Effect of Aralia chinensis on serum TNF-alpha, IL-4 and IL-10 level in rats with adjuvant-induced arthritis
Abstract
Our study investigated the effect of Aralia chinensis on serum tumor necrosis factor-alpha (TNF-alpha), Interleukins (IL-4, IL-10) in rats with adjuvant-induced arthritis. Adjuvant-induced arthritis rats had more slowly increased body weight, dramatically increased TNF-alpha level but reduced IL-4, IL-10 levels compared with control group. A. chinensis, Tripterygium wilfordii and control group had significantly increased body weight, IL-4, IL-10, and markedly reduced arthritis index and TNF-alpha level compared with the adjuvant-induced arthritis group. A. chinensis and T. wilfordii group had significantly increased IL-4 and IL-10 levels and markedly reduced TNF-alpha level compared with control group. A. chinensis could significantly increase the IL-4 level compared with the control group and it can balance the cytokines in arthritis rats, which attributes to the improvement of adjuvant induced arthritis.
Introduction
Rheumatoid arthritis is an autoimmune disease characterized by symmetrical arthritis in multiple joints, chronic inflammation in the synovium and progressive destruction of joints. It may cause damage to multiple organs including heart, lung and kidney. Late stage may result in ankylosis, joint deformity and severe joint dysfunction, which significantly influence the health and quality of life. In the present study, the effect of Aralia chinensis L on the serum tumor necrosis factor-alpha (TNF-alpha), Interleukins (IL-4, IL-10) was investigated in rats with adjuvant arthritis aiming to explore the pathogenesis of rheumatoid arthritis.
Materials and Methods
Animals
Male SD rats (specific pathogen free) weighing 200 ± 20 g and aged 8-10 weeks were purchased from the Experimental Animal Center of Xi'an Jiaotong University. Animals were given ad libitum access to food and water.
Main reagent, drugs and instrument
chinensis (Shenlong Hospital of Traditional Chinese Medicine), liquid extract 1 g/mL), tripterygium wilfordii (Huangshi Feiyun Pharmaceutical Co. Ltd; Z33020422), incomplete Freund's adjuvant and detection kits for TNF-a, IL-4 and IL-10 (Sigma-ALDRICH) were used in this study. Vernier calliper (TESA, Swiss), GTZA precision balance (GTZA, Jiangsu, China), 721 grating spectrophotometer (Suzhou Qile Electronic Technology Co., Ltd), and refrigerated centrifuge (Xiangyi Centrifuge Co., Ltd) were used in the present study.
Preparation of the animal model
SD rats were allowed to accommodate to the environment for 2 weeks and then divided into 2 groups randomly. In the control group, 8 rats were included and received no treatment. Remaining rats received subcutaneous injection of incomplete Freund's adjuvant at the right hind paw (0.1 mL). From the second day, edema of the paw was present. A second injection was done at day 8. At 14 days after first injection, adjuvant arthritis animal model was induced.
Grouping and treatment
In the control group, rats received no treatment. The remaining rats were divided randomly into 3 groups (n = 8 per group): Adjuvant arthritis group, T. wilfordii group and A. chinensis extract group. In the T. wilfordii group, rats were intragastrically treated with T. wilfordii at 6.0 mg/kg (3 mL) once daily; in the A. chinensis extract group, rats were intragastrically treated with A. chinensis extract at 1 g/mL (3 mL) once daily. Treatment was done at 15 days after first injection of incomplete Freund's adjuvant and continued for 28 days. In the control and adjuvant arthritis groups, rats received intragastrically treatment with normal saline of equal volume once daily for 28 days.
Observations and detection
a) Observation of general condition: Before and after preparation of animal model and treatment, the spirit, hair, appetite and activity of these animals were observed, and the body weight, thickness of voix pedis and arthritis index were detected; b) Detection of biochemical parameters: At 28 days after treatment, blood was collected from the femoral artery and placed in a dry tube followed by centrifugation at 3,000 rpm for 8 min. The supernatant was obtained. The serum levels of TNF-a, IL-4 and IL-10 were detected according to the manufacturer's instructions.
Statistical analysis
Quantitative data were expressed as mean ± SD and statistical analysis was done with SPSS version 15.0. One-way analysis of variance was employed for comparisons among groups and "t"-test was used for comparisons between two groups.
