Osteoarthritis (OA) is characterized by progressive articular cartilage degradation that will lead to joint pain, immobility, and other functional impairments [1
]. Arthritis affects an estimated 54.4 million adults in the US, with projections of 78.4 million by the year 2040 [2
]. Although there have been significant advances in OA management, to date, there are no effective treatment options to modify progression of the disease. The current approach mainly targets to relieve symptoms by reduction of pain and improvement of joint function using oral or topical over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs). This strategy is known to mask the primary cause leading to irreversible damage to the articular structure. Chronic uses of NSAIDs for symptomatic relief of OA are also limited due to their gastrointestinal, renal, and cardiovascular side effects [3
]. Hence, these unmet needs could be bridged with natural products.
It has been a common practice to standardize the extract of a medicinal plant to be used alone or in combination with other functional bioactives or nutrients for multiple human ailments. For instance, in traditional and contemporary medicine, Morus alba
root-bark extract has been reported to have antibacterial [4
], antioxidant, hypoglycemic [5
], hypolipidemic, neuroprotective, antiulcer, analgesic [7
], and anti-inflammatory activities [10
]. Extracts and prenylated flavonoids from Morus are known to inhibit nitric oxide and interleukin-6 (IL-6) production, downregulate inducible nitric oxide synthase [11
], inhibit activation of Nuclear Factor kappa light chain enhancer of activated B cells (NF-κB) [12
], and inhibit a tumor necrosis factor α (TNF-α), [13
] and interleukin-1β (IL-1β) production [14
]. This suggests its use in inflammatory conditions. Similarly, Acacia catechu
extract has been widely used for its anti-oxidation [15
], free radical scavenging [16
], DNA damage protection [17
], antiproliferative, cytotoxic [18
], antidiabetic [19
], hepatoprotective [21
], analgesic [22
], chemoprotective [24
], anti-microbial [25
], and anti-inflammatory activities [26
These properties of A. catechu and M. alba were indeed translated into beneficial applications for OA when their standardized composition, UP1306, was tested in vitro and in vivo. To mention a few tests, UP1306 was found to cause (a) suppression of inflammation and pain sensitivity in carrageenan induced rat paw edema model [27
], (b) modulation of cyclooxygenase and lipoxygenase activities [27
], (c) synergistic inhibition of glycosaminoglycan release ex vivo [27
], and (d) increased cartilage sparing activities in monoiodoacetate-induced rat OA model [28
]. In a randomized and double-blinded placebo-controlled clinical trial, UP1306 administered at 400 mg/day to arthritic subjects showed significant reduction in urinary C-telopeptides of type II collagen (CTX-II), when compared to placebo after 12 weeks of daily supplementation [29
]. In each of these studies, the effect of UP1306 on pro-inflammatory cytokines and matrix degrading enzymes were implied, although it was not directly measured. Herein, we designed a study that utilizes the collagen induced arthritis model to address these gaps.
The collagen induced arthritis model is known to cause autoimmunity to type-II collagen that could lead to autoimmune arthritis which encompass inflammation of synovial joint, cartilage destruction, and bone erosion [30
]. Both cellular and humoral immunity are involved in the pathogenesis of the disease. The pro-inflammatory cytokines interleukin-1 (IL-1), IL-6, and TNF-α are heavily involved in the etiology of arthritis [31
]. It has been known that TNF-α has an early and crucial role in the cascade of pro-inflammatory cytokine production and subsequent inflammatory process. Previous studies showed increase in arthritis severity when TNF-α works in synergy with IL-1β. With the concept of TNF-α as the tip of pro-inflammatory network in early Rheumatoid Arthritis (RA) pathogenesis, anti-TNF-α antibodies (e.g., infliximab, etanercept, and adalimumab) were developed as prescription drugs for the treatment of rheumatoid arthritis by neutralizing TNF-α [32
]. Those biologics showed remarkable clinical benefit validating the hypothesis that TNF-α plays a major role in the pathology of RA. While patients receiving anti-TNF therapy have shown significant improvement in arthritic signs and symptoms, not all patients were equally responsive for anti-TNF therapy indicating the need for additional cytokine inhibitions, such as IL-6 and IL-1β [33
]. Similar efficacies have also been achieved with IL-6 and IL-1β inhibitors (e.g., Tocilizumab and canakinumab, respectively) for RA patients [34
]. These pro-inflammatory cytokines play key roles in disease initiation and progression by triggering other inflammatory cytokines and inducing cartilage degrading enzymes, such as metalloproteinases and aggrecanases [35
]. Considering its application in arthritis, commonly used natural compounds, such as curcumin, Boswellia extracts, and others, have employed this model to address mechanic and functional based activities of products [36
]. Considering the collagen induced arthritis as a typical model for rheumatoid arthritis, we used Methotrexate as a reference compound in our study. It is an anti-neoplastic immunosuppressant drug that is widely used for treating rheumatoid arthritis at lower dosages. Its low cost, extensive safety record, and weekly treatment regimen makes it an attractive option in early RA.
