Celastrus aculeatusMerr. suppresses the induction and progression of autoimmune arthritis by modulating immune response to heat-shock protein 65

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder that affects about 2.1 million Americans [1‐5]. The drugs commonly in use for the treatment of RA include glucocorticoids (for example, cortisone and prednisone), non-steroidal anti-inflammatory drugs (NSAIDS; for example, ibuprofen and naproxen), disease-modifying anti-rheumatic drugs (DMARDs; for example, methotrexate (MTX) and leflunomide), and biological response modifiers (for example, tumor necrosis factor-αblocking agents) [6, 7]. However, besides their high cost, the prolonged use of many of these drugs is associated with severe adverse reactions and toxicity, including some risk of infections in subsets of patients being treated with biological response modifiers [6, 7]. As a result, alternative treatments based on natural plant products and herbal mixtures belonging to the realm of complementary and alternative medicine (CAM) are becoming increasingly popular in the US and other countries [2‐5, 8]. However, there is skepticism about CAM products in the minds of both the public as well as the scientific community, mostly because the mechanisms of action of many of these products are poorly defined, or not at all. Thus, there is a need to systematically study and define the mechanisms underlying the activity of CAM products that have been used for the treatment of rheumatic diseases in folk medicine around the world for centuries.

Celastrus aculeatus Merr. (Celastrus) [9‐15] is a Chinese medicine that belongs to the family Celastraceae and the genus Celastrus. The roots, stem, and leaves of Celastrus have been used in folk remedies in China for centuries to treat RA, osteoarthritis, lower back pain, and so on. Celastrus and some of its defined constituents possess anti-inflammatory, anti-oxidant, and anti-cancer properties [9‐15]. However, the mechanisms underlying the anti-arthritic activity of Celastrus have not been fully examined. Considering that RA is an autoimmune disease resulting from a dysregulated immune system [6, 7, 16, 17], it is imperative to examine the immunological basis of Celastrus or any other new potential anti-arthritic therapeutic agent under consideration.

Animal models of RA have contributed significantly both to our understanding of the pathogenesis of autoimmune arthritis as well as to the testing of new therapeutic agents of natural or synthetic origin [18‐21]. As a pre-requisite to unraveling the mechanisms underlying the beneficial effects of Celastrus in RA, we set out to first validate the anti-arthritic activity of Celastrus under controlled experimental conditions using a well established model of RA, adjuvant-induced arthritis (AA), which can be induced in the Lewis (LEW; RT.1^l) rat by subcutaneous (s.c.) immunization with heat-killed M. tuberculosis H37Ra (Mtb) [18, 22‐26]. Thereafter, we examined in LEW rats with AA the effects of Celastrus on the T cell and antibody responses to the disease-related antigen mycobacterial heat-shock protein 65 (Bhsp65) [18, 22‐25], which also is the target of T cell and antibody response in RA patients [27, 28].

Our results show that Celastrus can induce protection against arthritis both in the preventive as well as in the therapeutic setting, and that this beneficial anti-arthritic effect of Celastrus is attributable in part to modulation both of the immune response to the disease-related antigen Bhsp65 [22‐25] and one of the mediators of inflammation and tissue damage, nitric oxide (NO) [29, 30].

Our results show that Celastrus aculeatus Merr. (Celastrus) suppresses the induction of AA when fed to LEW rats prior to Mtb challenge, as well as down-modulates the progression of AA when administered to arthritic rats at the onset of the disease. The significant reduction in the severity of clinical AA following Celastrus feeding was further validated by limited histological changes in the joints. Furthermore, the level of suppression of ongoing AA by Celastrus was comparable to that of MTX, a standard anti-arthritic agent used for the treatment of arthritis. This attribute of Celastrus is an important one because many regimens based on synthetic or natural compounds can successfully prevent the induction of arthritis, but they often fail to control the course of the ongoing disease. In this regard, Celastrus is a promising anti-arthritic agent that could be further explored as a therapeutic modality in controlled pilot clinical trials on RA patients. As this is our first study on the effect of Celastrus on AA, we have used the unfractionated ethanol extract of the roots and stems to preserve as much of the natural proportion of different constituents in the mixture as possible. Accordingly, the dose of Celastrus fed to rats is apparently high. However, in subsequent follow up studies, we plan to use one or more of the purified components of the crude extract. It has been reported by others that various components of Celastrus possess anti-inflammatory and anti-tumor properties, and these include a variety of sesquiterpene esters (for example, celastrol, celaphanol, celasdin, orbiculin, esters with the β-dihydroagarofuran skeleton) and flavonoids (for example, epiafzelechin) [9‐15]. Some of the reported pathways inhibited by these components are mediated by nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX) [12‐15, 35].

