Objective To observe the effect of osteopontin (OPN) and integrin αtvβ3 in collageninduced arthritis (CIA) and the possible mechanism of Tripterygium wilfordii polyglycoside (TWP) in the treatment of rheumatoid arthritis (RA). Methods CIA rats model were developed and were randomly divided into the experimental group and the TWP group.And tissue samples were obtained 4 weeks later.Then the expressions of OPN and integrin αvβ3 in the synovium,synovium fluid and serum of each group were determined by immunohistochemical stain and ELISA.Variance analysis was used for data analysis.Results The concentrations of OPN of the normal controls,experimental group and the TWP group in the serum were (5.7±2.9), (7.8±6.2), (5.0±1.9) ng/ml respectively and there were significant differences between these 3 groups (F=6.74,P=0.016).The concentration of OPN (measured by mean grey value) in the synovium and cartilage of the three groups were 229±15,81±15,93±13 and 211±17,91±19,100±15 and there were significant differences between the three groups (F=52.48,P＜0.01; F=18.98,P=0.01).The concentrations of protein αvβ3 (measured by mean grey value) in the synovium and cartilage were 235±16,91±16,131±14 and 198±10,99±15,113±14,respectively and there were significant differences between the three groups (F=23.03,P=0.002; F=12.04,P=0.008).The expressions of OPN and integrin αvβ3 in the synovium,synovium fluid and serum of the experimental group were markedly higher than that of the controls.The expressions of OPN and integrin αvβ3 in the synovium,synovium fluid and serum of the treatment group were obviously lower than the experimental group.Conclusion OPN and integrin αtvβ3 are involved in the hyperplasia of the synovium,cartilage and bone destruction in CIA rats.The underlying molecular mechanism that TWP is effective in treating synovitis and bone destruction of RA is possibly related to down-regulation of the expression of OPN protein and integrin αvβ3.

The effect of triptolide (TP) on the expression of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) was explored in rat adjuvant induced arthritis (AA). AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate (MTX) at the onset (day 9) of arthritis. On the peak of arthritis (day 24), the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells (PBMC) was detected. TNF-alpha and IL-1beta levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group (P < 0.01); The expression levels of RANKL in the synovium (P < 0.01) and bone (P < 0.05), and OPG level in synovium (P < 0.05) were lower in TP group than in AA group (P < 0.05). In TP group, the expression levels of RANKL mRNA and TNF-alpha, IL-1beta in PBMC were lower than in AA group (all P < 0.01). It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

The relationship between tumour necrosis factor-alpha (TNF-alpha) gene polymorphism and inhibitory effects of triptolide on TNF-alpha production from peripheral blood mononuclear cells (PBMC) of healthy humans was investigated. Genomic DNA from 41 healthy people was typed for TNF-alpha--308 polymorphism by allele-specific polymorphism chain reaction (AS-PCR). The TNF-alpha concentration in the supernatant was measured by ELISA. The results showed that the production of TNF-alpha from TNF-alpha--308 non-G/G genotype PBMC was higher than that from TNF-alpha--308 G/G genotype PBMC after stimulated by LPS. Triptolide could lower the production of TNF-alpha from G/ G genotype PBMC, but had no effect on the level of TNF-alpha from non-G/G genotype PBMC. It was concluded that TNF-alpha gene polymorphism was related to the TNF-alpha production from triptolide-inhibited PBMC culture in healthy humans.

AIM: To explore whether the inhibitory effect of triptolide on IL - 1β production by PBMC is asso ciated with IL - 1β gene polymorphisms. METHODS: IL - 1β gene polymorphism was analyzed in 31 healthy volunteers. From genomic DNA, the C - T polymorphism at IL - 1 β - 511 was typed by PCR - RFLP. Meanwhile the IL - 1 β was also measured in the supernatants of the cultured and stimulated peripheral blood mononuclear cells (PBMC) by ELISA. RE SULTS: After LPS stimulation in PBMC cultures of healthy subjects, the secretion levels of IL - 1 β in 9 volunteers who carried IL - 1β -511 T/T genotype were higher than in volunteers who are not T/T genotype (P ＜0.05). Triptolide sup pressed the production of IL - 1β significantly in LPS - treated human PBMC carried C/C and C/T genotype ( P ＜ 0.05 ), but this significant inhibitory effect of triptolide was not seen in T/T genotype ( P ＞ 0.05 ). CONCLUSION: The gene polymorphism at IL - 1β - 511 was related to the production of IL - 1β, and the inhibitory effect of triptolide on the produc tion of IL - 1β was different in C/C, C/T, T/T genotype of IL - 1β -511, which may be one of the reasons for the phe nomenon that people respond differently to triptolide.

