Effects of small nucleolar Ribonuclei Acid host gene 1 on the proliferation of fibroblastlike synoviocytes in rheumatoid arthritis / 中华风湿病学杂志

Objective:To preliminary study the effects of long noncoding Ribonuclei Acid (RNA) small nucleolar RNA host gene 1 (SNHG1) on proliferation of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and the possible mechanism.Methods:The FLS from RA and trauma group were primarily cultured by the explant culture, and the expression of SNHG1 were detected by quantitative polymerase chain reaction (qPCR). Transfection of siRNA was used to interfere the expression of SNHG1. Cell viability was measured using CCK-8 assay and cell cycle distribution was detected by flow cytometry. And the protein level of cyclinD1 was detected by Western blotting. Independent sample t test was used for the comparison between two groups, and the one-way analysis of variance (ANOVA) analysis was used to compare the samples of multiple groups. Results:Compared with FLS from trauma group, SNHG1 expression in RA-FLS was up-regulated [(2.13 ±0.55) vs (1.00 ±0.01)] ( t=-5.87, P=0.004). In RA-FLS, after silencing the expression of SNHG1, the cell viability of SNHG1-siRNA treatment group was down-regulated [(0.930 ±0.033) vs (0.759 ±0.027)]( t=6.879, P=0.002), the proportion of G 2/M+S cells was down-regulated [(28.2 ±1.5)% vs (9.7 ±2.6)%]( t=10.715, P<0.01), and thelevelof cyclinD1 protein was down-regulated ( t=6.168, P=0.004) compared with the negative control group. Conclusion:The SNHG1 is abnormally expressed in RA-FLS, and SNHG1 may participate in the regulation of FLS proliferation by affecting the expression of cyclinD1 protein, thereby contributing to synovial hyperplasia.

Effects of fibrobl ast growth factor 4 on the proliferation of fibroblast-like synoviocytes in rheumatoid arthritis / 中华风湿病学杂志

Objective To investigate the expression of fibroblast growth factor 4 (FGF4) in serum of active rheumatoid arthritis (RA) and its role in RA synoviocyte proliferation. Methods The serum level of FGF4 were detected by protein arrays in 20 patients with RA, and 20 age and gender matched healthy controls. FLSs were isolated from RA synovium,and were co-cultured with recombinant human FGF4 (rhFGF4). Cell proliferation was quantified by Cell Counting Kit-8 assay and cell cycle distribution was evaluated by flow-cytometry. The protein levels of cyclin D1, phospho-Akt (p-Akt) and phospho-p38 (p-p38) were measured by western blot. Results The serum expression of FGF4 in RA group was higher than that in control group (P=0.041). After being treated with different concentrations of rhFGF4 (12.5, 25, 50, and 100 ng/ml), RA-FLS showed significant increase in cell proliferation, with different rates of [(121 ±8)％], [(126 ±12)％], [(129 ± 12)％], a nd [(134 ±14)％] respectively, comparing with that of the controls [(100 ±0)％, (P12.5=0.049, P25=0.009, P50=0.004, P100=0.001).]. Among them, the percentage of G2/M+S phase cells were [(12.6±3.6)％], [(15.3±4.5)％], [(17.1±5.1)％], [(19.6±4.1)％] respectively, and except the lowest rhFGF4 concentration treatment group of 12.5 ng/ml, G2/M+S phase cells in other groups was significantly increased compared with the controls [(5.4±2.4)％] (P12.5=0.159, P25=0.042, P50=0.018, P100=0.005). And the protein expression of cyclin D1 was up-regulated after being treated with 50 ng/ml and 100 ng/ml rhFGF4 (P50=0.035, P100=0.027). FGF4 transiently increased the expression of p-Akt and p-p38 protein at the concentration of 50 ng/ml. Comparisons of data between groups were performed by independent sample Student's t-test. Statistical significant differences among groups were tested by one-way analysis of variance (ANOVA) or the Kruskal-Wallis test. The Dunnett's t-test was used for multiple comparisons. A P-value of <0.05 was considered statistically significant. Conclusion Our results suggest that FGF4 is highly expressed in the serum of active RA patients. FGF4 may promote the proliferation of RA-FLS via modulating PI3K/Akt and p38-MAPK signaling pathways, which subsequently contributs to synovial hyperplasia.

Sonic Hedgehog promotes fibroblast-like synoviocytes proliferation via modulating the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway in rheumatoid arthritis / 中华风湿病学杂志

Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

Effects of Sonic Hedgehog pathway on apoptosis of endothelial cells / 中华风湿病学杂志

Objective To investigate Smoothened (Smo) expression in endothelial cells of synovial tissues in active rheumatoid arthritis (RA),and the expression of Sonic Hedgehog (Shh) signaling pathwayassociated factors after TNF-α treatment in EA.hy926 cells,and the effects of specific inhibitor of Smo (cyclopamine) on the apoptosis of EA.hy926 cells.Methods The Smo expression in endothelial cells in synovial tissue from 4 RA patients and 4 patients with traumatic or meniscal injury (with no arthritis,act as control group) were detected by immunohistochemistry assay.EA.hy926 cells were treated with different concentrations of TNF-α or TNF-α together with different concentrations of cyclopamine,and Shh,Ptch1,Smo,Gli1 mRNA expression levels were detected by real time-PCR.EA.hy926 cells were co-cultured with three different concentrations of cyclopamine for 24 hours before the addition of TNF-α and ActinomycinD (ActD).The cell survival rate was detected using CCK-8,and the population of apoptotic cells was detected using a flow cytometry.T-test and one-way ANOVA were used for statistical analysis.Results The positive expression rate of Smo in endothelial cells of synovial tissue in RA group was (81±23)％,which was higher than that in the control group (20±17)％ (P＜0.05).After being treated with TNF-α,the expressions of Shh and Smo mRNA in EA.hy926 cells increased,while the expression of Gli1 mRNA decreased (P＜0.05),and the expression of Ptch1 mRNA did not change significantly (P＞0.05).The expressions of Shh,Smo and Gli1 mRNA were down-regulated (P＜0.05).EA.hy926 cells treated with different concentrations of cyclopamine (2,4 and 8 μmol/L) showed a significant decrease in cell viability,in cell survival rates (57±6)％,(44±8)％ and (32±5)％ compared with that of TNF-α/ActD group (64±6)％ (P＜0.05),and cell apoptosis rates [(12.4±3.3)％,(14.5±2.7)％ (15.7±2.4)％] compared with that of TNF-α/ActD group (7.1±1.3)％ (P＜0.05).Conclusion Shh pathway is activated in endothelial cells of synovial tissue in active RA.The apoptosis in endothelial cells is promoted after cyclopamine treatment.Shh pathway may play an important role in the antiapoptotic regulatory mechanism of endothelial cells.

The Dynamic Changes of LAK,NK and T Lymphocytes Subsets Before and After Operation in Patients with Hepatic Cancer / 中国医师杂志

To investigate the changes of immunological function of LAK,NK cell activity and T lymphoctyes subsets before and after operation in patients with hepatic cancer.The activity of Lymphokine-activated Killer Cell (LAK|,Natural Killer Cell(NK) and T lymphocytes subsets were detected in 31 patients with hepatic cancer of preoperation and postoperation,so did the 30 normal volunteers(as control group).The results showed that the activities of LAK,NK cell and T lymphocytes subsets of CD3,CD4 and the ratio of CD4/CD8 in patients with hepatic cancer were lower than that in normal control group(P