Results
At 4 days after first injection, rats developed lassitude, less activities, loss of appetite and lameness, and significant edema was found in the right hind paw. At 9 days after first injection, the hair lost luster, inflammatory nodules were present sequentially at the tail, and congestion of nasal mucosa, mental fatigue, loss of appetite and edema of left hind paw were also observed. At 12 days after treatment, the mental status and appetite in the control group, A. chinensis extract group and T. wilfordii group were improved gradually, but remained unchanged in the adjuvant arthritis group. None died during the experiment.
After introduction of adjuvant arthritis, the body weight gain was slower than that in the control group. After treatment with A. chinensis extract or T. wilfordii, the body weight gain was significantly higher than that in the adjuvant arthritis group (p<0.05). There was no marked difference in the body weight gain among A. chinensis extract group, T. wilfordii group and control group (p>0.05; Table I).
Table I: Body weight of rats in different groups before and after treatment
| Group | n | Before(g) | After(g) |
|---|---|---|---|
| Control | 8 | 255.5 ± 10.5 | 298.6 ± 9.3c |
| Adjuvant arthritis | 8 | 255.5 ± 11.4 | 264.0 ± 11.3a |
| T. wilfordii | 8 | 250.1 ± 11.1 | 277.1 ± 13.1ab |
| A. chinensis | 8 | 252.8 ± 3.6 | 279.0 ± 9.7ab |
| ap<0.01 vs control group; bp<0.05; cp<0.0 vs AA group | |||
There was no significant difference in the arthritis index among groups before treatment (p>0.05). After treatment, the arthritis index in the A. chinensis extract group and T. wilfordii group was markedly reduced (p<0.05). No significant difference was observed between T. wilfordii group and A. chinensis extract group (p>0.05; Table II).
Table II: Arthritis index in different groups before and after treatment
| Group | n | Before | After |
|---|---|---|---|
| Adjuvant arthritis | 8 | 7.9 ± 0.8 | 10.0 ± 1.5 |
| T. wilfordii | 8 | 8.6 ± 1.3 | 8.3 ± 1.6a |
| A. chinensis | 8 | 7.8 ± 1.4 | 7.5 ± 0.8a |
| Data are mean ± SD; ap<0.05vs AA group | |||
When compared with control group, the TNF-alpha level increased significantly but the IL-4 and IL-10 reduced markedly (p<0.05 or <0.01). In the T. wilfordii group and A. chinensis extract group, the TNF-alpha level reduced and the IL-4 and IL-10 increased (p<0.01). Moreover, the increased in IL-4 in the AT group was higher than that in the T. wilfordii group (p<0.01; Table III).
Table III: Changes in serum levels of TNF-alpha, IL-4 and IL-10 after treatment
| Group | n | TNF-alpha (pg/mL) | IL-4 (pg/mL) | IL-10 (pg/mL) |
|---|---|---|---|---|
| Control | 8 | 83.1 ± 11.5d | 54.4 ± 4.5c | 25.1 ± 1.7d |
| Adjuvant arthritis | 8 | 268.8 ± 22.9b | 49.3 ± 0.7a | 15.8 ± 1.9b |
| T. wilfordii | 8 | 159.5 ± 12.0bd | 65.1 ± 2.1bd | 31.1 ± 1.5bd |
| A. chinensis | 8 | 178.5 ± 11.2bd | 76.1 ± 2.8bde | 29.2 ± 1.7bd |
| Data are mean ± SD; Note: ap<0.05, bp<0.01 vs control group; cp<0.05, dp<0.0 vs Adjuvant arthritis group; ep<0.01 vs T. wilfordii group | ||||
Discussion
The etiology and pathogenesis of rheumatoid arthritis is still poorly understood and has been found to be related to multiple factors. Some studies support that rheumatoid arthritis is an inflammatory disease due to the regulatory dysfunction of immune system. The inflammatory response in rheumatoid arthritis is very complex and has involvement of multiple genes (Springer, 1994; Murdoch and Finn, 2000), and a lot of cytokines have been found to participate in the regulation of complicated immune network. Among numerous cytokines in rheumatoid arthritis, TNF-alpha and IL are found to play important roles in the pathology of rheumatoid arthritis (Yu, 2007). TNF-alpha has potent biological activity. Studies have confirmed that TNF-alpha can induce the inflammatory response and mediate the killing of targeted cells (virus infected cells) and cancer cells, may cause fever, metabolic disorder and dyscrasia and has found to be related to the damage to articular cartilage in rheumatoid arthritis. IL-4 and IL-10 are two anti-inflammatory factors. IL-4 can promote the migration of macrophages into the inflammatory sites; IL-4 can inhibit the secretion and activities of TNF-alpha, IL-1 and IL-6 to antagonize the effect of IL-1; IL-4 may promote the proliferation of Th2 cells to suppress the Th1 mediated immune response (Wang and Chen, 1994). IL-10 can down-regulate Th1 mediated immune response and increase the IL-10/TNF-alpha producing regulatory T lymphocytes, which may inhibit the inflammation and the damage to the bone and cartilage and reduce the production of TNF-alpha and IL-1 (Gonzalez-Rey et al., 2007). There is evidence showing that IL-4 and IL-10 can synergistically inhibit the secretion of proteases that can degrade the cartilages, and reverse the function of decomposition factors, both of which may inhibit the damage to cartilages (Palmer et al., 2002). In healthy subjects, there is a balance between anti-inflammatory cytokines and pro-inflammatory cytokines. The disruption of this balance may induce the occurrence of rheumatoid arthritis.