BACKGROUND: Common threewingnut root has the functions of anti-inflammation and immune inhibition, etc., and it has been used at present to treat various autoimmune diseases including rheumatoid arthritis.. Common threewingnut root has complex components, and triptolide is acknowledged as one of the important effective components of common threewingnut root.OBJECTIVE: To establish rat models of type Ⅱ collagen induced arthritis, and observe the effects of triptolide on the contents of interleukin-6,interleukin-10 and tumor necrosis factor alpha (TNF-α) in peripheral serum and synovial fluid.DESIGN: A randomized control animal experiment.SETTING: Department of Integrated Traditional and Western Medicine,Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiments were carried out in the Tongji Hospital from November 2004 to July 2005. Fifty healthy male Wistar rats of clean degree were purchased from the experimental animal center of Tongji Medical College, Huazhong University of Science and Technology [qualification number of animal [scxk(E)2004-2007]. Triptolide (nobatch number because of temporary production) was bought from Fujian Institute of Medical Sciences, and the purity was above 98.5%.METHODS: ① Ten of the 50 rats were randomly selected as the normal controls, and the others were made into models. Type Ⅱ collagen emulsion was injected intradermally at five points along the back and tail of the rats,0.05 mL for each point, and injected intradermally at two points after 15 days. The rats in the normal control group were treated with saline in the same way. The effects of the model establishment were evaluated according to the scoring standards of arthritis index at 30 days after the first immunity, and the rats scored 6 points or above were taken as successful models and enrolled in the experiments. Twenty successful rat models were randomly divided into arthritis model group (n=10) and triptolide treated group (n=10). ② Triptolide (100 mg/L)was dispensed into parenteral solution with propylene glycol (0.05 in volume fraction), and then intramuscularly injected into hindlimb of rats in the triptolide treated group (0.04 mL/100 g), once every three days for 30 days. The rats in the normal control group were given isovolume saline, and those in the arthritis model group were treated with isovolume propylene glycol (0.05 in volume fraction). ③ The materials were removed at 30 days after administration. The contents of interleukin-6, interleukin-10 and TNF-α in peripheral serum and synovial fluid were detected with enzyme-linked immunosorbant assay(ELISA).MAIN OUTCOME MEASURES: The effects of triptolide on contents of TNF-α, interleukin-6 and interleukin-10 in peripheral serum and synovial fluid were observed.RESULTS: Fifty male Wistar rats of clean degree were selected, 10 were used as normal controls, and 20 of the other 40 rats were successfully made isto models and enrolled in the analysis of results. ① The TNF-α contents in peripheral serum and synovial fluid were the highest in the arthritis model group, and obviously decreased after treatment of triptolide [(35.09±8.82), (15.35±3.56) ng/L; (44.17±8.94), (22.54±4.76) ng/L; P＜ 0.01], which were similar to those in the normal control group (P ＞ 0.05).② The contents of interleukin-6 in peripheral serum and synovial fluid were the highest in the arthritis model group, and were obviously decreased after treatment of triptolide [(76.58 ±6.81), (42.45 ±5.72) rig/L;(88.69±10.56), (48.67±5.97) ng/L; P ＜ 0.01], but did not recover to the levels in the normal control group (P ＜ 0.05). ③ The contents of interleukin-10 in peripheral serum and synovial fluid were the lowest in the arthritis model group, and obviously increased after treatment of triptolide[(17.53±2.07), (21.23±2.91) ng/L; (10.59±2.96), (14.74±1.85) ng/L; P＜ 0.01], which were similar to those in the normal control group (P ＞ 0.05).CONCLUSION: Triptolide can treat arthritis by modulating the contents of cytokines.

Objective:To study the relation between triptolide inhibit peripheral blood mononuclear cell to secret TNF-? and tumour necrosis factor-? gene polymorphism.Methods:Genomic DNA from 41 healthy people was typed for TNF-? -308 polymorphism by allele-specific polymorphism chain reaction(AS-PCR); the TNF-? concentration in the supernatant was measured by ELISA.Results:The TNF-? production of TNF-? -308 non-G/G genotype in LPS-inhibited peripheral blood mononuclear cell(PBMC) culture was more than that of G/G genotype; Compared with TNF-? -308 non-G/G genotype peripheral blood mononuclear cell(PBMC), triptolide can lower the production of TNF-? in G/G genotype peripheral blood mononuclear cell(PBMC).Conclusion:Tumour necrosis factor ?(TNF-?) gene polymorphism might influence the TNF-? secretion of peripheral blood mononuclear cell(PBMC) in healthy humans. We speculate that it may be relative to the different curative effect of Tripterygium Wilfordii Hook.F.(TWHF) to RA patients.

AIM:To explore whether the inhibitory effect of triptolide on IL-1? production by PBMC is associated with IL-1? gene polymorphisms.METHODS:IL-1? gene polymorphism was analyzed in 31 healthy volunteers.From genomic DNA,the C-T polymorphism at IL-1?-511 was typed by PCR-RFLP.Meanwhile the IL-1? was also measured in the supernatants of the cultured and stimulated peripheral blood mononuclear cells(PBMC)by ELISA.RESULTS:After LPS stimulation in PBMC cultures of healthy subjects,the secretion levels of IL-1? in 9 volunteers who carried IL-1?-511 T/T genotype were higher than in volunteers who are not T/T genotype(P0.05).CONCLUSION:The gene polymorphism at IL-1?-511 was related to the production of IL-1?,and the inhibitory effect of triptolide on the production of IL-1? was different in C/C,C/T,T/T genotype of IL-1?-511,which may be one of the reasons for the phenomenon that people respond differently to triptolide.

