Role of GDC-0449 in a rat model of liver fibrosis induced by carbon tetrachloride combined with 2-acetylaminofluorene / 临床肝胆病杂志

Objective To investigate the intervention effect of GDC-0449, a hedgehog signaling pathway inhibitor, on rats with liver fibrosis induced by carbon tetrachloride (CCl 4 ) combined with 2-acetylaminofluorene (2-AAF). Methods A total of 18 female Fisher344 rats were randomly divided into normal group, CCl 4 /2-AAF group, and GDC-0449 group, with 6 rats in each group. The rats in the CCl 4 /2-AAF group and the GDC-0449 group were given subcutaneously injected 30% CCl 4 -olive oil solution at a dose of 2 mL/kg twice a week for 6 weeks to induce liver fibrosis; since week 7, in addition to the injection of CCl 4 -olive oil solution, the rats in these two groups were given 2-AAF (100 mg/kg/d) by gavage, and the rats in the GDC-0449 group were given GDC-0449 (25 mg/kg/d) by gavage, while those in the normal group were given an equal volume of olive oil solution by injection and normal saline by gavage. All rats were sacrificed at the end of week 9, and related samples were collected. HE staining and sirius red (SR) staining were used to observe the changes in liver histopathology and collagen deposition, and the semi-quantitative analysis of SR-positive area and Ishak score were used to evaluate fibrosis degree; the alkaline hydrolysis method was used to measure the level of hydroxyproline (Hyp) in liver tissue; immunohistochemistry, Western blot, and qRT-PCR were used to measure the expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col-Ⅰ), type Ⅳ collagen (Col-Ⅳ), cytokeratin 19 (CK19), cytokeratin 7 (CK7), the epithelial cell adhesion molecule Epcam, and the hedgehog signaling pathway in liver tissue; double immunofluorescence staining was used to observe the colocalization of CK19 and the oval cell marker OV6. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the CCl 4 /2-AAF group had marked inflammatory cell aggregation and collagen deposition in liver tissue, with the formation of a pseudolobular structure, as well as significant increases in Hyp level and collagen positive area ratio in liver tissue ( P < 0.05), Ishak score ( P < 0.05), and the expression of α-SMA, Col-Ⅰ, Col-Ⅳ, Epcam, CK19, CK7, the transmembrane transporter Smoothened (Smo), Hedgehog ligand Desert Hedgehog (Dhh), the Indian Hedgehog membrane-binding receptor Patched (Ptch2), and glioma-related oncogenes Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed that CK19-positive cells also expressed OV6 in the liver tissue of rats in the CCl 4 /2-AAF group, with a significant increase compared with the normal group. Compared with the CCl 4 /2-AAF group, the GDC-0449 group had significant reductions in inflammatory cell aggregation and collagen deposition in liver tissue, Hyp level and collagen positive area ratio in liver tissue ( P < 0.05), Ishak score ( P < 0.05), and the expression of α-SMA, Epcam, CK19, CK7, Smo, Ptch2, Gli1, Gli2, and Gli3 (all P < 0.05); double immunofluorescence staining showed a significant reduction in the number of cells with co-expression of OV6 and CK19 in liver tissue. Conclusion The Hedgehog signaling pathway inhibitor GDC-0449 can significantly inhibit the progression of liver fibrosis induced by CCl 4 /2-AAF in rats, possibly by inhibiting hepatic stellate cell activation, collagen deposition, activation and proliferation of hepatic progenitor cells, and differentiation of hepatic progenitor cells into biliary epithelial cells.

Survival analysis for patients with T4bsquamous cell carcinoma of thoracic esophagus receiving concurrent chemoradiotherapy / 中华放射肿瘤学杂志

Objective We aimed to analyze the clinical efficacy and treatment-related complications in patients with T4besophageal squamous cell carcinoma (SCC) who received concurrent CRT,and to explore the potential prognostic factors related to survival. Methods Between 2010 and 2015,143 patients with T4b esophageal SCC treated with CRT were analyzed, including 71% patients with trachea and/or bronchus invasion and 44% patients with aorta and/or large vessel invasion. The median radiation dose was 60 Gy ( range, 44-68 Gy ) with conventional fractionation, including 69 patients ( 48%) treated with three-dimensional conformal radiotherapy and 74 patients ( 52%) treated with intensity-modulated radiotherapy. All patients received concurrent platinum-based chemotherapy during radiotherapy. Kaplan-Meier method was used to analyze the survival,the log-rank test was used to examine group differences,and the Cox regression model was used for multivariate analysis. Results The median overall survival ( OS) time for the whole cohort was 12. 2 months. The 2-and 3-year OS rates were 34% and 29%,respectively. A total of 51 patients experienced ≥2 severe non-hematological complications,including 42 esophageal fistula,6 pneumonia,and 3 esophageal hemorrhage. Patients with severe complications showed significantly worse survival than those without complications (6. 9 months vs.20. 4 months,P＜0. 01).Multivariate analysis revealed that TNM stage and severe complications were independent prognostic factors for OS. Conclusions Patients with T4b esophageal SCC who received CRT showed satisfactory survival but with high risk of severe complications. Therefore,prevention and treatment of severe complications is the key to improve efficacy.

