Effects of the Sri Lankan medicinal plant, Salacia reticulata, in rheumatoid arthritis
© Springer-Verlag 2009
Received: 27 July 2009
Accepted: 5 August 2009
Published: 2 September 2009
Salacia reticulata is a native plant of Sri Lanka. In the traditional medicine of Sri Lanka and India, Salacia reticulata bark is considered orally effective in the treatment of rheumatism, gonorrhea, skin disease and diabetes. We have investigated, both in vivo and in vitro, whether the leaf of Salacia reticulata (SRL) can ameliorate collagen antibody-induced arthritis (CAIA) in mice as the rheumatoid arthritis (RA) model. The mice were fed a lard containing chow diet (AIN-93G) or the same diet containing 1% (w/w) SRL powder. All mice were bred for 23 days. On day 7 or 14 after LPS injection, mice were killed, and tissue and blood samples were collected. Histological analysis was performed, and serum levels of inflammatory mediators and the mRNA levels of inflammation-related genes and osteoclast-related genes were measured. SRL treatment ameliorated the rapid initial paw swelling, inflammatory cells infiltration, skeletal tissues damage, osteoclast activation and the mRNA levels for osteoclast-related genes compared with the CAIA mice. However, the serum and mRNA levels of inflammatory mediators did not differ between the CAIA mice and the SRL-treated mice. SRL might reduce the inflammatory cells induction and skeletal tissue degradation by CAIA by the regulating osteoclastogenesis.
Salacia reticulata (SR) is a plant native to Sri Lanka. In the traditional medicine, called “Ayurveda,” the roots and stems of SR have been used for prevention of rheumatism, gonorrhea, skin disease and diabetes. The effects of SR on diabetes have already been reported. For example, it is known that SR contains such unique compounds as salacinol, kotalanol and mangiferin . Salacinol and kotalanol were reported to inhibit α-glucosidase activity , while Mangiferin was reported to decrease the expression of fructose-1,6-bisphosphatase (FBP), a key enzyme involved in gluconeogenesis in the liver . However, no studies are available on the effects of SR on rheumatoid arthritis (RA) at biochemical and biophysical level. Accordingly, we investigated if the leaf of SR (SRL) could be considered a possible prophylactic/therapeutic agent in RA.
Rheumatoid arthritis is a chronic inflammatory disease, characterized by inflammatory cell infiltration, synovial hyperplasia and destruction of cartilage and bone. Although the etiology of RA is not yet fully understood, recent studies have suggested an involvement of autoantibody production, immune complex formation, inflammatory cell infiltration and tumor-like proliferation of synovium “pannus” in the pathogenesis of RA [6, 16, 20]. In addition, activation of osteoclasts in RA is associated with skeletal tissue destruction [2, 7, 18, 19]. RA treatment is currently mainly based on the administration of non-steroidal anti-inflammatory drugs (NSAID), corticosteroid preparation, disease-modifying anti-rheumatic drugs and molecularly targeted drugs such as indomethacin, prednisolone, methotrexate and infliximab . However, many drugs only provide symptomatic treatment, and there is no fundamental treatment for RA.
When RA develops, inflammatory mediators such as interleukin (IL) and prostaglandin are released into the whole joint and blood. Especially, tumor necrosis factor-alpha (TNF-α), IL-1β and IL-6 have often been used as indices of the severity of RA symptoms. Moreover, there have been attempts to administer immunomodulators such as or interferon (IFN)-γ and anti-pro-inflammatory cytokines such as IL-10 . However, no effective medical treatment has been established.
A granulation tissue called “pannus” is involved in RA. The presence of osteoclasts in pannus of skeletal tissue in RA has been confirmed . Pannus releases several pro-inflammatory mediators, matrix metalloproteinase (MMP), immune modulatory factors and bone metabolism-related factors . It was thought that osteoclastogenesis is enhanced by release of factors such as ILs, TNF-α and receptor activator of nuclear factor-kappa B ligand (RANKL) from pannus . Activated osteoclasts have been shown to stain for tartrate-resistant acid phosphatase . Moreover, the transcription factor c-fos is essential for osteoclast differentiation. Expression of RANKL and c-fos genes is elevated in RA . Here, we report a study on RA in CAIA mice treated with SR by histological and the genes expression analyses.
Leaves of Salacia reticulata (Hippocrateaceae) plants cultivated in Sri Lanka were purchased from Ayurveda Kothalahimbutu Association (Tokyo, Japan). The plant species was identified by Dr. G. A. S. Premakumara, at Herbal Technology Section, Industrial Technology Institute (Colombo, Sri Lanka). Fresh leaves were sun-dried. After the removal of damaged leaves, dried intact leaves were pulverized in a food mill (Panasonic Corporation, Osaka, Japan) and filtered though a 150-μm mesh sieve to obtain a powder.
Animals and treatments
Mouse hind paw fingers were collected and sequentially immersion-fixed in 10% paraformaldehyde/0.1 M phosphate buffer (PB) pH 7.4, 12.5% glucose/0.1 M PB pH 7.4, and 25% glucose/0.1 M PB pH 7.4, for each two days. After the toes were decalcified using 5% ethylenediaminetetraacetic acid (EDTA)/25% glucose/0.1 M PB pH 7.4 for 2 days. They were routinely processed and embedded in 4% carboxy methyl cellulose. Standard sagittal sections measuring 5 μm in thickness were prepared from the frozen and fixed toe using a cryostat CM3050S (LIECA, Tokyo, Japan). The joint spaces were histologically assessed on the sections, and histological scores (0–3) were assigned based on a previously reported scoring method . The sections were stained with Mayer’s hematoxylin and eosin (HE), toluidine blue (TB) pH 7.0 and tartrate-resistant acid phosphatase (TRAP). Scoring of sections based on inflammatory cell infiltration in the joint space and skeletal tissue destruction was performed using a scale of 0–3 (0 with in normal limits; 1 minimal; 2 mild; 3 severe) essentially as described previously . A leukocyte acid phosphatase kit (Sigma-Aldrich Corp, Tokyo, Japan) was used for TRAP activity staining. Osteoclasts (TRAP-positive multinucleate cells containing three or more nuclei) located at the bone surface were counted in each slide .
