| Objective: Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease whose pathogeny and pathogenesis is still unclear. Although the pathogenesis and therapy strategy of RA have been studied extensively for many years, the therapeutic outcome is not satisfied. Bone erosions occur in account for about 50% patients within two years. Ultimately, the disease can result in joint destruction and deformity. The average life-span of patients with RA will be shortened 10 to 15 years. It has been one of the main diseases which cause loss of labor ability in China. Therefore, it is necessary for us to study pathogenesis and search for new therapy methods of RA to improve life quality of patients.It is well-known that the main pathologic change of articular tissues of RA patients is synovitis. Excessive local production of cytokines has been demonstrated to play a central role in the pathogenesis of RA. Recent evidence has revealed that high mobility group box chromosomal protein 1(HMGB1) is a new cytokine which can activate many kinds of biological function. Some research discovered that extracellular HMGB1 plays an important role in many diseases by promoting inflammatory cells chemotaxis, tumor cells and microvascular proliferation. But the mechanisms of HMGB1 in RA pathogenesis have not been clarified.A large body of researches have shown that the important role of Arsenic trioxide (ATO) is to inhibit cell proliferation and induce apoptosis. While recent study indicated it has exert immunosuppressive effects, for example, to lower lymphocytes differentiation and phagocytic activity of macrophages. So, some researchers try to use ATO to improve condition in experimental arthritis as a therapeutic method. Methods:1 The expression of HMGB1 in the peripheral blood of patients with RA Samples of peripheral blood were obtained from 40 patients with RA including 20 active and 20 inactive ones, and 12 healthy volunteers as controls. All RA patients met the 1987 revised diagnostic criteria of the American College of Rheumatology without smoking habits, malignant tumors and infection diseases. ELISA assay was used to detect serum concentration of HMGB1. Reverse transcriptase-polymerse chain reaction (RT-PCR) was used to detect the expression of HMGB1 mRNA and flow cytometry (FCM) was used to measure the expression of Toll-like receptor 4 (TLR4) on peripheral blood mononuclear cell (PBMC). Meanwhile, the related clinical and lab indexes such as ESR, CRP, RF, X-ray phase changes, were recorded.2 The role of HMGB1 in pathological articular tissues of Collagen-induced arthritis ratsAll 30 male Wistar rats were divided randomly into four groups, two control groups (each including 6 rats) and two model groups (each including 9 rats). Type II collagen (CII) was prepared for establishing collagen-induced arthritis (CIA) models. Joint swelling was evaluated twice a week as arthritis index. Articular tissue samples were obtained from control and model rats at 28th and 49th day separately after first immunity injection. H.E staining was used to observe the pathological changes of rat joint structure. The expression of HMGB1 and proliferation cell nuclear antigen (PCNA) proteins in synovium and articular tissues were analyzed by immunohistochemistry. FCM was performed to detect the expression of HMGB1, PCNA, p-JAK2, p-STAT1/3, SOCS1/3 proteins of atricular tissues.3 The possible mechanism of HMGB1 on the proliferation of rat RSC-364 cell linesRSC-364 synoviocytes were inoculated in the dose of 1×104mL-1. After 24h, cells were cultured with standard medium as control group or with medium supplemented with 10μg.L-1 human recombinant protein HMGB1 as trial group in vitro. Then cells were collected in 6h, 12h, 24h respectively, as well as normal control group cells. RT-PCR was used to detect p-STAT1/3 mRNA expressions. FCM was performed to analyse the changes of cell cycle distribution, proliferation index (PI) and PCNA, Cyclin D1, CDK4, p-STAT1/3, SOCS1/3 proteins expressions. Immunocytochemical staining was adopted to detect PCNA, P21, p-STAT1/3, SOCS1/3 proteins expressions of RSC-364 synoviocytes in different groups.4 The inhibitory effects of ATO on the proliferation of synoviocytes of RA and possible mechanism(1) The effects of ATO on the proliferation of rat RSC-364 cells induced by HMGB1The RSC-364 cell lines were inoculated in the dose of 1×104mL-1. After 24h, the cells were divided into control group, solution group, 10μg.L-1 HMGB1 group and 10μg.L-1 HMGB1 +various concentration ATO-treated group(0, 5, 10, 20, 40, 80μmol.L-1 ATO). MTT assay was used to study inhibitory effects of HMGB1on the cell growth. According to MTT results, the cells were divided into five group again which including control group, HMGB1 group and HMGB1 + 10, 20, 40μmol.L-1 ATO -treated group. Cells were collected in 24h respectively. The changes of cell cycle distribution and CyclinD1, CDK4, p-STAT1 protein expressions were analysed by FCM. P21 and p-STAT1 protein expressions were detected by immunocytochemical staining. p-STAT1 mRNA expressions were detected by RT-PCR.