ObjectiveTo investigate the effects and mechanisms of IL-32γ-shRNA-mediated gene silencing on the proliferation and apoptosis of fibroblast-like synoviocytes in patients with rheumatoid arthritis (RA).MethodsA eukaryotic expression plasmid of shRNA targeting IL-32γ was transfected into fibroblastlike synoviocytes by liposome in patients with rheumatoid arthritis.RT-PCR was used to determine the expression level of IL-32γ.Western blotting was used to detect the levels of cyclin D1 and p-Akt.The proliferation of RA-FLS was examined by MTT.Cell cycles were analyzed by flow-cytometry.The apoptosis of cells were measured by TUNNEL.Comparisons between groups were tested by t test.Results ① The expression of IL-32γwas significantly inhibited by shRNA-IL-32γ-expressing plamid PGCsi 3.0 targeting sequence 1,2 and 3,and the inhibition rate had reached 75.6％,66.2％ and 64.1％,respectively.② The absorbance value of proliferation of RA-FLS in EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group and normal group at day 3 [(0.23±0.03) vs (0.35±0.03) and (0.36±0.04),P＜0.05] and 5 [(0.27±0.03) vs (0.52±0.05) and (0.53±0.04),P＜0.01 ] after transfection.③ The rate of RA-FLS at phase G1 in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group respectively [(88±6)％ vs (69±5)％ and (68±4)％,P＜0.05],while those at phase S+G2 in the EASY-shRNA-IL-32γgroup was significantly lower than that in the shRNA-control group and normal group [ ( 13.6±3.0)％ vs (30.2±4.1)％ and(32.1±4.3)％,P＜0.01].④The rate of RA-FLS apoptosis in the EASY-shRNA-IL-32γ group was significantly higher than that in the shRNA-control group and normal group[(20.50±3.21 )％ vs (9.20±0.32)％ and (8.60±0.22)％,P＜0.01].⑤ The expression of cyclin D1(0.36±0.04) and p-Akt(0.31±0.03) in the EASY-shRNA-IL-32γ group was significantly lower than that in the shRNA-control group [ (0.59±0.08) and (0.53±0.06)] and normal group [(0.61±0.07) and (0.52±0.06),P＜0.01].ConclusionEASYshRNA-IL-32γ can inhibit RA-FLS proliferation by down-regulating the expression of cyclin D1 and induce RA-FLS apoptosis by down-regulating the expression of p-Akt.

By means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, the association between vitamin D receptor (VDR) genotypes and bone mineral density (BMD) in the patients receiving long-term glucocorticoid therapy was studied. The clinical data and blood of 71 patients with rheumatosis who received long-term glucocorticoid therapy were collected. BMD was measured by dual-energy X-ray absorptimometry. VDR gene fragment (about 185 bp) was amplified by PCR from the extracted genomic DNA, then digested with restriction endonuclease Bsm I. The genotypes were evaluated based on the fragment length following endonuclease digestion and the association between genotypes and BMD or Z-score values was analyzed. Among the 71 cases, the detected genotypes were Bb and bb with the distribution frequency being 11.3% and 88.7% respectively. The distribution frequency of the alleles was in agreement with the Hardy-Weinberg equilibrium. There was no significant difference between the two genotypes in age, gender, body mass index (BMI), disease duration, disease types, time of glucocorticoid administration and cumulative dosage (P > 0.05). Osteoporosis rate of the patients with Bb or bb genotype was 37.5% and 33.3% respectively, with the difference being not significant (chi 2 = 0.05, P = 0.8). The BMD and Z-score values at lumbar spine and femur in two genotypes were not similar, but the difference had no significant (P > 0.05). The distribution frequency of bb type of VDR genotypes in Han populations of China was more prevalent, followed by Bb and bb types in turn. In the patients receiving long-term glucocorticoid therapy, there was no significant difference in BMD between Bb and bb genotypes. The data suggest that the VDR genotypes may not be means of identifying patients at greater risk of glucocorticoid-induced osteoporosis, which await to be further confirmed by a large sample size.
Arthritis, Rheumatoid/drug therapy ; Bone Density ; Genotype ; Lupus Erythematosus, Systemic/drug therapy ; Osteoporosis/chemically induced ; Osteoporosis/*genetics ; *Polymorphism, Restriction Fragment Length ; Prednisolone/*adverse effects ; Prednisolone/therapeutic use ; Receptors, Calcitriol/*genetics