wilfordii is a common drug used in the clinical treatment of rheumatoid arthritis having favorable efficacy. However, T. wilfordii may cause reduction of white blood cells and platelets and influence the reproductive system leading to menstrual disorder, compromised sperm activity and reduction of sperms. Thus, in the present study, A. chinensis extract with relatively less side effects was used to treat rheumatoid arthritis and the therapeutic efficacy was evaluated and compared with that of T. wilfordii.
Rheumatoid arthritis is one of arthralgias in Traditional Chinese Medicine. The Inner Canon of Huangdi proposed that the combination of wind-cold-damp results in arthralgias. Thus, to dispel wind, disperse cold and eliminate damp have been used in the treatment of arthralgias. A. chinensis is a plant of araliaceae belonging to aralia genus. A. chinensis is mainly distributed in the mountain area of central and west China. The type specimen is mainly obtained from Taibai in Shaanxi Province. Non-governmentally, A. chinensis is also known as one of seven Taibaishan herbs in Qinling of Shaanxi. It is rich in China and the skin of root and stem, the stem, the leaf and fruit of A. chinesis can be used as drugs (Wang et al., 1998). AT has the characteristics of dispelling wind and eliminating damp, attenuating heat and relieving pain, activating blood circulation and dissipating blood stasis, inducing diuresis and reducing edema, invigorating qi for tranquilization and liver-preservation (Wang et al., 1997). Its bioeffects are consistent with the principles of treatment based on syndrome differentiation in Traditional Chinese Medicine. Our results demonstrated that A. chinensis could recover the energy, increase the body weight and attenuate the inflammatory response in rats with rheumatoid arthritis, exerting therapeutic effect on rheumatoid arthritis. This effect on rheumatoid arthritis may be related to the down-regulation of TNF-alpha and up-regulation of IL-4 and IL-10 as well as the recovery of balance between anti-inflammatory cytokines and pro-inflammatory cytokines.
References
Gonzalez-Rey E, Chorny A, Varela N, O'Valle F, Delgado M. Therapeutic effect of urocortin on collagen-induced arthritis by down-regulation of inflammatory and Th1 responses and induction of regulatory T cells. Arthritis Rheum. 2007; 56: 531-43.
Murdoch C, Finn A. Chemokine receptors and their role in inflammation and infectious diseases. Blood 2000; 95: 3032-43.
Palmer G, Guerne PA, Mezin F, Maret M, Guicheux J, Goldring MB, Gabay C. Production of interleukin-1 receptor antagonist by human articular chondrocytes. Arthritis Res. 2002; 4: 226-31.
Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 1994; 76: 301-14.
Wang TG, Chen WF. Advance in biological effect of interleukin-4. Foreign Med Sci (Section Immunol). 1994; 1: 13-16.
Wang Y, Zhu Z, Wang C, Yang J. [Determination of oleanolic acid and total saponins in Aralia L]. [Article in Chinese] Zhongguo Zhong Yao Za Zhi. 1998; 23: 518-21, 574.
Wang Z, Tang H, Su Z, Tan M, Hu J, Li C, Yi Y. [Resources survey of medicinal species from genus Aralia]. [Article in Chinese] Zhongguo Zhong Yao Za Zhi. 1997; 22: 3-6, 60.
Yu SS. Clinical significance of measurements of changes of serum IL-2, SIL-2R and TNF-α levels after treatment in patients with rheumatoid arthritis. J Radioimmunol. 2007; 20: 231-33.
