Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune arthritis affecting mainly synovial joints, along with a variety of other tissues and organs. The pathology findings in RA include synovial cell edema and hyperplasia, abundant inflammatory cell infiltration, and fibroblast formation. This challenging disease may cause bone and joint destruction. The process produces an inflammatory response of the synovium secondary to the 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. RA can also involves the lungs and lead to interstitial lung disease. The interstitial lung disease occurs in about 28% of the RA patients, either at the beginning or during their disease in china. The presence of rheumatoid lung disease is associated with severe active disease and increased mortality compared to the general population. However, the etiology of rheumatoid lung disease at this time is unknown. A prior study suggested that its pathogenesis may be related to CD4+ CD25+ regulatory T cells (Treg) and Foxp3.1,2 Tripterygium glycosides (TPG) are also known as triptolide (TP, Fig. 1). Tripterygium wilfordii is a traditional medicinal plant and has been used in China for many years to treat inflammatory joint diseases, including RA. In this study, we intended to detail whether the administration of TPG is superior to methotrexate (MTX) in improving the paw swelling degree, arthritis index (AI), pulmonary function, and other previously noted factors in adjuvant arthritis (AA) rats.
2.1. Experimental animals
A total of 48 male Wistar rats, weighing from 200 g to 220 g, were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China), certificate No. SCXK(SU)2008-0004. The study protocol was approved by the Ethics Committee of First Affiliated Hospital, Anhui University of Chinese Medicine (Hefei, China). All animals were housed under specific-pathogen-free (SPF) conditions, and given free access to water and standard rat chow.
MTX, in 2.5-mg tablet form, was produced by Shanghai Traditional Chinese Medicine Co., Ltd., Xinyi Pharmaceutical Factory, Shanghai, China (batch: 20090104). TPG, in 10-mg tablet form, was produced by Shanghai Medical Hongqi Pharmaceutical Factory, Shanghai, China (batch: 20091102).
Freund's complete adjuvant (FCA) was obtained through Sigma-Aldrich, St. Louis, MO, USA, (lot: 098k8729). Elisa kit was purchased from R&D Systems, Minneapolis, MN, USA. This also included interleukin-10 (IL-10, lot: 341225), tumor necrosis factor alpha (TNF-α, lot: 341012), and Endothelin-1 (ET-1, lot: 340918). Regulatory T cells kit anti-mouse CD4-FITC was purchased through eBioscience, USA (lot: 11-0040, Clone: OX35), and anti-mouse CD25-PE was purchased from BioLegend, San Diego, CA, USA (lot: 202105, Clone: OX-39). The following were also used in this study: PCR Master Mix Kit and M-MuLV Reverse Kit (Fermentas, Canada, lots: K0171, K1622, respectively), Anti-mouse FOXP3 monoclonal antibody (Bioworld Technology, Louis Park, Minneapolis, USA, lot: BS2163), and β-actin pAb (Bioworld Technology, Louis Park, Minneapolis, USA, lot: AP0060).
2.4. Model copy, grouping, and administrations
A total of 48 male Wistar rats were randomly divided into four groups: normal control (NC), model control (MC), MTX, and TPG groups, with 12 rats in each group. Except for the rats of the NC group, the others were intracutaneously injected with 0.1 mL of FCA in the right hind limb. From 19 days after infection, the NC and MC groups were treated with physiological saline (1 mL/100 g per day);. the MTX and TPG groups were treated with MTX (1 mL/100 g per week) and TPG (1 mL/100 g per day), respectively for 30 days.
2.5. Determination of the experimental index
2.5.1. Paw swelling and AI
The paw volume was measured in the right hind limb of rats, and the swelling degree was calculated.3 Twelve days after inflammation, the joints were observed and recorded, and thereafter once in every 3 days. Calculation of AI,4 score from 0 to 5 according to the swelling and involved joints was made using the following scale: 0 point, no swelling; 1 point, swelling from little toe joint; 2 points, swelling from toe joints and foot; 3 points, swelling from ankle and below; and 4 points, swelling from all of ankle.
