Objective To investigate the effects of 1,25-dihydroxyvitamin D3 [1,25 (OH) 2D3] on T helper cell type 17 (Th17) cytokines and therapeutic mechanism in patients with rheumatoid arthritis (RA).Methods Peripheral blood mononuclear cells (PBMCs) from healthy donors and RA patients were collected.The PBMCs were stimulated with anti-CD3/anti-CD28 monoclonal antibodies in the absence or presence of 1,25(OH)2D3 and methotrexate (MTX).After co-culture,the serum levels of Th17 cytokines interleukin (IL)-17,IL-6,tumour necrosis factor alpha (TNFα) were analyzed by cytometric bead array (CBA).The level of IL-22 was analyzed by enzyme-linked immunosorbent assay (ELISA).The independent samples t test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results The levels of cytokines IL-17,TNFα,IL-6 and IL-22 in RA group were significantly higher than those in the control group[(43 ± 6) ng/L,(5.91 ± 2.53) ng/L,(16.6 ± 12.0) ng/L,(51 ± 17) ng/L vs (21 ±3)ng/L,(2.63 ±0.27) ng/L,(4.2 ±2.3) ng/L,(22 ± 14) ng/L].Each of the three different 1,25 (OH) 2 D3 doses inhibited secretion of IL-17 [(533 ± 47) pg/ml,(426 ± 55) pg/ml,(319 ± 86) pg/ml],TNFα[(424 ± 82) pg/ml,(382 ± 79) pg/ml,(326 ± 87) pg/ml],and IL-6 [(5 513 ± 3 429) pg/ml,(4 555 ±3 157)pg/ml,(3 748 ± 1 919)pg/ml]in RA group (P ＜0.05),yet no statistical difference was found in IL-22 secretion with a trend of decrease after treatment of 1,25 (OH)2D3.Three different doses of MTX inhibited secretion of IL-17 [(452 ± 50) pg/ml,(372 ± 67) pg/ml,(315 ± 104) pg/ml] and TNFα [(319 ± 74) pg/ml,(292 ± 59) pg/ml,(266 ± 64) pg/ml] in RA group (P ＜ 0.05).However,levels of IL-6 and IL-22 were not affected after treated with MTX.Conclusion Our data indicated that 1,25 (OH)2D3 may play as an immune modulating agent to suppress Th17 cell cytokines.Supplement of vitamin D has the effective potential to treat patients with RA or other Th17 cell mediated autoimmune disorders.

Objective To study the expression of peripheral blood lymphocyte P-glycoprotein(P-gp)and related impact factors in rheumatoid arthritis(RA)patients,and explore whether the periodic combination of methotrexate(MTX)or leflunomide(LEF)with cyclophosphamide(CTX)could reverse P-gpinduced drug resistance.Methods This study was a cross-sectional study.Forty-two RA patients were enrolled(15 cases in the primary untreatment group,while 27 cases in the treatment group).In the treatment group,patients were divided into the improvement group(12 patients)and the refractory group(15 patients)respectively according to their disease activity scores(DAS 28).According to the medication used,42 patients were divided into 3 groups:the monotherapy group(MTX or LEF),the MTX+LEF combination treatment group and MTX or LEF+CTX treatment group.The expression of peripheral blood lymphocyte P-gp in each group was detected by flow cytometry.T test,ANOVA,LSD test,Bonferroni test,x2 test and Pearson correlation analysis were used for statistical analysis with software 13.0.Results ① Mean relative fluore scence intensity(RFI)of P-gp was 2.0±0.7,2.9±1.0,3.5±1.4,5.0±2.0 respectively in healthy control group,the primary untreatment group,the improvement group and the refractory group,the difference was significant(F=7.955,P＜0.01).in multiple comparisons,the fluorescence intensity was higher in the primary untrea-tment group as compared to the healthy group,but the difference was not statistically significant(P=0.137);however,it was significantly higher in the refractory group as compared to the improvement group(P=0.013).② There was no correlation between the expression of P-gp in RA patients and sex,age of onset,disease duration or positive rate of RF.But the expression of P-gp was significantly correlated with ESR and DAS28(r=0.447,P=0.002;r=0.398,P=0.012).③ RFI of the P-gp group was 5.1±2.2,6.0±1.2,3.4±1.1 respectively in monotherapy group,MTX+LEF group and MTX/LEF+ CTX group(P＜0.05).The expression of P-gp was significantly lower in the MTX/LEF+CTX group as compared to the monotherapy group(P＜0.05),but higher in the MTX+LEF group(P＞0.05).Conclusion The expression of peripheral blood lymphocyte P-gp in RA is correlated with disease activity.The loss of efficacy in DMARDs is due to the enhanced expression of P-gp.Combination of methotrexate or leflunomide with cyclo-phosphamide reverses P-glycoproteininduced drug resistance.