The present study examined the functional profile of dendritic cells (DCs) in patients with rheumatoid arthritis (RA) and the effects of simvastatin on the function of DCs. A total of 40 patients who was recently diagnosed as having RA were equally assigned to two groups: the routine treatment group (group R) and the routine treatment plus simvastatin group (group R+S). Twenty healthy individuals served as control. The peripheral blood mononuclear cells (PBMCs) were isolated before and 4 weeks after the treatment and then cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulatory factor (GM-CSF) to prepare mature DCs. The expression of co-stimulating factor CD86 on the surface of DCs was assessed by flow cytometry. And the stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR). The contents of cytokines in culture supernatants of DCs in MLR were detected by ELISA. Blood lipids and high-sensitivity C-reactive protein (hs-CRP) were detected. The relationship between the expression of CD86 and the blood CRP level was also investigated. The results showed that, as compared with the control group, the CD86 expression and the level of cytokines secreted by DCs were significantly increased in RA patients and greater stimulating capacity of DCs in MLR was demonstrated in RA patients. T lymphocytes in MLR secreted higher levels of proinflammatory cytokines (IL-2, IL-17, TNF-α and INF-γ) and lower level of anti-inflammation cytokine (IL-10). The function of DCs was markedly weakened and the level of hs-CRP and low-density lipoprotein was substantially lowered in group R+S in comparison to group R. The CD86 expression was positively correlated with hs-CRP. It was concluded that DCs in RA are highly activated and DC-initiated immune reaction may play an important role in the pathogenesis of RA. Simvastatin administration can significantly inhibit the DCs function and reduce the level of hs-CRP, indicating the suppression on inflammatory reaction may be one of the mechanisms by which simvastatin exerts its effect in treating RA.

Objective To investigate the expression level of CD226 mRNA in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) and explore the relation between the gene expression and disease activity, and the relation between the gene expression and Gly307Ser polymorphism of CD226 was also examined. Methods CD226 gene was measured with real-time polymerase chain reaction (qRT- PCR) in PBMCs. The expression levels of CD226 gene in PBMCs were compared between 90 SLE patients and 30 healthy individuals. One-way ANOVA and pearson correlation were used for statistical analysis. Results The expression level of CD226 in the PBMCs of SLE patients (6.8±1.1) was significantly decreased compared to healthy individuals (26.5±6.7) (P＜0.01), while there was no association between mRNA level and genotype (P＞0.05). No correlation between ESR, CRP, ANA, SLEDAI scores, C3 and the expression level of CD226 gene was discovered. Conclusion In Hubei Chinese Han population, CD226-Gly307Ser locus is associated with the development of SLE, while T allele does not impact the expression of CD226 gene, thus the role of CD226 gene in autoimmune diseases should be explored in the future.

Systemic lupus erythematosus (SLE) is a multiple organ autoimmune disorder, including the liver, but the possible reason in impairment in the liver is still unclear. Our present study assessed alterations of transcription factor Foxp3(+) regulatory T cells (Tregs) and several other immune molecules [programmed cell death 1 and its ligand (PD1 and PD-L1), and interleukin 10 (IL-10) and transform growth factor β (TGF-β)] in the liver and other major organs of lupus-prone BXSB mice by flow cytometry, real-time quantitative reverse transcription PCR, and enzyme-linked immunosorbent assay. Results showed that both frequency and number of Foxp3(+) Tregs were dramatically reduced in the thymus, spleen and kidney of the BXSB mice (P<0.05), but those in the liver were kept in nearly normal range, when compared to negative control C57BL/6 mice. In comparison to control mice, the mRNA levels of Foxp3, PD1 and PD-L1 were significantly decreased in the kidneys of BXSB mice (P<0.05), but there was no significant difference in the livers of the BXSB mice (P>0.05). Protein levels of IL-10 and TGF-β in serum showed no significant difference between BXSB and C57BL/6 mice, but were significantly increased in the kidneys and livers of BXSB mice as compared with those in C57BL/6 mice (P<0.05). These results suggest that reduced Foxp3(+) Tregs are involved in the pathogenesis of SLE in BXSB mice, and relatively higher number of these cells in the livers than in the other target organs could constitute a protective mechanism against hepatic lesions in lupus-prone mice, which may provide insights into development of new therapeutic approaches in SLE patients.