2.5.2. Paw swelling degree
E (%) = (Vt − Vn)/Vn × 100% (Vn and Vt represent the volume prior to and after modeling, respectively).
2.5.3. Lung index (LI)
LI = Lung wet weight (mg)/body weight (g) × 100%.
2.5.4. Morphology of lung tissues
Thirty days after infection, all the rats were killed. The lung tissues were remove, and then fixed in 4% paraformaldehyde solution for 8 hours, followed by dehydration, waxing, and embedding. The sliced lung tissues were stained with hematoxylin-eosin (HE). The extent of alveolitis integration was determined using Szapiel's method5: without alveolitis (-); mild alveolitis (+): lesions was confined to below 20% of the total lung tissue; moderate alveolitis (++): lesions reach about 4–50% of the total lung tissue; and severe alveolitis (+++): lesions were greater than 50% of the total lung tissue. These results scored 0, 1, 2, and 3, respectively.
2.5.5. Electron microscopy of the ultrastructure of lung tissues
Lung tissue was washed in phosphate buffer solution (PBS) and then cut into the samples sized 1 mm × 1 mm × 1 mm for each piece. It was fixed in glutaraldehyde (100 mL/L), immediately followed by 1% osmic acid. Samples then underwent alcohol dehydration and were embedded into an Epon812 resin using standard procedures. Ultrathin sections (50 nm) were stained with uranyl acetate and lead citrate. All specimens were observed under the electron microscope (magnification: 8000×).
2.5.6. Pulmonary function
Pulmonary function tests included: forced vital capacity (FVC), 1 second 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), and peak expiratory flow (PEF). The pulmonary function was tested by the animal lung function analysis system (Beijing Bei Lanbo Technology Co., China; model: AniRes 2003). The procedures for pulmonary function tests were done as follows6: the rats were anesthetized with chloral hydrate (10%, 0.35 mL/100 g) through intraperitoneal injection; they underwent tracheotomy endotracheal intubation; the head was kept lower; and the ventilator tube was connected to the mechanical ventilation device to test the pulmonary function. The computer measured each test automatically, quickly, and accurately.
2.5.7. Detection of CD4+ CD25+ Treg by flow cytometry
Fresh blood samples were collected from the rats, and whole blood samples were taken into K3-EDTA-containing tubes. Then, anti-mouse CD4-FITC (0.25 μg) and anti-mouse CD25-PE (1.0 μg) were added to each tube of blood (106 cells per tube) successively. The tubes were kept in dark for about 20–30 minutes at room temperature (20–25°C). We then added 1-mL red blood cell (RBC) lysate to the tube and incubated it for 15–25 minutes again in the dark. Thereafter, the tube contents were washed with PBA twice and centrifuged, wherein the supernatant was discarded. The expression of CD4+ CD25+ Treg was measured by flow cytometry (Beckman Coulter, Inc., USA, model: EPICS Altra).7
Total ribonucleic acid (RNA) was extracted by TRIzol (TAKARA BIO, Otsu, Shiga, Japan) from 5 × 105 cells or 100-mg lung tissues and quantified using a spectrophotometer (Eppendorf, Hamburg, Germany). Three micrograms of total RNA was reverse transcribed into cDNA using Murine Moloney Leukemia Virus Reverse Transcriptase (M-MLV RT; Promega, Madison, WI, USA). PCR was carried out according to the manufacturer's instructions.
The following sequence specific primers were used for PCR amplification. (1) The housekeeping gene GAPDH (GenBank accession: NM-017008): sense: 5′-TCC ACC ACC CTG TTG CTG TAG-3′, antisense: 5′-CCA CAG TCC ATG CCA TCA CT-3, and amplified fragment 258 bp. (2) 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′, and amplified fragment 450 bp.