AIM: To determine whether triptolide induce apoptosis of synovial cells in collagen - induced arthritis(CIA) in rats. METHODS: The male Wistar rats were used to make CIA models by immunized with Bovine collagen Ⅱ ( BC Ⅱ )in Freund's complete adjuvant (FCA). A total of 20 CIA rats were randomly divided into 2 groups, triptolide group (10 rats) and CIA control group (10 rats). Triptolide group were administered with triptolide at 40 μg/kg body weight intramuscularly every three days. CIA control group andanother 10 age - matched normal rats were given normal saline instead. The rats were sacrificed on the 31st day after the triptolide administration. The pieces of synovium of the rat knee joints were harvested. The synovium was examined by HE staining and electron microscope. The apoptosis was tested by TUNEL and flow cytometer. RESULTS: The earlier phase of apoptotic synoviocytes were observed under the electron microscope. The flow cytometry showed that the percentage of the apoptotic cells was (3.98 ± 1.16)% in the triptolide group, (1.83 ± 0.82)% in the CIA control group, and (0.87 ±0.24)% in the normal group (P＜0.01: triptolide vs control group). While the percentage of the cells in DNA synthesis phase was (3.3± 1.2)% in the triptolide goup, (8.0± 1.4)% in the CIA control group, and (3.4 ± 0.7)% in the normal group.There is significantly different in the apoptosis changes between the triptolide group and the CIA control group ( P ＜ 0.01: triptolide vs CIA control group). The TUNEL labeling demonstrated that the percentage of the apoptotic cells was (4.5 ± 1.0)% in the triptolide group, (2.2 ± 1.0) % in the CIA control group, and ( 1.0 ± 0.4) % in the normal group. The difference of apoptotic rate between the triptolide group and the CIA control group is significant ( P ＜ 0.01). CONCLUSION: This study demonstrates that triptolide can induce apoptosis in CIA rats, which may be one of the mechanisms that triptolide treats the rheumatoid arthritis.

ObjectiveTo study the regulatory effect of Tripterygium wilfprdii polyglycoside (TWP) on the expression of EGFR and ErbB-2 induced arthritis rats.The effect of TWP on arthritis was also explored.MethodsAfter the model of CIA rats were established,the expression of EGFR and ErbB-2 in the synovium and articular cartilage were tested by immunohistochemical stain and real time PCR.ANOVA was used for statistical analysis.ResultsThe protein and mRNA expression of EGFR and ErbB-2 in the synovium (EGFR 0.268±0.059,ErbB-2 0.25±0.04,EGFR mRNA:14.2±0.55,ErbB-2 mRNA 23.46±3.64) and articular cartilage (EGFR 0.193±0.018,ErbB-2 0.217±0.033,EGFR mRNA:4.16±0.50,ErbB-2 mRNA 9.23±0.66) of the model group were significantly higher than those of the control group(P＜0.01).After being treated with TWP and MTX,the protein and mRNA expression of the EGFR and ErbB-2 decreased markedly (P＜0.01).Conclusion EGFR and ErbB-2 may play an important role in the pathogenesis of arthritis development.The molecular mechanism that TWP can treat synovitis and bone destruction of RA is related to the inhibition of EGFR and ErbB-2.

Objective To study the regulatory effect of triptolide(TP)on the angiogenesis of coll-agen-induced arthritis(CIA) rats.The effect of TP on arthritis is also explored.Methods After the model of CIA was established,the articular volume was measured and the synovium was examined with regular HE stainand the inflammation and pathological changes were evaluated.In addition,the vascular endothelial growth factor (VEGF),basic fibroblast growth factor (bFGF)and endostatin protein expressions in synovium and serum were tested.The micro-vessel density (MVD) of synovium was also measured by caulating CD34 level.Results The expressions of VEGF,bFGF and MVD in CIA rats'synovium and serum were evidently higher than the control group(X2=65.3,31.6,q=9.2,P＜O.01,respectively),while the expression of endostatin showed no statistical difference with controls (X2=0.8,P＞0.05).After treated with TP,the expressions of VEGF,bFGFand MVD decreased markedly(X2=19.7,6.0,q=6.5,P＜O.01,respectively),but the pmtein expression of endostatin significantly increased (X2=3.9,P＜O.05).However,only the expression of endostatin increased significantly after treated with MTX (X2=17.9,P＜0.01).Conclusion Imbalance in growth factors prnduction may play an important role in the process of arthritis development.Re-establishing the balance of growth factors maybe one of the mechanisms of TP in the treatment of arthritis.