To explore the effect of technetium-99 conjugated with methylene diphosphonate (99 TcMDP) on IgM-RF, IgG-RF and IgA-RF (RFs), 47 cases were selected for study, including 33 patients with rheumatoid arthritis (RA) and 15 patients with joint pain/arthritis. After 99Tc-MDP for drips model being given to the patients by intravenous drip 0.2 g daily for 5 days, the injection A and B models of 99Tc-MDP were used to the patients by intravenous injection one set daily for 10 days, that was one course of treatment. The next course started after 10 days. Each case used it from 2 to 4 courses of treatment. The RFs in serum were determined by the method of enzyme-linked immunoabsorption assay (ELISA) before and after 2 and 4 courses of treatment. In the patients with RA, the concentrations of IgM-RF were 296.2 +/- 108.4 IU/ml, 189.5 +/- 92.3 IU/ml and 107.8 +/- 72.5 IU/ml; the concentrations of IgG-RF were 325.6 +/- 126.2 IU/ml, 209.7 +/- 98.2 IU/ml and 160.2 +/- 80.8 IU/ml; the concentrations of IgA-RF were 330.4 +/- 136.3 IU/ml, 210.7 +/- 89.2 IU/ml and 148.8 +/- 72.2 IU/ml before and after 2 and 4 courses of treatment, respectively. The concentrations of the above RFs were significantly lower after 2 and 4 courses than those before treatment (P < 0.05 and P < 0.01). There was no significant difference in RFs concentrations in the patients with joint pain/arthritis before and after use of 99Tc-MDP. In the patients with positive RFs before treatment, the RFs concentrations were decreased significantly after 2 and 4 courses of treatment (P < 0.05 and P < 0.01). There was no obvious change of RFs concentrations in the patients with negative RFs after treatment of 99Tc-MDP. It was concluded that 99 Tc-MDP could obviously reduce the abnormally high concentrations of RFs, but not influence the normal RFs, which indicated that 99Tc-MDP has an important effect on controlling the activities of RA.
Adult ; Arthritis, Rheumatoid ; immunology ; therapy ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Immunoglobulins ; blood ; Male ; Rheumatoid Factor ; blood ; Technetium Tc 99m Medronate ; therapeutic use

The present study examined the functional profile of dendritic cells (DCs) in patients with rheumatoid arthritis (RA) and the effects of simvastatin on the function of DCs. A total of 40 patients who was recently diagnosed as having RA were equally assigned to two groups: the routine treatment group (group R) and the routine treatment plus simvastatin group (group R+S). Twenty healthy individuals served as control. The peripheral blood mononuclear cells (PBMCs) were isolated before and 4 weeks after the treatment and then cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony stimulatory factor (GM-CSF) to prepare mature DCs. The expression of co-stimulating factor CD86 on the surface of DCs was assessed by flow cytometry. And the stimulating capacity of DCs was measured by mixed lymphocyte reaction (MLR). The contents of cytokines in culture supernatants of DCs in MLR were detected by ELISA. Blood lipids and high-sensitivity C-reactive protein (hs-CRP) were detected. The relationship between the expression of CD86 and the blood CRP level was also investigated. The results showed that, as compared with the control group, the CD86 expression and the level of cytokines secreted by DCs were significantly increased in RA patients and greater stimulating capacity of DCs in MLR was demonstrated in RA patients. T lymphocytes in MLR secreted higher levels of proinflammatory cytokines (IL-2, IL-17, TNF-α and INF-γ) and lower level of anti-inflammation cytokine (IL-10). The function of DCs was markedly weakened and the level of hs-CRP and low-density lipoprotein was substantially lowered in group R+S in comparison to group R. The CD86 expression was positively correlated with hs-CRP. It was concluded that DCs in RA are highly activated and DC-initiated immune reaction may play an important role in the pathogenesis of RA. Simvastatin administration can significantly inhibit the DCs function and reduce the level of hs-CRP, indicating the suppression on inflammatory reaction may be one of the mechanisms by which simvastatin exerts its effect in treating RA.
Adult ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; immunology ; C-Reactive Protein ; metabolism ; Cytokines ; metabolism ; Dendritic Cells ; drug effects ; immunology ; Female ; Humans ; Male ; Middle Aged ; Simvastatin ; pharmacology ; therapeutic use

