New herbal composition (OA-F2) protects cartilage degeneration in a rat model of collagenase induced osteoarthritis

Osteoarthritis (OA) is a complex multifactorial disease of whole joint [1]. Pathological loss of cartilage reflects the imbalance between catabolic and anabolic mechanism of cartilage remodeling, which is influenced by oxidative and inflammatory changes in the surrounding tissues especially synovium and subchondral bone. The prevalence of OA in Indian population is estimated to be 22–39%; it is a major cause of joint pain and disability in aging population imposing a high social burden [2‐4]. However, mechanism involved behind this degenerative disease is poorly understood that reflects in the current lack of effective medical therapies [5].

The cascade starting from reactive oxygen species (ROS) leading to inflammation and increased matrix metalloproteinases are the important factors of degradation of matrix components [6]. They have been found to be overproduced in OA cartilage and synovium [6]. Previous reports have shown that, ROS scavengers can slow-down cartilage loss [6]. ROS contribute to cartilage degradation by sustaining the activity of catabolic cytokines and by reducing cartilage repair capacities. When ROS production exceeds the antioxidant capacities of the cell, an “oxidative stress” occurs that contributes to structural and functional cartilage damages. Oxidative stress is found to be elevated in cartilage and synovial tissues, which contributes to disease progression by increasing production of inflammatory mediators and matrix degrading enzymes [7]. To prevent toxicity by ROS, chondrocytes express a well co-ordinated anti-oxidant enzyme system in the form of SOD, CAT and GPX [6]. Matrix metalloproteinases (MMPs), which contribute to the accelerated degradation of extracellular matrix and tissue inhibitors of metalloproteinases (TIMPs) that regulates the MMP activity, play an important function in cartilage matrix turnover [8]. However, in OA affected joint tissues, up-regulation of MMPs is reported higher compared to TIMPs [8]. Altogether, these observations support the concept of antioxidant therapy along with modulation of MMP-TIMP might also decrease the progression of OA.

Unfortunately, at present there is no medicine or therapy that can target cartilage protection. Consequently, there is a growing interest in complementary and alternative medicines (CAM) either in the form of nutraceuticals or in the form of herbals that primarily thought to be chondroprotective and may even repair cartilage. It is therefore interesting to explore traditional systems of medicine for a potential drug that may help to reduce inflammation, protect cartilage damage, improve joint functions and restore patient’s activity levels.

In our earlier studies, anti-osteoarthritic potential of six herbs was shown, which are commonly used by Ayurvedic physicians for the treatment of OA viz. Sida cordifolia L., Piper longum L., Zingiber officinale Rosc., Ricinus communis L., Vitex negundo L. and Tribulus terrestris L., in collagenase type II induced OA (CIOA) rat model [9]. Sida cordifolia L. has been proven to be effective in balancing Vata dosha, which is one of the basic principals in Ayurveda [10]. S. cordifolia is shown to inhibit cyclooxygenase (COX) leading to the inhibition of prostaglandin (PGE) synthesis [11]. Zingiber officinale Rosc. is a common spice with several ethnomedicinal and nutritional values. It has been used traditionally for treating arthritic conditions [12]. It decreases interleukin-1 (IL1), nitric oxide (NO) and PGE2 and inhibit leukotriene B4 (LTB4) production in osteoarthritic cartilage [11, 13], it also inhibit production of tumor necrosis factor-α (TNF-α) in human osteoarthritic synoviocytes and chondrocytes [14]. Srivastsava et al. [15] reported 6-gingerol, active compound from its rhizome inhibit nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), TNF-α, interleukin 12 (IL12), inducible NO synthase and COX-2 [16]. Z. officinale inhibits biotransformation of arachidonic acid into inflammatory prostaglandin. In a clinical study on knee OA, using formulation containing Z. officinale as one of its ingredients, there was significant reduction in pain score [17].

In the present study, a novel herbal formulation named as OA-F2 was designed comprising a proportionate combination of S. cordifolia and Z. officinale, these herbs have shown appreciable anti-osteoarthritic potential in our previous studies [9]. We have studied efficacy of oral administration of OA-F2 in alleviating OA associated symptoms by using collagenase-induced OA (CIOA) in rats and its underlying molecular mechanism.

Osteoarthritis (OA) is a major cause of disability throughout the world, it causes pain due to inflamed knee joints, which involves progressive degeneration of articular cartilage, synovitis, formation of osteophyte, increased fibrillation due to increased denaturation and loss of collagen fibers [27]. Current therapeutic options for OA are mainly palliative and have little influence on progression of the disease and are also associated with many adverse effects [28]. Therefore, there is an increasing merit in use of compounds derived from natural plants or herbs for the treatment of OA [28]. In OA, cartilage degeneration is the result of combined biochemical and mechanical factors. Therefore, restoration of MMP-TIMP balance with the help of natural products that can effectively control oxidative stress and inflammation may provide an important therapeutic approach for the treatment of OA [29].