The targeted DNA was amplified using PCR amplification (Biometra Inc., Jena, Germany, model: T-Gradient Thermoblock), and confirmed by electrophoresis (Amersham Biosciences, Piscataway, USA, model: EPS-301) and sequencing. PCR products were analyzed using gel image analysis system (Bio-RAD Inc., Hercules, USA, model: Gel Doc XR) after scanning the ethidium bromide-stained 1.5% agarose gel.
2.5.9. Western blotting
The protein concentration was determined using the Bradford dye-binding assay with bovine serum albumin as the standard. Cells were lysed in gel-loading buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol (DTT), 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, and 10% glycerol. Fifty micrograms of total protein was resolved by SDS polyacrylamide gel electrophoresis (PAGE) and electrically blotted onto a nitrocellulose membrane (NC, millipore, Boston, MA, USA, lot:HATF00010). 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 using the mouse anti-Foxp3 monoclonal antibody (1:500, Bioworld Technology) or the rabbit anti-beta-Actin polyclonal antibody (1:5000, Bioworld Technology) as the primary antibody, and goat anti-mouse or goat anti-rabbit IgG-conjugated horseradish peroxidase (Bioworld Technology, Louis Park, Minneapolis, USA) as the secondary antibody. The bands were visualized by using the enhanced chemiluminescence system (Pierce, Rockford, IL, USA).
2.5.10. Enzyme-linked immunosorbent assay (ELISA)
The levels of TNF-α, IL-10, and ET-1 in the supernatant of different groups were measured using commercially available ELISA kits (R&D System) according to the test protocols. Values were expressed as pg/mL. The ELISA standard curve was prepared using a serial dilution of TNF-α (IL-10 and ET-1) standard protein concentrations. Absorbance was measured at 450 nm using an absorbance microplate reader (BioTek, Vermont, USA, model: ELx800). The levels of recombinant TNF-α (IL-10 and ET-1) in the supernatant of the cell culture were calculated from the OD450 values according to the ELISA curve within commercial TNF-α (IL-10 and ET-1) standards.
Lung tissues were obtained during operation, snap frozen in liquid nitrogen, and stored at −80°C. All slides were processed for immunohistochemistry as previously described. In brief, the tissues were 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 minutes 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 the molecular variables. After washing in PBS, the horseradish peroxidase (HRP) polymer-linked secondary antibody was added for 60 minutes at room temperature. The sections were then visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. Sections for negative control were prepared using rabbit IgG1 or mouse IgG1 instead of the primary antibody under the same experimental conditions.
2.6. Statistical analyses
Continuous variables were expressed as mean ± standard deviation (SD). All samples were tested to ascertain if they followed a normal distribution. Data comparison among groups was carried out using analysis of variance (ANOVA). Comparison between the groups was carried out using the independent samples t test. SPSS version 13.0 software (International Business Machines Corporation, Armonk, New York City, USA) was used for data analyses. A p value <0.05 was considered significant.
3.1. Effects of TPG on paw swelling degree and AI
After the FCA injection, paw swelling developed 12 hours later, and there was no significant difference among three groups (MC, MTX, and TPG groups). For the NC group, however, there was no apparent paw swelling. When we treated those rats with MTX or TPG, the level of paw swelling and AI was significantly reduced when compared with the untreated MC group (Fig. 2A and B).
3.2. Effects of TPG on pulmonary function and lung tissues in AA rats
Forty-nine days after the FCA injection, the pulmonary function was found to change significantly. It showed that the parameters of the pulmonary function (FVC, FEV1/FVC, FEF25, FEF50) were significantly reduced in the MC group. However, when treated with MTX or TPG after 30 days, we found that the values of FVC, FEV1/FVC, FEF25, FEF50, FEF75, MMF, and PEF in the TPG and MTX groups were higher than those in the MC group. When compared with the MTX group, FVC, FEF25, FEF50, FEF75, MMF, and PEF were increased in the TPG group. Meanwhile, the morphology of lung tissues were improved, and these characteristics showed that the levels of lung coefficient and alveolitis integration had reduced (p < 0.05 or p < 0.001; Table 1).