(2) The inhibitory effects of ATO on the proliferation of synovitis in CIA ratsAll 42 male Wistar rats were divided randomly into five groups: I as control group (including 6 rats); II~V as model control group and 1.0, 2.0, 4.0 mg.kg-1.d-1ATO -treated group (each including 9 rats). ATO were administered for 28 days in CIA rats with a minimum arthritis index of 2. All rats were killed at 49th day. H.E staining was used to observe the pathological changes of rat joint structure. Immunohistochemical staining of synovial tissue sections was used to study local effects of ATO on expressions of HMGB1 and PCNA proteins. FCM was performed to detect the expression of HMGB1, PCNA, p-STAT1, SOCS1 proteins.Results:1 Expression of HMGB1 in the peripheral blood of patients with RA and its clinical significance(1) Expression of HMGB1 protein and mRNA in the peripheral blood of patients with RAThe expression of HMGB1 in active RA was higher than healthy control group and inactive RA [(10.60±1.29 vs 8.39±1.53, 8.10±2.38)ng.mL-1] (P<0.01). There was no statistically significant difference between inactive RA and healthy controls. The changes of HMGB1 mRNA detected by RT-PCR in PBMC were unanimous with those of HMGB1 proteins.(2) Change of TLR4 expression on PBMC in different groups CD14+TLR4+cells were higher in active RA than those in healthy controls and inactive RA(P<0.05)significantly. The expression of TLR4 on the lymphocytes had no statistical difference in different groups.(3) Level of HMGB1 protein in serum of RA was positively correlated with ESR, CRP, the numbers of tender joints and swollen joints. It was not correlated with age, course of diseases, RF and radiographic phase. 2 Effects of HMGB1 on proliferation of pathological articular tissues in CIA rats(1) Histological changes in articular tissues of CIA rats by microscopic examination hematoxylin stainingAbnormal histological feature was not visible in articular specimens from normal control rats. In contrast, specimens from model groups displayed a strong destruction with synoviocyte proliferation, inflammatory cells infiltration, pannus formation, even accompanied by cartilage or bone erosion. These changes became more and more severe with arthritis aggravation.(2) Expression of HMGB1 protein in articular tissuesImmunohistochemical staining revealed that HMGB1 protein expression primarily confined to the nuclear of synoviocytes, and little expression localized in cytoplasm in normal control specimens. Both nuclei and cytoplasm of cartilage cells were positive expression in normal rats. This pattern differed distinctly from model groups which was significantly increased in the nuclei, cytoplasm and extracellular milieu. Moreover, expression of HMGB1 was positive also in microvascular endothelial cells, some inflammatory cells, even lots of cells in bone marrow. FCM also revealed that HMGB1 protein expression increased as arthritis being serious.(3) The expression of PCNA protein in articular tissuesImmunohistochemical staining showed that the positive expression of PCNA was expressed in nuclei of synovicytes, no expression was in cartilage cells and microvascular endothelial cells. With arthritis being worse, the number of positive expression cells of PCNA increased. FCM also revealed that PCNA protein expression increased as arthritis being serious.(4) The expression of p-JAK2, p-STAT1/SOCS1, p-STAT3/SOCS3 proteins in articular tissuesFCM showed that the expression of p-JAK2 only enhanced in early model group(P<0.05). Compared with normal group, the expression of p-STAT1/SOCS1, p-STAT3/SOCS3 proteins were higher in model groups(P<0.01). But compared with early model group, the expression of SOCS1 decreased; p-STAT1,p-STAT3 / SOCS3 proteins were no statistical change in later model groups.(5) There were positive correlation between the expression of HMGB1, p-STAT1, p-STAT3 and PCNA protein(r =0.638, P=0.000;r =0.529, P=0.005;r =0.670, P=0.000), as well as HMGB1 and p-STAT1,p-STAT3(r =0.474, P=0.014;r =0.480, P=0.013). There was no correlation between the expression of HMGB1and p-JAK2(r =0.222, P=0.276).3 The possible mechanism of HMGB1 to promote the proliferation of rat RSC-364 cell lines(1) The effects of HMGB1 on the proliferation of rat RSC-364 cell lines By FCM, there was no significant effect on cell cycle distribution and proliferation index in 6h, but the number of cells in G0/G1 phase was down-regulated, which in G2/M phase were up-regulated in 12h and 24h. Thus PI increased in time-dependent.Immunocytochemical staining showed the number of positive cells of PCNA increased and that of p21 decreased in time-dependent. By FCM, HMGB1 could up-regulate the expression of PCNA and Cyclin D1 proteins in time-dependent, also increased the expression of CDK4, but not in time-dependent manner.