Intra-articular injection of collagenase significantly increased inflammation and swelling of the knee joint and thus increased diameter of the knee. Oral administration of OA-F2 significantly attenuated this inflammation and has reduced knee swelling and paw edema compared to osteoarthritic control group (Figs. 1 and 2). MMPs are the catalytic enzymes, which degrade the cartilage and in turn enhance the inflammatory process by releasing more cytokines and MMPs [27]. OA-F2 treatment to osteoarthritic rats showed significant improvement in paw withdrawal latency time compared to untreated osteoarthritic rats (Fig. 3). Serum CRP, probably the most widely used clinical marker of systemic inflammation, has been shown to correlate well with CRP in synovial fluid in patients with OA or RA [30]. In OA patients, elevated levels of CRP and ALP were recorded [25, 31]. Interestingly in the present study, CRP and ALP levels were significantly decreased by OA-F2 as compared to osteoarthritic control and diclofenac treated group (Figs. 4 and 5). Glycosaminoglycan is a major component of joint cartilage, joint fluid and other soft connective tissues. GAGs are released from the degrading cartilage matrix in a large amount. Administration of OA-F2, lowered GAG release in which means cartilage is protected as seen in the improved parameter of the joint space (Figs. 6 and 7), which was also confirmed in radiography. H and E staining showed that OA-F2 protected synovium and cartilage degeneration during OA (Fig. 8). Modification of ECM and increased articular chondrocyte proliferation are characteristics of OA [32], OA-F2 also protected chondroid matrix and prevented proteoglycan loss from ECM (Fig. 8). Histopathological observation suggests correlation of OA-F2 intervention with improved muscle degeneration, fibrillation, erosion of cartilage and chondroid matrix.

As observed in histopathology, studied biochemical and physiological parameters, deteriorating changes seen in OA induced rats are may be outcomes of elevated release of MMPs or these focal changes are might be due to secretion of pro-inflammatory cytokines with reactive oxygen species (ROS) from the inflamed synovium and from activated chondrocytes [27, 29]. ROS are also involved in the regulation of IL-1 effects mediated by NF-κB. In the progression of OA, NF-κB is a transcriptional factor that can be triggered by a number of stimuli, such as cytokines, excessive mechanical stress and degradation products of ECM [28]. Activated NF-κB regulates several catabolic enzymes involved in matrix degradation, including MMPs [28, 33]. MMP-3 has been shown to break down a number of ECM proteins, including fibronectin, laminin, denatured collagens and proteoglycans [28]. In addition to ECM degradation, MMP-3 is involved in the activation cascades of MMP-13 and gelatinases [28]. Since MMP-1 and MMP-3 play vital roles in ECM turnover, their regulation has been suggested to be useful therapeutic target [28]. On the other hand, TIMPs are the endogenous regulators of MMPs, which play important roles in maintaining homoeostasis with MMPs [28]. Imbalance between MMPs and TIMPs is a salient feature of OA progression, leading to disruption of the balance between ECM biosynthesis and degradation [28]. Therefore, exploration of the mechanisms through which the proteolytic activity, production and expression of MMPs are inhibited by plant-derived natural products may lead to new therapeutic strategies [34]. To determine whether OA-F2 may affect the destruction of articular cartilage, we examined the expression of the OA biomarkers MMP-3, MMP-9 and TIMP-1 in the synovium of collagenase-induced OA rats. Interestingly, our results demonstrated that the elevated MMP-3 and −9 expression in osteoarthritic control rats were decreased by OA-F2 supplementation. Present results demonstrated up-regulation of TIMP-1 by OA-F2L and OA-F2H in osteoarthritic rats. S. cordifolia extract was shown to inhibit COX enzyme and prostaglandin synthesis [11], whereas, Z. officinale decrease IL1, NO and PGE2 and LTB4 production as well as inhibit TNF-α production [11, 13, 14].

In summary, we evaluated the efficacy of orally administered OA-F2, a composition containing a blend of two medicinal plants, roots of S. cordifolia and rhizomes of Z. officinale, in collagenase induced OA model in rats. OA-F2 notably tackled the physiological problems and decreased levels of inflammatory markers such as CRP, ALP and GAG in the serum. We demonstrated at the mRNA level that OA-F2 inhibited the expression of MMP −3 and −9 and increased the expression of TIMP-1, all of which are classical biomarkers of inflammation and cartilage degradation in OA.

Our study revealed chondroprotective effect of OA-F2, which is explained through modulation of biochemical parameters studied as well as expression of MMPs-TIMP genes in knee synovial tissue. OA-F2 thus merits consideration as an alternative therapy from natural sources for the treatment of OA. However, further preclinical and human clinical studies are necessary.