Objective:To study the expression of receptor activator of nuclear factor-κB ligand (RANKL) induced by interleukin-6 (IL-6) in fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLS) and its signal pathway, to clarify the synergistic mechanism of methotrexate (MTX) combined with cyclophosphamide (CTX) in inhibiting RA bone erosion.Methods:The synovial tissues of 6 patients with active RA were collected, and RA-FLS were cultured in vitro. IL-6/sIL-6R and drug intervention were given successively. All RA-FLS were divided into five groups, including blank control group, IL-6/sIL-6R group, CTX group, MTX group and MTX+CTX group. The expression levels of RANKL and signal pathway protein in RA-FLS were detected by real-time quantitative polymerase chain reaction (qPCR), Western blotting and cell-based enzyme-linked im-munosorbent assay (ELISA). Single factor analysis of variance was used to compare in the comparison of multiple groups, LSD- t test or Dunnett's T3 test was used in the comparison of two groups, and 2×2 factorial analysis of var-iance was used in the interaction of two drugs. Results:① There were statistically significant differences in the mRNA and protein levels of RANKL in each group ( F=26.246, P<0.01; F=4.565, P=0.023). The highest level of RANKL mRNA (2.14±0.40) and protein (2.33±0.39) was found in IL-6/sIL-6R group, and the lowest level of RANKL mRNA (0.10±0.08) and protein (0.75±0.21) in MTX+CTX group. ② The difference of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) level in each group was statistically significant ( F=18.151, P<0.01). The level of p-STAT3 (0.328±0.073) was the highest in IL-6/sIL-6R group and the lowest (0.178±0.022) in MTX+CTX group. There was no significant difference in STAT3 in each group ( F=3.173, P=0.051). ③ MTX combined with CTX had interaction on the expression of RANKL mRNA and protein ( F=33.932, P<0.01; F=16.265, P<0.01), and had interaction on the expression of p-STAT3 ( F=16.477, P=0.01), but had no interaction on the expression of STAT3 ( F=0.471, P=0.500). Conclusion:IL-6 can up-regulate the expression of RANKL in RA-FLS through JAK2/STAT3 signal pathway. MTX combined with CTX can down-regulate the expression of RANKL by inhibiting the phosphorylation of STAT3, and this combination is synergistic.

