Rheumatoid arthritis (RA) is an autoimmune arthritis affecting joints mainly, chronic inflammatory, along with many other tissues and organs. RA is an inflammatory disorder that principally attacks synovial joints. The process produces an inflammatory response of the synovitis secondary to hyperplasia of synovial cells, excess synovial fluid, and the development of pannus in the synovium. The pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs. Owing to the lung tissue has redundant connective tissue and the close relation with blood vessels, and also has a link-intensive cycle system, the lung is one of the primary target organs. Interstitial lung disease (ILD) is the most common manifestation of rheumatoid lung disease [
Tripterygium glycosides tablet comprises triptolide which is a traditional medicinal plant that has been used in China for many years to treat inflammatory conditions including RA. In this study we will be testing tripterygium glycosides tablet that is superior to methotrexate in improving the paw swelling degree, arthritis index (AI), pulmonary function, and so on.
Rats were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). All animals were housed under specific pathogen free (SPF) conditions and given free access to water and standard rat chow.
Tripterygium glycosides tablet (TPT), 10 mg per piece, was produced by the Shanghai Medical Hongqi Pharmaceutical Factory, batch number NO. 20110819. Methotrexate (MTX), 2.5 mg per piece, was produced by the Shanghai Traditional Chinese Medicine Co., Ltd. Xinyi Pharmaceutical Factory, batch number NO. 20111004.
Elisa kit was purchased from R & D company, USA. Interleukin-10 (IL-10, Lot number 341225), Tumor Necrosis Factor alpha (TNF- Freund’s complete adjuvant (FCA, Sigma, USA, Lot number NO. 098k8729). Regulatory T cells kit: anti-mouse CD4-FITC (eBioscience, USA, Lot: 11-0040, Clone NO. OX35), anti-mouse CD25-PE (BioLegend, USA, Lot number: 202105, Clone NO. OX-39). Anti-mouse Foxp3 monoclonal antibody (Santa Cruz, USA, Lot: SC-130666). PCR Master Mix Kit and M-Mulv Reverse Kit (Fermentas, Canada, Lot: K0171, K1622).
Microplate reader was produced by Bio-TEK Corporation, USA (Model: ELX800). PCR amplification was produced by Biometra Inc., Germany (Model: T1-Thermoblock, T-Gradient Thermoblock). AniRes 2003 animal lung function analysis system was produced from Beijing Bei Lanbo Technology Co., Ltd., China. Electrophoresis was produced by Amersham Corporation, USA (Model: EPS-301).
The rats were randomly divided into four groups, the norma control (NC) group, model control (MC), methotrexate (MTX), and tripterygium glycosides tablet (TPT) group, 12 rats in each group. Except for the rats of NC group, the others were intracutaneously injected with 0.1 mL of Freund’s complete adjuvant in the right hindlimb. Administration from 19th after inflammation, NC group and MC group were treated with physiological saline (1 mL/100 g per day). MTX group and TPT group were treated with MTX, 1 mL/100 g per week, and TPT, 1 mL/100 g per day.
Paw swelling was measured in the right hindlimb of rats and calculated swelling degree [
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30th after administration, remove the lungs tissue, and tissue was fixed in 4% paraformaldehyde 8 hr. Dehydration, transparent, dipping wax, embedding, slicing and HE staining successively. According to Szapiel method to determine the extent of alveolitis [
Pulmonary function parameters have forced vital capacity (FVC), 1 SEC. average expiratory flow (FEV1/FVC %), 25% vital capacity of the peak expiratory flow (FEF25), 50% vital capacity of the peak expiratory flow (FEF50), 75% vital capacity of the peak expiratory flow (FEF75), maximum mid-expiratory flow (MMF), peak expiratory flow (PEF). Process of the operation [
Whole blood samples were taken into K3-EDTA containing tubes and added to each tube with blood (106 cells per tube), anti-mouse CD4-FITC (0.25 ug), anti-mouse CD25-PE (1.0 ug) successively. Keep them in dark place about 20–30 minutes at room temperature (20–25°C). Add 1 mL RBC lysate to tube and incubate for 15–25 minutes in dark again. Washing with PBA twice, centrifugation, the supernatant was given up. Detection of the expression of CD4+ CD25+ Treg was by flow cytometry [
RNA was extracted by TRIZOL (TaKaRa Co., Japan) from 100 mg lung tissue and quantified using a spectrophotometer (Eppendorf Co., German). Three micrograms of total RNA were reverse transcribed into cDNA using Murine Moloney Leukemia virus (M-MLV) reverse transcriptase (Promega, USA). PCR was carried out according to the manufacturer’s instructions. The house keeping gene GAPDH (GenBank accession: NM-017008): sense: 5′-TCC ACC ACC CTG TTG CTG TAG-3′, and antisense: 5′-CCA CAG TCC ATG CCA TCA CT-3′. amplified fragment 258 bp. The Foxp3 gene (GenBank accession: NM-001108250): sense: 5′-GCA AAC GGA GTC TGC AAG TG-3′, and antisense: 5′-GCA GGA GCT CTT GTC CAC TGA-3′, amplified fragment 450 bp. The targeted DNA amplified specifically was confirmed by electrophoresis and sequencing. PCR products were analyzed using Gel Works software after scanning the ethidium bromide-stained 1.5% agarose gel.
