ABSTRACT
Objective:To investigate the effect of B7-H3 gene on the biological function of fibr-oblastlike synoviocytes (FLS) in osteoarthritis (OA).Methods:Synovial tissue of five cases of OA and synovial tissue of 4 normal knee were obtained, and the primary cell lines were isolated and cultured. The expression of B7-H3 in OA synovial tissue and primary OA-FLS were studied by immunohi-stochemistry, real time-poly merase chain reaction (PCR) and FACS. According to sites 996 and 1041 of B7-H3, corresponding siRNA was designed and the expression of B7-H3 in FLS was silenced and down-regulated. The inhibition of B7-H3 and its protein in target cells was determined by Western blot and FACS. The migration and invasion ability of B7-H3 in target cells were analyzed by scratch assay and Transwell assay. CCK8 assay was used to detect cell proliferation ability, and CBA assay was used to detect cytokines and chemokines in cell culture supernatant. GraphPad Prism 8.0 software was used to analyze the experimental data. The normal distribution data was expressed as mean±standard deviation ( Mean± SD). The comparison between data was performed by T test, and P<0.05 was considered statistically significant. Results:The abnormally high expression of B7-H3 in fibro-blast-like synoviocytes of OA was detected. Compared with siNC, si996 and si1041 inhibited the expression of B7-H3 in OA-FLS. In the Transwell migration experiment, the mean cells number of random view in the siNC group, the si996 group, and the si1041 group indicating decreased migration ability of OA-FLS [siNC vs si996 (100.3±3.7) /view vs (48.7±1.2) /view, t=13.24, P<0.001; siNC vs si1041 (100.3±3.7) /view vs (59.7±1.9) /view, t=9.80, P<0.001). In the Transwell invasion experiment, the mean cells number of random view in the siNC group, in the si996 group, and in the si1041 group indicating decreased invasion ability of OA-FLS [siNC vs si996 (127.3±5.6) /view vs (39.7±3.3) /view, t=13.49, P<0.001; siNC vs si1041 (127.3±5.6) /view vs (57.3±1.9) /view, t=11.85, P<0.001]. The secretion of IL-6 [siNC vs si996 (248±21) pg/ml vs (111±12) pg/ml, t=24.08, P=0.002; siNC vs si1041 (248±21) pg/ml vs (46±5) pg/ml, t=13.21, P=0.006], IL-8 [siNC vs si996 (118.1±15.6) pg/ml vs (47.1±5.4) pg/ml, t=6.68, P=0.022; siNC vs si1041 (118.1±15.6) pg/ml vs (10.0±1.3) pg/ml, t=13.08, P=0.006], CXCL8 [siNC vs si996 (178.8±6.4) ng/ml vs (83.2±2.7) ng/ml, t=13.77, P=0.005; siNC vs si1041 (178.8±6.4) ng/ml vs (93.5±2.8) ng/ml, t=12.23, P=0.007] and CCL2 [siNC vs si996 [(184.1±5.1) ng/ml vs (109.4±5.9) ng/ml, t=9.57, P=0.011; siNC vs si1041 (184.1±5.1) ng/ml vs (97.1±1.5) ng/ml, t=16.39, P=0.004] was decreased . Conclusion:B7-H3 may regulate the migration, invasion, cytokine secretion and other biological functions of OA-FLS, providing clues for further study of B7-H3's involvement in the pathogenesis of OA.
ABSTRACT
Objective To investigate the expression of T cell immunoglobulin and mucindomain-containing molecule-3 (TIM3) on PBMCs,and the plasma concentrations of soluble forms of Galetcin9 and their clinical relationship with rheumatoid arthritis (RA).Methods Peripheral blood samples were collected from 39 patients,25 osteoarthritis (OA) patients and 20 healthy subjects (HC).The expressions of TIM3 on peripheral blood mononuelear cells (PBMCs) were detected by flow cytometry.The concentrations of soluble Galetcin9 were assessed by enzyme linked immunosorbent assay (ELISA).And the relationship between their expression levels and clinical manifestations were analyzed.Levene F test was used for statistical analysis,normal distribution data were compared by t test and Pearson correlation analysis,while Mann-Whitney U test and Spearman correlation analysis were used for non-normal distribution data.Results The expression of TIM3 on CD4+ T cells was significantly higher than that of the HC [(14.7±3.2)％ vs (5.1±0.8)％,t=2.339,P=0.022 7],while there was no statistical difference between the RA group and the OA group [(14.7±3.2)％ vs (5.8±0.4)％,t=1.928,P=0.058 9].The expression of TIM3 was significantly correlated with the concentration of RF in the serum and the corresponding DAS28 score (r=0.325 8,P=0.043 0;r=0.407 5,P=0.010 0).The expression of TIM3 on CD8 + T cells in RA patients was significantly higher than that in the HC and OA [(21.1±3.4)％ vs (8.3±1.5)％,t=2.531,P=0.0142;(21.1±3.4)％ vs (10.7±1.0)％,t=2.314,P=0.024 0] which was significantly correlated with the concentration of RF in serum and the corresponding DAS28 score (r=0.451 5,P=0.003 9;r=0.524 1,P=0.000 6) as well.However,the expression of TIM3 on CD56+ NK cells was not significantly different from that of either OA or HC[(56.4±3.4)％ vs (50.6±3.8)％,t=1.047,P=0.299 8;(56.4± 3.4)％ vs (56.1±3.4)％,t=0.048,P=0.961 9],the concentration of serum RF and the corresponding DAS28 score were not significantly related.We also found that plasma Galectin 9 concentrations in RA patients were significantly higher than those of OA patients [(4.24±0.22) ng/ml vs (3.15±0.18) ng/ml,t=3.187,P=0.024] and healthy subjects [(4.24±0.22) ng/ml vs (2.55±0.14) ng/ml,t=5.567,P＜0.01],which was correlated with RF and DAS28 (r=0.479 2,P=0.002 0;r=0.353 0,P=0.027 5) while there was no correlation with CRP (r=0.176 3,P=0.283 1).Conclusion The upregulated expressions of TIM3 on peripheral lymphocytes and the high levels of plasma concentration of soluble Galectin 9 are closely correlated with the severity of the disease,suggesting that TIM3/Galectin 9 pathway may play a critical role in the pathogenesis of RA.