Objective:To explore the effect and mechanism of different concentrations of metformin on bleomycin (BLM)-induced systemic sclerosis (SSc) mice model.Methods:C57BL/6 mice were divided into the normal group, the model group, the high, the medium and the low metformin (MET) treatment groups randomly. All mice were sacrificed after BLM and metformin treatment for 4 weeks. Local skin was exminedby histopathological staining method to measure the thickness of dermis and collagen, and immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) were used to detect the protein and mRNA levels of Interleukin (IL)-17, forkhead box P3 (Foxp3) and α-smooth muscle actin (α-SMA). Flow cytometry was used to detect the percentage of effector T cell (Teff) and regulatory cells (Treg) in splenic mononuclear cells. The data such as dermal collagen thickness, α-SMA, IL-17, Foxp3, Teff and Treg levels were statistically analyzed by one-way analysis of variance. The data such as dermal collagen thickness, α-SMA, IL-17, Foxp3, Teff and Treg levels were analyzed by one-way analysis of variance, and least significant difference (LSD)- t or Kruskal-Wallis test was used for comparison between groups. Results:Compared with the normal group, remarkable fibrotic lesions appeared in the skin of mice in the model group, and the levels of T-helper cells (Th)1, Th2, Th17, and T follicular helper cells (Tfh) cells were increased, accompanied by a significant decrease in the level of Treg cells. After high-dose metformin treatment, the dermal thickness [(131±25) μm], collagen thickness [(119±18) μm], and α-SMA [(3.0±0.5)/HPH] were significantly reduced( F=14.390, P<0.01; F=40.245, P<0.01; F=44.626, P<0.01). Th1[(27.00±6.68)%], Th17[(0.56±0.20)%], Tfh[(6.4±1.6)%] cells ware significantlyreduced ( F=32.390, P<0.01; F=16.083, P<0.01; F=16.546, P<0.01), and Treg[(11.23±1.52)%] cells were significantly increased ( F=10.171, P<0.01). Conclusion:Metformin can effectively reverse the local skin changesin BLM-induced SSc mouse model, and show immune regulation and anti-fibrosis effects by restoring the Teff/Treg balance.

Objective:To investigate the characteristics of lymphocyte subsets and the correlation between the immune imbalance of T helper 17 cell/regulatory T cell (Th17/Treg) and cytokines in peripheral blood of patients with Beh?et's disease(BD).Methods:From January 2018 to November 2019, 82 outpatient and inpatient with BD of the Department of Rheumatology and Immunology of the Second Hospital of Shanxi Medical University with complete data were enrolled. The disease activity was evaluated according to BD Current Activity Form(BDCAF), and 66 age and matched healthy people were selected as the control group. The absolute numbers of lymphocyte subsets and T cell subsets dominated by Th17 and CD4 +CD25 +Foxp3 + Treg in patients with BD and healthy controls were detected by Flow cytometry. The levels of interleukin (IL-2), IL-4, IL-6, IL-10, IL-17, Interferon (IFN)-γ and Tumor necrosis factor-α (TNF)-α in patients with BD were measured by Cytometric Beads Array (CBA). The correlations between the ratio of Th17/Treg with inflammatory index, the number of organ involved and the levels of cytokines were analyzed. Data were analyzed by Mann-Whitney U test, Kruskal Wallis H test, Spearman correlation analysis and multiple linear regression. Results:①The absolute numbers of Th17 cells in peripheral blood of patients with active BD [13.9(7.7, 21.1) cells/μl] and patients with stable BD [8.7(6.1, 14.0) cells/μl] were higher than those of healthy controls [6.8(4.4, 8.5) cells/μl] ( P<0.01); The ratio of Th17/Treg was significantly increased ( P<0.01). The absolute co unts of Treg cells in BD group [24.79(15.64, 37.91) cells/μl] were sign-ificantly lower than those in healthy controls [30.59(23.04, 42.08) cells/μl], the difference was statistically significant ( P=0.016). ② The ratio of Th17/Treg was positively correlated with BDCAF ( r=0.298, P=0.007) and the number of organ involved ( r=0.304, P=0.006) was negatively correlated with age ( r=-0.254, P<0.05), and not correlated with the duration of disease and ESR ( P>0.05) in patients with BD. In addition, multiple linear stepwise regression showed that the ratio of Th17/Treg was positively correlated with BDCAF ( β=0.228, P=0.036) and negatively correlated with age ( β=-0.219, P=0.043), R2=0.101. ③ The levels of IL-2, IL-6, IL-10, IFN-γ in patients with BD were stati-stically higher than those of healthy controls ( P<0.01). The ratio of Th17/Treg was positively correlated with the levels of IL-2 ( r=0.307, P<0.01) and IL-4 ( r=0.301, P<0.01) in patients with BD. Conclusion:There is immune imbalance of Th17/Treg in patients with BD, which is closely related to disease activity, the number of organ involved, and the levels of cytokines such as IL-2 and IL-4. IL-2 and IL-4 may play an important role in the immune imbal-ance of Th17/Treg in patients with BD.