Protein was lysed in gel-loading buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, and 10% glycerol. Fifty micrograms of total protein was resolved by SDS polyacrylamide gel electrophoresis and electrically blotted onto a nitrocellulose membrane. The filters were blocked with phosphate buffered saline (PBS) containing 15% nonfat milks. Detection of Foxp3 or beta Actin was carried out by western blot analysis with the mouse anti-Foxp3 monoclonal antibody (1 : 500) or the rabbit anti-beta Actin polyclonal antibody (1 : 5000) as the primary antibody, and goat anti-mouse or goat anti-rabbit IgG-conjugated horseradish peroxidase as secondary antibody. The bands were visualized by using the enhanced chemiluminescence system.
Levels of TNF-
Lung tissue was dewaxed and rehydrated followed by antigen retrieval through microwaving in 2 mM EDTA (pH 9.0) for Foxp3 antigen. Sections were blocked with 5% bovine serum albumin (diluted in PBS) for 30 min and then incubated with each primary antibody in a moist chamber at 4°C overnight. Parallel sections from the same tissue block were used for the staining of all molecular variables. After washing in PBS, HRP polymer-linked secondary antibody was added for 60 min at room temperature. The sections were then visualized with DAB and counterstained with hematoxylin. Sections for the negative control were prepared using rabbit IgG1 or mouse IgG1 instead of primary antibody under the same experimental conditions.
Continuous variables are the mean ± standard deviation. All samples were tested to ascertain if they followed a normal distribution. Data comparison among groups was carried out using ANOVA. Comparison between groups was carried out using the independent samples
Before inflammation, there was no obvious difference between paw swelling degree and arthritis index in each group. Before administration, the level of paw swelling degree and arthritis index of MC group, MTX group, TPT group were significantly higher than that in the NC group (
After administration, compared to the NC group, the level of paw swelling degree and arthritis index of MC group were increased significantly (
Comparisons of pulmonary function, pulmonary coefficient, and alveolitis in rats (
Index | Group | |||
---|---|---|---|---|
NC | MC | MTX | TPT | |
Pulmonary function | ||||
FVC (mL) | 6.09 ± 2.01 | 5.07 ± 0.27b | 5.25 ± 0.25b | 5.89 ± 0.30ce |
FEF25 (mL/s) | 45.2 ± 6.21 | 30.7 ± 2.81b | 36.8 ± 3.67bc | 42.2 ± 4.71cd |
FEF50 (mL/s) | 39.4 ± 6.84 | 27.0 ± 6.07b | 30.3 ± 4.94b | 40.2 ± 4.36ce |
FEF75 (mL/s) | 37.7 ± 5.87 | 37.7 ± 5.87 | 24.1 ± 5.80b | 34.4 ± 10.5cd |
MMF (mL/s) | 39.2 ± 5.72 | 39.2 ± 5.72 | 31.7 ± 4.36b | 37.1 ± 4.62cd |
PEF (mL/s) | 39.1 ± 4.87 | 39.1 ± 4.87 | 33.1 ± 3.12b | 37.8 ± 4.33cd |
Pulmonary coefficient | 2.61 ± 0.08 | 0.36 ± 0.36b | 2.83 ± 0.11bc | 2.74 ± 0.12bcd |
Alveolitis points (point) | 0.37 ± 0.74 | 2.75 ± 0.70b | 1.87 ± 0.35bc | 1.62 ± 0.51bcd |
Note: compared with NC group, a
30th after administration, pulmonary function parameters such as FEF50, FEF25, FVC, FEF75, MMF, and PEF were significantly decreased, then FEV1/FVC, LI and score of alveolitis were increased in MC group. FVC, FEF25, FEF50, FEF75, MMF, and PEF were decreased and the score of alveolitis was increased with treatment of TPT.