Screening and Identification of Antigenic Proteins from the Hard Tick Dermacentor silvarum (Acari: Ixodidae)

In order to explore tick proteins as potential targets for further developing vaccine against ticks, the total proteins of unfed female Dermacentor silvarum were screened with anti-D. silvarum serum produced from rabbits. The results of western blot showed that 3 antigenic proteins of about 100, 68, and 52 kDa were detected by polyclonal antibodies, which means that they probably have immunogenicity. Then, unfed female tick proteins were separated by 12% SDS-PAGE, and target proteins (100, 68, and 52 kDa) were cut and analyzed by LC-MS/MS, respectively. The comparative results of peptide sequences showed that they might be vitellogenin (Vg), heat shock protein 60 (Hsp60), and fructose-1, 6-bisphosphate aldolase (FBA), respectively. These data will lay the foundation for the further validation of antigenic proteins to prevent infestation and diseases transmitted by D. silvarum.

Morphological Characteristics of Normal and Gynandromorphic Hyalomma asiaticum Schulze and Schlottke, 1930

Gynandromorphic ticks are extremely rare, and often attract parasitologists' attention. During our examination of tick specimens, an engorged gynandromorph of Hyalomma asiaticum was noticed. This is the first record of gynandromorphic ticks from China. In this study, several important morphological structures of normal and gynandromorphic H. asiaticum were analyzed. Comparing to the normal H. asiaticum, the gynandromorphic specimen was a typical bipartite protogynander. Its right side showed normal female characteristics, whereas the left side had normal male traits. Different from other gynandromorphic ticks containing 1 anus, this tick reported here had 2 complete anuses, and the anus of the male part had a single adanal plate.

Gene Cloning, Expression and Immunogenicity of the Protective Antigen Subolesin in Dermacentor silvarum

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.

Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an alpha-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

Regulatory effect of tripterygium wilfordii polyglycoside on expression of epidermal growth factor receptor family in collagen induced arthritis rats / 中华风湿病学杂志

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.

Prediction of response of collagen-induced arthritis rats to methotrexate: An (1)H-NMR-based urine metabolomic analysis / 华中科技大学学报（医学）（英德文版）

Over one half the patients with rheumatoid arthritis (RA) are being treated with methotrexate (MTX). Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis (CIA) and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M (1)H-nuclear magnetic resonance ((1)H-NMR) for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats (n=20) was different from that from the non-responsive rats (n=11). Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis (PLS-DA) (R(2)=0.812, Q(2)=0.604). In targeted profiling, 13 endogenous metabolites (uric acid, taurine, histidine, methionine, glycine, etc.) were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that (1)H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

Prediction of response of collagen-induced arthritis rats to methotrexate: an (1)H-NMR-based urine metabolomic analysis / 华中科技大学学报（医学）（英德文版）

Over one half the patients with rheumatoid arthritis (RA) are being treated with methotrexate (MTX). Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis (CIA) and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M (1)H-nuclear magnetic resonance ((1)H-NMR) for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats (n=20) was different from that from the non-responsive rats (n=11). Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis (PLS-DA) (R(2)=0.812, Q(2)=0.604). In targeted profiling, 13 endogenous metabolites (uric acid, taurine, histidine, methionine, glycine, etc.) were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that (1)H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.

The effect of Tripterygium wilfordii polyglycoside on the expression of osteopontin and integrin αvβ3 in collagen-induced arthritis in rats / 中华风湿病学杂志

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.

Association between discoidin domain receptor 2 and cartilage damage / 中华风湿病学杂志

Objective To detect the changes in the expression of discoidin domain receptor 2(DDR2)and matrix metalloproteinase (MMP)-13 in different stages of cartilage and synovium damage of osteoarthritis rats.The relation between DDR2 and the degree of cartilage damage was explored.Methods Modified papain knee joint injection approach was adopted to establish animal model of OA.The expression and distribution of protein of DDR2 and MMP-13 were checked in articular cartilage and synovium at different stages of OA.Results The expressions of DDR2 in articular cartilage and synovium of experimental groups were different from those of the normal group (P＜0.01).They were higher in cartilage than those in the corresponding synovium.The expressions of MMP-13 demonstrated the same characteristics with those of DDR2,r=0.93(P＜0.01).Conclusions The important role of DDR2-MMP-13 in cartilage damage has been proven in the pathogenic process of OA.The upregulated expressions of DDR2 in articular cartilage and synovium have a detrimental effect on cartilage degeneration.

Regulatory effect of triptolide on angiogenesis in synovium of collagen-induced arthritis rats / 中华风湿病学杂志

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.

Inhibitory effect of triptolide on production of IL -1β from PBMC is associated with IL -1β gene polymorphism / 中国病理生理杂志

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.

Relationship between single nucleotide polymorphism in TNF-alpha gene promoter region and inhibitory effects of triptolide on TNF-alpha production in peripheral blood mononuclear cells of healthy humans / 华中科技大学学报（医学）（英德文版）

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.

Effect of triptolide on expression of receptor activator of nuclear factor-kappaB ligand in rat adjuvant induced arthritis / 华中科技大学学报（医学）（英德文版）

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.

Triptolide induces apoptosis of rat synoviocytes in collagen- induced arthritis / 中国病理生理杂志

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.

Interventional effects of triptolide on the levels of cytokines in peripheral serum and articular cavity of rats with collagen induced arthritis / 中国组织工程研究

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.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis / 华中科技大学学报（医学）（英德文版）

The effect of triptolide (TP) on the expression of receptor activator of nuclear factor-κB 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-α and IL-1β 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-α, IL-1β 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.

Relationship between Single Nucleotide Polymorphism in TNF-α Gene Promoter Region and Inhibitory Effects of Triptolide on TNF-α Production in Peripheral Blood Mononuclear Cells of Healthy Humans / 华中科技大学学报（医学）（英德文版）

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