Objective:To investigate the level and clinical significance of peripheral blood CD4 +T cell subpopulations in late-onset systemic lupus erythematosus (SLE) patients. Methods:This study included 260 SLE patients hospitalized in the Rheumatology and Immunology Department of the Second Hospital of Shanxi Medical University from January 2016 to December 2021: of whom 58 and 202 were late- (≥50 years) and adult-(18~49 years) onset patients. This study also included 160 subjeces as healthy controls(HCs), of whom 35 and 125 were Control Group 1 (≥50 years) and Control Group 2 (18~49 years). Peripheral blood CD4 +T lymphocyte subsets of these participants were assessed by flow cytometry. The clinical data of all patients and healthy controls (HCs)were recorded. The differences between the groups were analyzed by Mann-Whitney U test or χ2 test. Results:(1)The time of diagnosis of late-onset SLE was longer than that of adult-onset SLE [Median time: 5.0 (2.0, 24.0)months vs 3.0 (1.0, 7.3)months, Z=-3.13, P=0.002]. Compared with adult-onset SLE, the SLEDAI score of late-onset SLE was lower [12.0 (8.0, 15.2) vs 14.0 (10.0, 18.0), Z=-2.12, P=0.034]. Some manifestations occurred more frequently in late-onset SLE, such as weight loss, nausea, abdominal pain, cerebral infarction, interstitial pneumonitis, Sj?gren′s syndrome and infection. The manifestations of skin and mucos a occurred less frequently in late-onset SLE. (2)CD4 +T cell subpopulations: ①The absolute counts of Treg, Th17, Th1 and Th2 cells in the peripheral blood of patients with late-onset SLE were significantly lower than those of HCs [Treg: 10.94 (6.14, 19.23) vs 32.65 (28.07, 41.65), Z=-6.79, P<0.001; Th17: 3.43 (0.94, 5.64) vs 6.13 (3.77, 7.82), Z=-3.24, P=0.001; Th1: 36.02 (10.80, 76.38) vs 128.70(89.82, 159.89), Z=-5.29, P<0.001; Th2:3.56 (1.56, 6.06) vs 8.25 (4.69, 12.98), Z=-4.57, P<0.001]. The ratio of Th17/Treg cells was higher than that of HCs[0.28(0.13, 0.59) vs 0.17 (0.12, 0.28), Z=-2.38, P=0.017].②The absolute counts of Treg, Th17, Th1 and Th2 cells in peripheral blood of patients with adult-onset SLE were significantly lower than those of HCs [Treg: 10.28 (5.37, 17.04) vs.30.19 (21.20, 39.75), Z=-11.28, P<0.001; Th17: 3.44 (1.84, 6.14) vs 6.48 (4.23, 10.66), Z=-6.53, P<0.001; Th1: 29.59(15.14, 56.81) vs 90.75(42.67, 162.00), Z=-7.01, P<0.001; Th2: 2.74 (1.62, 4.77) vs 8.25 (4.75, 11.99), Z=-9.91, P<0.001]. The ratio of Th17/Treg was higher than that of HCs[0.35 (0.17, 0.65) vs 0.23(0.14, 0.37), Z=-3.89, P<0.001].③The ratios of Th17/Treg in patients with late-and adult-onset SLE were higher than those of HCs. The ratio of Th17/Treg was the highest in adult-onset SLE patients. Conclusion:Patients with late-onset SLE have reduced numbers of Treg cells and the immune imbalanced of Th17/Treg. However, the immune imbalance of Th17/Treg in late-onset SLE patients is milder than that in adult-onset SLE patients, which may be related to lower disease activity.