3.3. Effects of TPG on type II cells of lung tissue in AA rats
NC group: The structure of alveolar type II cells was intact, there was no swelling in the mitochondria, and few lamellar bodies were emptied (Fig. 3A). MC group: Alveolar type II cells showed proliferation and swelling, the membrane is not complete, there was swelling in the mitochondria, the lamellar body was reduced and evacuated, and the mononuclear macrophage was infiltrated and accumulated (Fig. 3B). MTX group: The structure of alveolar type II cells was intact mostly, there was no swelling in the mitochondria, and some lamellar bodies were emptied, indicating macrophages granulocyte acid (Fig. 3C). TPG group: There were obvious neutrophils, monocytes, macrophages within and around the structure of alveolar type II cells. The most mitochondria were intact, while few lamellar bodies were emptied in the type II cells (Fig. 3D).
3.4. Effects of TPG on TNF-α, IL-10, and ET-1 in AA rats
Forty-nine days after the FCA injection, compared to the NC group, the concentrations of TNF-α, ET-1 in serum, and ET-1 in the lung tissue were significantly increased; IL-10 in serum had decreased noticeably in the MC group (p < 0.001). Thirty days after administration, there were a lot of differences between the treated and untreated groups. The concentrations of TNF-α and ET-1 in the TPG group were significantly lower than those in the MC group, and the concentrations of IL-10 were significantly higher than those in the MC group (p < 0.001). The differences in concentrations of TNF-α and ET-1 between the TPG and MTX groups were statistically significant. Compared to the MTX group, the levels of TNF-α (serum) and ET-1 (lung tissue) had significantly reduced in the TPG group (p < 0.001; Table 2).
3.5. Effects of TPG on Treg in peripheral blood and Foxp3 expression in lung tissue
In order to further analyze the reasons of pulmonary function injury and the effect of TPG on the treatment of pulmonary function in AA rats, we detected Treg expression by flow cytometry (FCM) in peripheral blood (Fig. 4A–E); immunohistochemistry (Fig. 5A–D), reverse transcription polymerase chain reaction (RT-PCR) (Fig. 6A–C), and western blot (Fig. 7A–C), which were used to detect the expression of Foxp3 in the lung tissue. Flow cytometry results showed that the expression of CD4+ CD25+ Treg had decreased significantly in the MC group (5.78 ± 0.85%) and increased in the TPG group (8.64 ± 1.88%). Immunohistochemistry results showed that the expression of Foxp3 in the lung tissue was concentrated in the nucleus and cytoplasm. There was low expression in the MC group, whereas high expression in the TPG group. RT-PCR and western blot results showed that the expressions of Foxp3 mRNA and protein had decreased significantly in the MC group. Again, the TPG group has higher expression than that in the MC group. TPG could improve the level of CD4+ CD25+ Treg and Foxp3, when compared with the control drugs.
Acute lung injury is a pulmonary characteristics of uncontrolled systemic inflammatory response syndrome (SIRS), in which the activation of numerous inflammatory effector cells, such as polymorph nuclear leukocytes and macrophages, triggers the excessive release of inflammatory mediators and cytokines. The mortality is still high in cases with severe acute lung injury.
After inducing AA in the rats, the expressions of paw swelling, AI, LI, FEV1/FVC%, alveolitis points, TNF-α, and ET-1 were found to increase significantly (p < 0.001), whereas the levels of FVC, FEF25, FEF50, FEF75, MMF, PEF, IL-10, CD4+ Treg, CD25+ Treg, CD4+ CD25+ Treg, and Foxp3 in the lung tissue were found to decrease significantly (p < 0.001). Our studies identified inflammatory reaction was not only involved in the joint but also in the lung after FCA injection. Pulmonary function change was indicated by the observation of pulmonary ventilation function disorders, particularly restrictive ventilatory disorder, which was accompanied by a small airway obstruction. It should be noticed that an imbalance between proinflammatory and anti-inflammatory cytokine existed, suggesting that the inflammation occurred in both joints and lungs. The abnormal expression of regulated T cells and Foxp3 showed that Treg was probably involved in RA pathogenesis of lung injury.8,9 It was shown that Treg reduced pulmonary function parameters, particularly for rats that did not receive any treatment. By contrast, MTX and TPG could improve the pulmonary function.