By means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, the association between vitamin D receptor (VDR) genotypes and bone mineral density (BMD) in the patients receiving long-term glucocorticoid therapy was studied. The clinical data and blood of 71 patients with rheumatosis who received long-term glucocorticoid therapy were collected. BMD was measured by dual-energy X-ray absorptimometry. VDR gene fragment (about 185 bp) was amplified by PCR from the extracted genomic DNA, then digested with restriction endonuclease Bsm I. The genotypes were evaluated based on the fragment length following endonuclease digestion and the association between genotypes and BMD or Z-score values was analyzed. Among the 71 cases, the detected genotypes were Bb and bb with the distribution frequency being 11.3% and 88.7% respectively. The distribution frequency of the alleles was in agreement with the Hardy-Weinberg equilibrium. There was no significant difference between the two genotypes in age, gender, body mass index (BMI), disease duration, disease types, time of glucocorticoid administration and cumulative dosage (P > 0.05). Osteoporosis rate of the patients with Bb or bb genotype was 37.5% and 33.3% respectively, with the difference being not significant (chi 2 = 0.05, P = 0.8). The BMD and Z-score values at lumbar spine and femur in two genotypes were not similar, but the difference had no significant (P > 0.05). The distribution frequency of bb type of VDR genotypes in Han populations of China was more prevalent, followed by Bb and bb types in turn. In the patients receiving long-term glucocorticoid therapy, there was no significant difference in BMD between Bb and bb genotypes. The data suggest that the VDR genotypes may not be means of identifying patients at greater risk of glucocorticoid-induced osteoporosis, which await to be further confirmed by a large sample size.
Adolescent ; Adult ; Arthritis, Rheumatoid ; drug therapy ; Bone Density ; Female ; Genotype ; Humans ; Lupus Erythematosus, Systemic ; drug therapy ; Male ; Middle Aged ; Osteoporosis ; chemically induced ; genetics ; Polymorphism, Restriction Fragment Length ; Prednisolone ; adverse effects ; therapeutic use ; Receptors, Calcitriol ; genetics

Systemic lupus erythematosus (SLE) is a multiple organ autoimmune disorder,including theliver,but the possible reason in impairment in the liver is still unclear.Our present study assessed alterations of transcription factor Foxp3+ regulatory T cells (Tregs) and several other immune molecules [programmed cell death 1 and its ligand (PD1 and PD-L1),and interleukin 10 (IL-10) and transform growth factor β (TGF-β)] in the liver and other major organs of lupus-prone BXSB mice by flow cytometry,real-time quantitative reverse trinscription PCR,and enzyme-linked immunosorbent assay.Results showed that both frequency and number of Foxp3+ Tregs were dramatically reduced in the thymus,spleen and kidney of the BXSB mice (P＜0.05),but those in the liver were kept in nearly normal range,when compared to negative control C57BL/6 mice.In comparison to control mice,the mRNA levels of Foxp3,PD1 and PD-L1 were significantly decreased in the kidneys of BXSB mice (P＜0.05),but there was no significant difference in the livers of the BXSB mice (P＞0.05).Protein levels of IL-10 and TGF-β in serum showed no significant difference between BXSB and C57BL/6 mice,but were significantly increased in the kidneys and livers of BXSB mice as compared with those in C57BL/6 mice (P＜0.05).These results suggest that reduced Foxp3+ Tregs are involved in the pathogenesis of SLE in BXSB mice,and relatively higher number of these cells in the livers than in the other target organs could constitute a protective mechanism against hepatic lesions in lupus-prone mice,which may provide insights into development of new therapeutic approaches in SLE patients.

Objective:To investigate the rates of low disease activity and clinical remission in patients with systemic lupus erythematosus(SLE)in a real-world setting,and to analyze the related factors of low disease activity and clinical remission.Methods:One thousand patients with SLE were enrolled from 11 teaching hospitals.Demographic,clinical and laboratory data,as well as treatment regimes were collec-ted by self-completed questionnaire.The rates of low disease activity and remission were calculated based on the lupus low disease activity state(LLDAS)and definitions of remission in SLE(DORIS).Charac-teristics of patients with LLDAS and DORIS were analyzed.Multivariate Logistic regression analysis was used to evaluate the related factors of LLDAS and DORIS remission.Results:20.7％of patients met the criteria of LLDAS,while 10.4％of patients achieved remission defined by DORIS.Patients who met LLDAS or DORIS remission had significantly higher proportion of patients with high income and longer disease duration,compared with non-remission group.Moreover,the rates of anemia,creatinine eleva-tion,increased erythrocyte sedimentation rate(ESR)and hypoalbuminemia was significantly lower in the LLDAS or DORIS group than in the non-remission group.Patients who received hydroxychloroquine for more than 12 months or immunosuppressant therapy for no less than 6 months earned higher rates of LLDAS and DORIS remission.The results of Logistic regression analysis showed that increased ESR,positive anti-dsDNA antibodies,low level of complement(C3 and C4),proteinuria,low household in-come were negatively related with LLDAS and DORIS remission.However,hydroxychloroquine usage for longer than 12 months were positively related with LLDAS and DORIS remission.Conclusion:LLDAS and DORIS remission of SLE patients remain to be improved.Treatment-to-target strategy and standar-dized application of hydroxychloroquine and immunosuppressants in SLE are recommended.