Objective We investigated the correlation between inflammatory cytokines and drugresi-stant proteins. Methods Fourteen DBA1 mice were successfully induced by collagen and Freund's adjuvant. According to the scores of synovial pathology, the collagen-induced arthritis (CIA) group was divided into mild, moderate-severe groups, another four mice were selected as controls. The mRNA expressions of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MRP1) in splenic lymphocyte cells were measured by reverse transcription-polymerase chain reaction (RT-PCR). The concentrations of inter-leukin (IL)-1β, IL-2, IL-6, IL-10, umor necrosis factor (TNF)-α, IL-17 in serum were detected by Cytometric Bead Array (CBA). The correlation between different inflammatory cytokines and these proteins were analyzed, then one of the proteins which were most related with cytokines by immunohisto-chemical (IHC) in the synovium was studied. Data were analyzed by the Mann-Whitney U test, Kruskal-Wallis H test and spearman analysis. Results ①Compared with normal controls, the levels of IL-6 and TNF-α in the serum of mild CIA group, moderate-severe CIA group were significantly increased {IL-6 controls: [7.75 (5.14, 9.17) pg/ml];mild CIA: [25.31 (15.15, 29.27) pg/ml]; modeate-severe CIA: [45.03 (38.87, 64.02) pg/ml]. TNF-α: controls: [22.81 (20.84, 28.17) pg/ml]; mild CIA: [45.00(32.76, 58.51) pg/ml]; modeate-severe CIA: [45.00(39.78, 8.95) pg/ml]}(Z=14.383, P<0.05; Z=8.375, P<0.05). Compared with the mild CIA group, the level of IL-6 in serum was significantly increased in the moderate-to-severe CIA group (P<0.05), but there was no signigicant difference in the TNF-α level (P>0.05). ② In the spleen lymphocytes, there was no significant difference in the mRNA expression level of P-gp and BCRP among the groups, but the mRNA expression level of MRP1 was significantly increased (Z=12.634, P<0.05). Compared with the mild CIA group, the MRP1 mRNA in the moderate-severe CIA group was higher, the difference was significant (Z=12.634, P<0.05). There was a correlation between mRNA expression of MRP1 and P-gp (r=0.635, P=0.015). ③ The mRNA expression of MRP1 was positively correlated with IL-6 level (r=0.711, P=0.004). ④ The expression of MRP1 in normal group, low-level IL-6 group and high- IL-6 level group were as follows: [1.08 (0.65, 1.30)], [1.32 (1.08, 1.49)], [2.07 (1.77, 2.22)] respectively.⑤Compared with the controls, the cytoplasm/membrane of the knee and ankle joint synovial tissue in the CIA group was yellowish-brown, which indicated that MRP1 expression was positive. Conclusion In the CIA arthritis model, synovial tissue lesion is not only related to inflammatory cytokines, but also related to MRP1 expression in the ATP-binding cassette (ABC) transport protein family, and it is proved that IL-6 is highly correlated to MRP1.

