blood was collected from the tail of the rat at 2 weeks,and the rats were sacrificed at 4 weeks,and blood samples were collected.At the same time,knee synovial,ankle,and spleen specimens were retained.The serum 25 hydroxyvitamin D level in rats were measured with ELISA.We detected rat serum calcium,total cholesterol(CHOL),high density lipoprotein cholesterol(HDL-C),and low density lipoprotein cholesterol(LDL-C)Triglyceride(TG)with fully automatic biochemical analyzer.Hematoxylin-eosin(HE)staining of synovial and ankle joints in rats was used to assess inflammation and joint damage.Tumor necrosis factor alpha(TNF-?),and interleukin 6(IL-6)levels in rats were measured with ELISA.Suspension of rat spleen lymphocytes was prepared,and the ratios of CD3 + CD4 + / CD3 + CD8 + cells,regulatory T cells(Treg),and T helper cell 17(Th17)cells were analyzed by flow cytometry.3.The expression of VDR in synovium of rats was detected by immunohistochemistry.Real-time Quantitative PCR Detecting System(q PCR)was used to detect VDR expression in synovium.Western blot(WB)was used to detect the expression of p-I?B? and I?B? protein in rat synovium.Considering the possible lag of vascular disease,we prolong the observation period of CIA rats.We prepared 60 female 4 weeks age Wistar rats with a body weight of about 100 grams.After adaptive breeding,they were grouped by weight: 12 rats in the blank control group,12 rats in the blank administration group,and the remaining 36 rats were subjected to CIA modeling.Two weeks later,12 successful models were selected and divided into a control group and model administration group.At 2 weeks after modeling,the rats in the blank administration group and the model control group were given 30,000 u / kg of vitamin D3.Rats were sacrificed at 4 and 6 weeks of immunization.We tested serum 25-hydroxyvitamin D,total NO,renin,and angiotensin ?(ANG-?)in rats,detected renin and ANG-? in aortic tissue,did HE staining of rat aortic tissue,detect renin and VDR in rat aorta with immunohistochemistry and q PCR,at 4 and 6 weeks of immunization.Synovial tissues of RA patients were cultured in vitro,intervention with 25-hydroxyvitamin D,and ELISA method was used to detect 25-hydroxyvitamin D,TNF-?,IL-6,interleukin 6 receptor alpha(Interleukin 6 recepter-?,IL-6 R?),interleukin 8(Interleukin 8,IL)-8),monocyte Chemotactic Protein 1(MCP-1),vascular Endothelial Growth Factor(VEGF).Results:1.A total of 268 newly diagnosed RA patients in the experimental group and 295 health examiners in the control group were included from October 2014 to October 2018,in the Rheumatology and Immunology Department from Shengjing Hospital of China Medical University.There was no difference in age and gender between the two groups.According to the month of enrollment,the difference of 25 hydroxyvitamin D levels before intervention between the two groups of subjects was compared.In January,February,June,July,August,and September,there was a statistical difference between the two groups.The 25-hydroxyvitamin D level in the experimental group was lower than that in the control group.Subjects with an insufficient serum 25-hydroxyvitamin D level were given a supplement equivalent to 300,000 units of vitamin D3,and the serum 25-hydroxyvitamin D levels in the two groups of subjects were retested after the intervention 4 weeks later.After intervention,the levels of 25-hydroxyvitamin D were significantly different each month throughout the year.Comparing the 25-hydroxyvitamin D changes before and after the intervention,the difference between the two groups of data is obvious.Using Spearman's correlation coefficient for ranked data,the initial 25-hydroxyvitamin D level was weakly correlated with DAS28,CRP,GH,tender joints,and swelling joints,but it was not correlated with RF and ACCP levels.The 25-hydroxyvitamin D changes before and after the intervention was strongly correlated with DAS28,CRP,GH,the number of tender joints,and the number of swollen joints,and not correlated with RF and ACCP levels.A total of 90 RV patients were recruited,matching 180 RA control groups.Vitamin D deficiency was present in 78.9% of RV patients.Losgist regression analysis found that the risk factors for RV were smokers(OR 2.56,CI 1.23,5.32),rheumatoid nodules(OR 2.48,CI 1.04,5.88),radiation erosion(OR 2.33,CI 1.31,4.17),and autoimmunity Thyroiditis(OR 3.14,CI 1.22,58.13),peripheral vascular disease(OR 3.14,CI 1.22,8.13),and malignant tumors(OR 3.19,CI 1.05,9.8),protective factors were higher HDL(OR 0.31,CI 0.13,0.7),and higher Alb levels(OR 0.89,CI 0.83,0.95).The recurrence rate of RV at 12 months' follow-up was 17.2%.The risk factors for RV recurrence were males(HR5.49,CI 1.55,19.61)(HR 4.05,CI 1.03,15.87),cutaneous vasculitis(HR 5.05,CI 1.28,20),and smoking(HR 3.12,CI 1.25,7.79)and hypertension(HR 4.05,CI 1.03,15.87).A total of 52 patients with RA and coronary heart disease were recruited,and 104 patients in the 1: 2 ratio RA control group were recruited.Multivessel disease were seen in 73.1% of RA patients with coronary heart disease.Combined hypertension(OR 3.73,CI 1.51,9.21)and higher ESR(OR 1.03,CI 1.01,1.04)were risk factors for RA with coronary heart disease.Tripterygium wilfordii(OR 0.36,CI 0.15,0.9)and higher serum 25-hydroxyvitamin D(OR 0.86,CI 0.79,0.94)were protective factors.2.The general state of the rats in the control group and the model administration group was worse than that of the blank control group and the blank administration group.The weight