We observed that the suppression of clinical arthritis in Celastrus-fed LEW rats was associated with significant changes in the immune response to Bhsp65. Furthermore, both the cell-mediated and the antibody responses to Bhsp65 were affected. AA is driven by pro-inflammatory cytokines (IFN-γ and tumor necrosis factor-α); in this context, Celastrus treatment facilitated the secretion of the anti-inflammatory cytokine IL-10 over the pro-inflammatory cytokine IFN-γ, resulting in the overall skewing (immune deviation) of the cytokine response to an anti-inflammatory type [26]. This relative deviation of the cytokine response, caused either by decreased Th1-type cytokines and/or by enhanced Th2-type cytokines leading to the regression of an autoimmune disease, is reminiscent of other compounds of synthetic (for example, peptides of antigenic proteins or cytokines) [25, 36, 37] or natural origin [38, 39] that can successfully control disease in animal models of arthritis.

Celastrus feeding to LEW rats immunized with Mtb led to enhanced production of antibodies to Bhsp65 compared to the control water-fed rats. Thus, a decrease in inflammatory arthritis in LEW rats was associated with an increase in the anti-Bhsp65 antibody response. This inverse association is supported by previous work by others [25] and us [31] demonstrating that anti-Bhsp65 antibodies produced during the course of AA are disease-protective rather than being pathogenic in nature. Unlike in other animal models of RA in which antibodies are arthritogenic [40, 41], in the AA model certain subsets of anti-Bhsp65 antibodies generated either during the course of AA [25, 31] or following AA-protective tolerization with Bhsp65 [42] contribute to disease regulation. It has been proposed that the protective effect of antibodies in AA is probably mediated by the induction of IL-10 production from mononuclear cells [25]. In this regard, our finding of a Celastrus-induced deviation of the cytokine response of arthritic LEW rats towards IL-10 correlates very well with our observation of enhanced anti-Bhsp65 antibody response in Celastrus-treated rats, and the observed immune deviation towards IL-10 might be attributable, in part, to the increased antibody response to Bhsp65. We further suggest that the anti-Bhsp65 antibodies might also contribute to the protection against AA by modulating antigen processing and presentation [43] and, thereby, facilitating the induction of the immune response to one or more of the regulatory T cell determinants within Bhsp65 previously identified by others [23, 25, 36] and us [24]. Thus, changes in both the cell-mediated and the antibody responses to Bhsp65 following Celastrus feeding might cooperate to down-regulate the severity of AA in the LEW rat.

In addition to the disease-regulating changes in the immune response to Bhsp65, the beneficial effect of Celastrus in AA was also related to inhibition of the production of a well known mediator of inflammation, namely NO [20, 29, 30, 35]. We observed antigen specificity in the production of NO; Bhsp65-restimulated LNCs of Mtb-immunized water-fed (control) LEW rats produced significantly higher levels of NO than those restimulated by the control antigen, HEL. Furthermore, Celastrus treatment significantly reduced the levels of NO in both LNC culture supernatant and sera of Mtb-immunized rats. Taken together, these results document not only a direct association between the levels of NO and the severity of AA, but also provide insight into the in vivo anti-inflammatory activity of Celastrus. These results of Celastrus-mediated suppression of NO production in vivo are further corroborated by reports by other investigators showing a similar effect of Celastrus in vitro using macrophage cell lines (for example, RAW cells) [12, 15]. Furthermore, it has been reported that oral feeding of B6 mice with the ethyl acetate extract of Tripterygium wilfordii Hook F (TWHF) or its active component, triptolide, led to the inhibition of both NO production and iNOS mRNA expression by macrophages [44], and this decrease in NO production was implicated in mediating the anti-inflammatory effects of TWHF. One of the mechanisms by which Celastrus leads to decreased NO production might involve NF-κB, which controls the expression of genes encoding inducible enzymes, such as iNOS and COX, which in turn generate some of the critical mediators of the inflammatory response [14, 15, 35]. In fact, some of the active components of Celastrus have been shown to serve as inhibitors of the NF-κB pathway (for example, celastrol and celaphanol A) [12, 15] and the COX pathway (for example, epiafzelechin) [13]. In addition, NF-κB activity is inversely related to that of the heat-shock response as the induction of heat-shock proteins is associated with a decrease in NF-κB activity [14]. Celastrol can lead to the induction of heat-shock protein gene expression by activation of heat-shock factor-1 [14], and the enhanced response to self hsp65 can, in turn, contribute to protection against AA [23, 32]. Thus, by regulating the activity of NF-κB, Celastrus apparently influences multiple inter-connected pathways that participate in the regulation of autoimmune arthritis.

Our results suggest that the ethanol extract of Celastrus as well as its individual components should be explored further for the treatment of RA through double-blind, placebo-controlled preclinical and clinical trials in RA patients following the strategy employed successfully by other investigators for translational research on TWHF [8, 45]. There is a compelling need to fully examine multiple natural products such as TWHF and Celastrus for their potential as anti-arthritic agents because all RA patients may not respond equally well to any single herbal medicine owing to differences in body constitution and genetics, and each natural plant product may have unique compatibility with the standard mainstream medications when taken together. The availability of several different natural plant products having anti-arthritic activity would enlarge the scope of the use of CAM modalities for the treatment of RA in conjunction with conventionally used drugs.