PGE2 and IL-1β analyses
Blood was drawn from the heart of 36 mice on days 7 and 14. Blood was put on ice for 30 min and centrifuged for 20 min at 13,370×g in a centrifuge separator. The first supernatant was collected and centrifuged for 20 min at 13,370×g. The second supernatant was used as serum sample. Serum PGE2 level was measured by enzyme immunoassay (EIA) using a PGE2 kit (Prostaglandin E2 Express EIA kit; Cayman Chemical, Ann Arbor, MI, USA). Serum IL-1β level was measured by enzyme-linked immunosorbent assay (ELISA) using a murine IL-1β kit (Quantikine; R&D Systems, Minneapolis, MN, USA).
RNA extraction and reverse transcription (RT)-PCR
Sequences of PCR primers used
Cathepsin K forward
Cathepsin K reverse
Results are expressed as mean with standard deviation (SD) of six mice. Statistical analysis was carried out with StatMate III Version3.18 (ATMS Co., Ltd. Tokyo, Japan). Distributed data were compared via paired t test.
First of all we investigated whether SRL affected paw swelling observed in CAIA arthritis. As expected, normal control mice developed no symptoms of arthritis (data not shown). In both CAIA mice and CAIA + SRL mice, the first visual sign of paw swelling appeared on day 1 after LPS injection, and the swelling gradually increased thereafter (Fig. 1). The swelling was quantified by gross observation and measurement of the foot thickness with slide calipers from day 0 through day 14 after LPS injection. The swelling score in CAIA mice was 2.3 ± 1.2 on day 1, and the maximum paw swelling score was 15.6 ± 0.8 on day 12. In CAIA + SRL mice, paw swelling score was of 2.3 ± 1.0 on day 1 rising to a maximum of 15.5 ± 0.8 on day 10. The statistical analysis indicated that SRL treatment affected the increase in paw swelling from day 2 through day 14, with the suppression being statistically significant on days 3, 4 and 6. On the other hand, SRL did not affect the paw swelling from day 7 to day 14.
RA is a systemic inflammatory autoimmune disease. The main symptoms of RA are inflammation, inflammatory cell infiltration, destruction of the joints cartilage and bone tissue. RA is usually treated with NSAIDs or corticosteroids. However, these treatments often cause severe adverse effects depending on the patient’s sensitivity or the drug dosage. For example, indomethacin is an NSAID that may adversely affect gastric tissue and function , while prednisolone is a corticosteroid that can adversely affect various organs .
Therapeutic drugs for RA include not only anti-inflammatory drugs but also anti-rheumatic and molecular-targeted drugs. However, these drugs also often have adverse effects. For example, methotrexate is an anti-rheumatic drug that has been reported to increase the risk of interstitial pneumonia . Infliximab, a molecular-targeted drug, was reported to have decreased its effect due to production of neutralizing antibody .
Traditional herbal therapy of RA takes advantage of using plants extracts such as SR, Uncaria tomentosa (UT) and Tripterygium wilfordii (TW) as an effective treatment alternative to established pharmacology. Moreover, herbal medicines such as SR, UT and TW have been reported to cause fewer adverse effects. UT is a significant anti-inflammatory activity and an antioxidant or free radical scavenger capacity . TW shows a significant immunomodulatory activity through the regulation of IL-1 and IL-2 secretion . The mechanism of SR in relation to RA has not been reported yet. However, it is known that mangiferin contained in SR, regulates several gene expression in mouse liver .
The present study contributes to clarify the mode of action of SRL in ameliorating the signs and symptoms of RA. CAIA mice is an established model for RA. In this model, DBA/1 J mice are treated with an anti-type II collagen antibody and LPS, and fed with SRL containing diet. Our study indicates that SRL ameliorates the swelling and redness due to inflammation in paws and the inflammatory cell infiltration in joints of CAIA mice. However, SRL treatment did not affect the serum concentrations of PGE2 or IL-1 and the levels of mRNA encoding TNF-α, IL-1, IL-6, IL-10 and IFN-γ in CAIA mice.
Osteoclastogenesis associated with RA was also beneficially affected by SRL. In this study, the treatment with SRL was associated with a decrease of the levels of mRNA encoding RANKL, MMP-2, MMP-3, cathepsin K and c-fos. Osteoclasts activation was also suppressed by SRL. We can hypothesize that SRL directly affects osteoclasts activity, and decrease the expression of mRNA encoding specific genes involved in osteoclastogenesis. Alternatively, it can be proposed that SRL decreases mRNA level for RANKL though stromal cells in the pannus and suppresses TRAP positive osteoclast numbers in the mice.
In conclusion, we investigated whether the symptoms of RA could be positively affected by SRL. SR contains various potentially active ingredients, other than mangiferin (MGF), such as triptotriterpenic acid B and (−)-epicatechin . Detailed studies of these active ingredients targeted to clarify the mechanism of SRL on RA are warranted.
We also demonstrated that the effect of SRL is not due to a generic quenching of the inflammatory response, but it suppressed the production of degradative enzymes and osteoclastogenesis. Thus, SRL appears to have potential as a functional food or herbal medicine for RA.
We thank Mr. L. Stiver, Quality Translation Co, Ltd, for useful English proofreading.
Conflict of interest statement
There is no author’s conflict of interest which must be stated here.
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