(2) The effects of HMGB1 on STAT/SOCS signal pathway of rat RSC-364 cell linesRT-PCR, immunocytochemical staining and FCM indicated that HMGB1 could significantly up-regulate the expression of STAT1 mRNA and protein in time-dependent. SOCS1 protein expression was enhanced in 6h and 12h, but gradually became weaken in 24h by immunocytochemical staining and FCMThere were no marked changes in STAT3 mRNA and protein expressions by RT-PCR, immunocytochemical staining and FCM from 6h to 24h. Immunocytochemical staining and FCM showed SOCS3 only increased in 6h, but there was no statistical difference among various groups.(3) There were markedly positive correlation between the expression of Cyclin D1, CDK4, p-STAT1 and PCNA protein(r =0.717, P=0; r =0.514, P=0.001;r =0.621, P=0), as well as Cyclin D1, p-STAT1 and PI(r =0.815, P=0.001;r =0.424, P=0.010). There was no correlation between the expression of p-STAT3 and PCNA(r =0.244, P=0.151).4 The inhibitory effects of ATO on proliferation of synoviocytes in RA and possible mechanism(1) The effects of ATO on the proliferation of rat RSC-364 cells induced by HMGB1MTT showed that ATO significantly inhibited the proliferation of RSC-364 cells in a time- and dose-dependent fashion. After 24h, ATO, 80μmol.L-1, inhibited the proliferation of RSC-364 cells by 88.18 %. FCM indicated that the number of cells in G0/G1 phase was up-regulated, which in S and G2/M phase were down-regulated after 24h, and PI decreased in dose-dependent. It also showed that ATO could down-regulate cyclin D1 and CDK4 protein expressions in RSC-364 cells induced by HMGB1. But P21 protein expression was improved in dose-dependent. RT-PCR, FCM and immunocytochemical staining indicated that ATO could significantly inhibit the expression of STAT1 mRNA and protein in dose-dependent.(2) The inhibitory effects of ATO on the synovitis of CIA ratsRats treated with ATO had a significant alleviation of the severity of arthritis compared with the model rats and the whole condition was improved as well. Microscopic examination after hematoxylin staining revealed that there were less synovicytes proliferation, inflammatory cells infiltration, pannus formation and much less destruction of cartilage or bone in rats treated with ATO. Immunohistochemical staining revealed that HMGB1 protein expression markedly decreased in the nuclear, cytoplasm and extracellular matrix in dose-dependent, consistent with results obtained of PCNA protein detection. By FCM, the expression of HMGB1 and PCNA were also lowered in dose-dependent. Low- and mid-dose ATO treated rats after 4W, p-STAT1 protein expression decreased(P<0.01, P<0.05), but there was no significant change in high-dose group(4mg.kg-1.d-1) compared with model group. Different-dose ATO had no effects on the expression of SOCS1.(3) There were markedly positive correlation between the expression of p-STAT1 and Cyclin D1, CDK4 protein in RSC-364 cells induced by HMGB1 of various concentration ATO group(r =0.854 or r =0.681, P=0). p-STAT1, Cyclin D1,CDK 4 positively correlated with P(Ir =0.833,P=0;r =0.735, P=0; r =0.414, P=0.023) respectively. In low- and mid-dose ATO treated rats after 4W, there were positive correlation between the expression of HMGB1, p-STAT1 and PCNA protein(r =0.425,P=0.038;r =0.413, P=0.045); there was no correlation in high-dose group(r =0.334,P=0.071;r =0.252, P=0.179).From the above results, the following conclusions can be drawn:1 HMGB1, as a new cytokine, could be actively synthesized and released from PBMC of active RA patients. Once released, HMGB1 could bind to cell-surface receptors—TLR4 of monocytes and effectively promote inflammatory responses. The level of HMGB1 in serum is positively correlated with ESR, CRP, the numbers of tender joints and swollen joints, which indicats HMGB1 may be a new clinical activity observation index of RA.2 HMGB1 plays an important role to promote synovicytes proliferation and bone destruction of CIA rats by up-regulating the expression of STAT1/SOCS1 and STAT3/SOCS3 proteins.3 HMGB1 strongly promotes synovicytes proliferation by activating STAT1/SOCS1 protein expression, elevating cyclin D1 and CDK4 protein expressions and lowering P21 protein expression.4 ATO efficiently inhibits synoviocyte proliferation by blocking STAT1 signal transduction, down-regulating CyclinD1 and CDK4 expression and up-regulating P21 protein expression, further blocking cell cycle.In conclusion, the present experiment first shows that HMGB1 promotes synoviocyte proliferation by JAK/STAT/SOCS signal transduction and elevating expression of cell cycle proteins. This study also first reveles that the mechanism of ATO inhibiting synoviocyte proliferation is to down-regulate HMGB1 expression and block STAT1 signal transduction pathway. HMGB1 could be a considerable new therapeutic target molecule in RA. But ATO is a poisonous substance, whose exact mechanism in RA treatment needs further investigation.
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