TP has been widely used in China to treat a broad spectrum of autoimmune and inflammatory diseases, including RA. The direct anti-inflammatory effects of TP were observed in the croton oil-induced ear swelling, carrageenan-induced paw edema, and air pouch model of carrageenan-stimulated acute inflammation in animals. Significantly lower volumes of the air pouch exudate, lower white blood cell counts with lower percentages of neutrophils, and lower concentrations of inflammatory mediators, including TNF-α, were found in the animals treated orally with the TP extract. Modern pharmacological studies show that TP exhibits anti-inflammatory and/or immunomodulatory properties in experimental animals. Pharmacological and clinical experiments also show that the major effective component of TP is alkaloids, euonymus. Alkaloids and euonymus can reduce capillary permeability, inhibit exudation of inflammatory cytokines or chemokines, inhibit or/and regulate the release of inflammatory mediators. These effects can reduce the lung tissue damage.10 The immunomodulatory effect of tripterygium ranged widely from the humoral immunity to cellular immunity. TP (monomer) has an inhibitory effect on inflammatory joint edema induced by carrageenan, croton oil, and FCA. A study also showed that TP could improve the recovery of alveolitis and fibrosis. TP can reduce the area of alveolitis and pulmonary fibrosis, and increase alveolar space.
In our results, compared with the AA rats, FVC, FEF25, FEF50, FEF75, MMF, PEF, IL-10, and the levels of CD4+ CD25+ Treg and Foxp3 had significantly increased in the TP-treated group, whereas paw swelling, AI, LI, FEV1/FVC%, TNF-α, and ET-1 decreased, indicating that AA rats the rats, arthritis induced by adjuvant, have swollen joints and damaged lung tissue. The results manifested that the pulmonary function decreases and there were disorders in the immunological parameters. It should be noted that an imbalance between proinflammatory and anti-inflammatory cytokine existed, suggesting that the inflammation occurred in both joints and lungs. We also found the treatment effect of tripterygium was more noticeable than that of MTX.9,10 The levels of CD4+ CD25+ Treg in peripheral blood of the AA rats had clearly decreased. It may be correlated to the breakdown of the autoimmune balance and the development of RA-induced lung injury, for a 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 to secrete IL-10 in the peripheral blood of AA rats, thus promoting the expression of Foxp3 in the lung tissue, then increasing the expression of anti-inflammatory cytokine and decreasing the expression of proinflammatory cytokines. Finally, the immunosuppression of CD4+ CD25+ Treg is exemplified exclusively, as our data implied. Our results showed that tripterygium can apparently improve the pulmonary function in AA rats, and the mechanism may be through the inhibition of the expression of TNF-α, ET-1, upregulation of the level of CD4+ CD25+ Treg, and promotion of the expression of IL-10 and Foxp3.11–15 In conclusion, it is suggested that tripterygium can upregulate CD4+ CD25+ Treg and Foxp3 expression, and this may explain why tripterygium is effective in the treatment of RA.16
The authors express their gratitude to Xiao-Yin Lv, Wen Hu, Ke Cheng, and Qing lin Li for their excellent technical assistance. This work was supported by grants from the National Natural Science Foundation Project (81173211), Medical key subjects Chinese paralysis disease in the national school construction projects [Traditional Chinese Medicine (2009) No. 30]; research projects of the Department of Science and Technology in Anhui Province (09020304046); Anhui Provincial Health Department of Traditional Chinese Medicine Projects (2009ZY05); Anhui Traditional Chinese Internal Applied Basic Research and Development at the Provincial Level Laboratory Construction Projects, 2009-2011, ID: Branch Article (2008) No. 150; and Anhui Traditional Chinese Medical Science and Technology Innovation Team Project (2010TD005).
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