of the model control group and model-administered group measured weekly from the 1st week of the model was lower than that of the blank control group,and was statistically significant.At 4 weeks,the weight of rats in the model administered group was higher than that in the model control group,and was statistically significant.There was no difference in the arthritis score between the model control group and the model administration group.Before administration,the level of serum 25 hydroxyvitamin D in model rats was significantly lower than that in the blank control group and was statistically significant.After 2 weeks of drug intervention,the model control group remained at a lower level compared with the blank control group.The level of 25-hydroxyvitamin D in the model administration group was higher than that in the model control group,which was equivalent to the blank control group.The 25-hydroxyvitamin D level in the blank administration group was significantly higher than that in the blank control group.At 4 weeks after modeling,the blood calcium and total cholesterol levels of the rats in the blank administration group were significantly increased,and there was no statistical difference in the other 3 groups.There were no differences in the triglyceride,low density cholesterol and high density cholesterol with the 4 groups.Compared with the blank control group,the model control group showed obvious abnormalities with the results of HE staining of the ankle joint and the synovium of the rats.There was slight improvement in the model administration group.After the drug intervention,the levels of TNF-? and IL-6 in the model control group and the model administration group were significantly increased,and the level of IL-6 in the model administration group was lower than that in the model control group,which was statistically significant.The proportion of CD4 / CD8 cells in the spleen lymphocyte subsets of rats in the model administration group was higher than that in the model control group.There was no difference in the percentage of Treg and Th17 in total T lymphocytes in the two groups.3.The expression of synovium VDR of the model rats were higher than those of the blank control group,for the immunohistochemical results of the VDR of the synovial membrane,and the model administration group slightly improved.Real-time fluorescence quantitative detection of synovial membrane VDR expression,the model groups were significantly higher than that of the blank control group,and the model administration group slightly improved.The p-I?B? / I?B? ratio of the control group was higher than that of the blank control group.The p-I?B? / I?B? ratio in the model-administration group decreased compared with the model-control group.We extended the observation time and analyzed the materials at 4 and 6 weeks.The levels of NO in the serum of the model rats were significantly higher than those of the blank control.The content of NO in rat serum at the same time point was not changed a lot in the model administration group than in the model control group.There was no difference in the levels of renin and ANG-? in the serum of different group.The levels of renin and ANG-? in the aorta of model rats were higher than those in the blank control group.The levels of renin and ANG-? in the model-administered group decreased after 4 weeks of administration compared with the model-control group.There were no significant differences in the aortic HE staining between the groups,and there was also no difference in the thickness of the aorta middle smooth muscle at 4 and 6 weeks.The expression of aorta VDR and renin of the model rats were higher than those of the blank control group,and the expression levels of the model administration group were slightly improved.The aortic renin and VDR transcription levels in model rats were significantly higher than those in the blank group,and the model administration group decreased compared with the model control group.Human synovial tissues were cultured in vitro,and the levels of 25-hydroxyvitamin D3 in the experimental group were significantly increased after the positive control with dexamethasone or the experimental group with vitamin D3.The levels of IL-6 and MCP-1 in the experimental group were lower than those in the blank control group.The levels of TNF-?,IL-6R,IL-8,and VEGF in the experimental group were similar to those in the blank control group.Conclusions:1.Insufficient serum 25-hydroxyvitamin D level in RA patients is difficult to rise.After vitamin D intervention,the increased level of 25-hydroxyvitamin D in RA patients is negatively correlated with disease activity indicators.2.Low 25-hydroxyvitamin D level may be one of the risk factors for RA patients with coronary heart disease.3.CIA modeling can make the rat synovium and blood vessels highly express VDR,and the serum 25-hydroxyvitamin D level can be reduced.4.After vitamin D intervention,serum 25-hydroxyvitamin D levels in CIA model rats rose,the synovial VDR was down-regulated,the activity of NF-?B signaling pathway was down-regulated,the serum IL-6 level was decreased,the synovial and joint pathological damage were mildly reduced,the aortic VDR of CIA model rats was down-regulated,the local aortic renin and angiotensin-? levels were decreased.5.The intervention of 25-hydroxyvitamin D can reduce the levels of IL-6 and MCP-1 secreted by the synovial tissue of RA patients. |
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