Comparisons of regulatory T cells, cytokines, and Foxp3 in rats (
Index | Group | ||||
---|---|---|---|---|---|
Case | NC | MC | MTX | TPT | |
CD4+CD25+Treg (%) | 10 | 8.64 ± 1.88 | 5.78 ± 0.85b | 5.92 ± 1.36a | 7.96 ± 2.31ce |
| |||||
Cytokine in serum | |||||
TNF- |
10 | 32.1 ± 7.40 | 80.5 ± 10.7b | 59.8 ± 20.3bc | 53.2 ± 19.7bd |
IL-10 (pg/mL) | 10 | 106.6 ± 14.7 | 51.5 ± 20.1b | 74.2 ± 23.6bc | 90.2 ± 21.8de |
ET-1 (pg/mL) | 10 | 19.0 ± 8.11 | 48.3 ± 16.8b | 32.2 ± 10.7bc | 30.8 ± 12.6ac |
| |||||
Cytokine in lung tissue | |||||
ET-1 (pg/mL) | 10 | 8.03 ± 5.76 | 27.2 ± 15.7a | 20.2 ± 11.9a | 11.7 ± 9.33cf |
Note: compared with NC group. a
30th after administration, compared to the NC group, the concentrations of TNF-
We detected Treg expression by flow cytometry in peripheral blood (Figures
CD4+ CD25+ Treg changes of peripheral blood in rats (%) (a) MC group; (b) NC group; (c) TPT group; (d) MTX group.
Comparisons of regulatory T cells in each group. Note: Compared with NC group,
Comparisons of Foxp3 mRNA expression in each group. Note: compared with NC group,
Comparisons of Foxp3 protein expression in each group. Note: compared with NC group,
We detected that paw swelling, AI, alveolitis points, TNF
Modern pharmacological studies show that triptolide has anti-inflammatory and immunomodulatory active ingredients effects in experimental animals. Pharmacological and clinical experiments also show that the major effective component of tripterygium is alkaloids, may directly lead to the results that reduce capillary permeability, inhibit infiltration of inflammatory exudation, inhibit or counter modulate various types of inflammatory mediators as well as the anticoagulant, antiembolism, and reduce the damage on lung tissue [
The levels of CD4+ CD25+ Treg decreased in peripheral blood of the AA rats obviously. It may be because of that the diminished CD4+ CD25+ Treg level is correlated to the breakdown of the autoimmune balance and the development of rheumatoid arthritis-induced lung injury, for low level of CD4+ CD25+ Treg cannot sufficiently convert CD4+ CD25− T cells into regulatory cells through immune induction. After the intervention of tripterygium, CD4+ CD25+ Treg was upregulated. We proposed that as a consequence, the CD4+ CD25− T cells can be converted into Treg that secretes IL-10 in peripheral blood of the AA rats, thus promoting the expression of Foxp3 in lung tissue, then making the high level expression of anti-inflammation cytokine and the low level expression of proinflammatory cytokines. Finally the immunosuppression activity of CD4+ CD25+ Treg is exemplified exclusively, as our data implied.
Our results showed that tripterygium can obviously improve pulmonary function in AA rats, and the mechanism may function through inhibiting the expression of TNF-
This work is supported by The National Natural Science Foundation Project (Grant no. 81173211); National Administration of Traditional Scientific Research Special Foundation of China (2004-2005 LP27); Eleventh Five-Year key Program of Anhui Province (07010300204); Anhui Science and Technology Key Research Program (no. 06023068); Anhui Traditional Chinese Medicine Applied Basic Research and Development of Provincial Experimental Room Program ((2008) 150); and Anhui Education Department Natural Science Key Research Program (KJ2008A165).