Objective:To investigate the level of peripheral blood regulatory T cells in rheumatoid arthritis (RA) patients with cardiovascular disease (CVD) and its clinical significance.Methods:A total of 191 patients with RA in the Department of Rheumatology and Immunology, the Second Affiliated Hospital of Shanxi Medical University and 86 healthy controls (HCs) were enrolled from January 2019 to January 2021. All peripheral blood CD4 + T lymphocyte subsets of participants were assessed by flow cytometry. Patients were divided into RA-CVD group ( n=71) and RA only group ( n=120) and their clinical data were recorded. The differences between the groups were analyzed by Independent-Samples t test, Mann-Whitney U test or χ2 test, and risk factors that affected CVD were analyzed using Logistic regression. Results:① The age of patients and the proportion of male patients in the RA-CVD group were significantly higher than those in the RA only group [age: (64±10) years old vs (56±12) years old, t=-4.16, P<0.001; male patients: 35 cases vs 31 cases, χ2=10.86, P=0.001]. ② The level of Treg cells in the peripheral blood of patients with RA only and RA-CVD groups was significantly lower than that of HCs ( Z=-4.14, P<0.001; Z=-6.27, P<0.001), while the numbers of peripheral Th17 cells in the two groups of patients were not significantly different from those of HCs ( P>0.05). The ratios of Th17/Treg cells in the two group patients were higher than those of HCs, but only the difference between RA-CVD patients and HCs was significant ( Z=-5.49, P<0.001). ③ Compared with the RA only group, the absolute number of Treg cells in peripheral blood of RA-CVD group was significantly lower [19.00(13.62, 26.73) vs 24.94 (19.32, 34.12), Z=-3.19, P=0.001], the level of Th17 cells was significantly higher [absolute number: 7.77 (3.86, 13.64) cell/μl vs 5.59 (3.49, 8.91) cells/μl, Z=-2.14, P=0.033; percentage: 1.37%(0.78, 2.00)% vs 0.80%(0.56, 1.24)%, Z=-4.20, P<0.001], and the ratio of Th17/Treg cells was significantly higher [0.40(0.24, 0.62) vs 0.23(0.14, 0.35), Z=-4.46, P<0.001]. ④ Logistic regression analysis showed that Treg cell [ OR(95% CI)=0.934 (0.903, 0.967)] was a protective factor, while elder age [ OR(95% CI)=1.038(1.003, 1.074), male [ OR(95% CI)=2.450(1.005, 5.973)], hypertension [ OR(95% CI)=2.654 (1.219, 5.779)] and Th17 cell [ OR (95% CI)=1.066 (1.019, 1.116)] were risk factors of RA complicated with CVD. Conclusion:The level of Treg cells in peripheral blood of RA patients with CVD decreases significantly, and the immune imbalance of Th17/Treg is more singificant than that of RA patients without CVD. It is suggested that the immune imbalance and dysfunction caused by the number and/or functional deficiency of Treg cells may be involved in the occurrence and development of RA complicated with CVD.

ObjectiveTo examine the clinical features of fractures and related risk factors in patients with rheumatoid arthritis(RA) in China.MethodsSix hundred and eighty-one RA patients were randomly selected from department of rheumatology of 18 hospitals of China.Data were obtained from the questionnaire,including age,sex,disease duration,the involvement of joints,treatment regimen,features of fractures etc.The possible risk factors of fracture in patients with RA were analyzed with a multi-variate Logistic regression analysis.Results① In 681 RA patients of the survey,48 patients had 54 fractures,and the incidence of fractures was about 8％.② Fractures occurred at various sites.Foot/ankle,femur,spine and wrist were the mostfrequent sites.③ The Logistic regression analysis showed that several factors increased the risk of fracture in RA patients,including long disease duration (OR:1.245,95％CI:0.987-1.570,P=0.065),male gender(OR:0.433,95％CI:0.199-0.942,P=0.035),more deformed joints(OR:1.042,95％CI:1.006-1.079,P=0.023),family history of RA (OR:2.201,95％CI:0.984-4.923,P=0.055),and high scores of SF-36(OR:1.017,95％CI:1.002-1.033,P=0.028).④ According to the degree of correlation from strong to weak,the risk factors of fracture were disease duration,SF-36,sex,number of deformed joints and family history of rheumatoid arthritis.ConclusionThe incidence of fracture is high in patients with rheumatoid arthritis.Several factors could increase the risk of fractures in RA patients,including long disease duration,male gender,more deformed joints,and family history of RA.In order to prevent the occurrence of fractures,cautions should be taken to prevent the development of fractures and treat the disease aggressively to suppress the disease activity of RA.