In the present study, we carried out collagen induced rat arthritis model to further investigate the mechanism, where UP1306 imposes its symptomatic relief and cartilage protection activities in arthritis. Key pro-inflammatory cytokines and matrix degrading enzymes were used as objective measures for data interpretations.
The CIA model in rats is the most commonly studied autoimmune model of RA with several pathological features resembling the immune-mediated polyarthritis in dogs and humans [41
]. Its short duration between immunization and disease manifestations makes the model feasible for therapeutic efficacy evaluations. Following inoculation of heterogenic type II collagen (CII), rats mount both humoral and cellular responses to the antigen [38
]. This sensitization subsequently leads to the host animal attacking its own type II collagen, which is predominantly present in the joint cartilage and hence results in erosive or non-erosive joint destruction. The pathophysiology of the disease is highly orchestrated and complex. Upon induction, rats will experience inflammatory pain and swelling, cartilage degradation, synovial hyperplasia, pannus formation, mononuclear cell infiltration, deformity, and immobility.
In the current study, rats started to show the pathognomonic signs of arthritis on day 12 post priming followed by a progressive increase in severity that approached near plateau on days 17 to 18. These symptoms were mitigated by oral treatment of an immune suppressant–Methotrexate and also a natural polyphenol composition-UP1306. Both treatment groups (Methotrexate and UP1306) showed measurable relief in arthritis severity, swelling, ankle width, and pain sensitivity when compared to the vehicle-treated diseased rats. When data for arthritis severity, paw thickness, and ankle diameter were pooled together for the duration of the study period from day 12–18 (where visible signs of arthritis were observed), CIA rats treated with Methotrexate and UP1306 showed statistically significant reduction in all of the cardinal signs of arthritis suggesting their application for symptomatic relief of arthritis.
TNF-α and IL-1β are the two primary cytokines involved in the initiation and progression of arthritis [42
], mainly through (a) downregulation of the synthesis of major extracellular matrix components by inhibiting anabolic activities of chondrocytes [43
]; (b) induction of additional cytokines (such as IL-6), chemokines, and extra cellular matrix degrading enzymes (MMPs and aggrecanases) [45
]; (c) inhibition of anti-oxidant activity of the host [47
]; and (d) induction of reactive oxygen species [48
]. These processes facilitate maintenance of chronic inflammation and perpetual joint destruction in arthritic patients. For example, while injection of IL-lβ into the knee joints of rats caused joint inflammation and marked proteoglycan depletion [49
], its blockade reversed the process [51
]. Besides direct involvement in the inflammation process and cartilage degradation, dysregulation of IL-6 levels is also linked to the common clinical manifestations associated with rheumatoid arthritis pathology such as fever, fatigue, and weight loss [54
]. Hence, modulating these pro-inflammatory cytokines at various stages of disease progression could alleviate the symptoms associated with arthritis and/or help to modify the disease. The anti-inflammation and cartilage protection activities of UP1306 observed in this study could be in part due to inhibition of these key pro-inflammatory cytokines.
Supplementation with UP1306 for three weeks resulted in significant reductions in the level of fundamental matrix proteolytic enzymes, such as MMP-13 and MMP-3. Along with aggrecan breakdown, degradation of collagen is a central feature of arthritis [55
]. Pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 are known to play important roles in cartilage matrix degradation in articular cartilage through a cascade of events that lead to stimulation of aggrecanase and matrix metalloproteinase production [42
]. When MMP enzymes are activated, MMP-3 is the first to be activated. It then triggers other MMPs, such as MMP1, MMP9, and MMP13, which leads to further degradation and amplification of extracellular matrix components. This leads to a simultaneously increased release and accumulation of degradants in the joints and causes joint inflammation. During the course of disease pathology, the major histocompatibility complex presents these fragments to T cells and promotes the activation and release of large amounts of inflammatory cytokines, such as IL-1β and IL-6, which in turn increases other MMP expression levels in the chondrocytes and synovial fibroblasts. Consequentially, all these processes result in augmented collagenase activity and worsening of joint inflammation. MMP-3 and MMP-13 have been found in increased levels at the sites of cartilage erosion in cases of rheumatoid arthritis and osteoarthritis [56
]. Previous studies have shown that these MMP levels in OA patient’s blood and synovial fluid were higher than in healthy people and the level was consistent with the extent of cartilage damage [57
]. In fact, MMPs secreted into the synovial fluid can directly degrade the cartilage and bone composition leading to enhanced damage of surrounding articular structures [59
]. In the current study, there was significant suppression of serum MMP-3 and MMP-13 levels by UP1306, which could provide a wholesome protection of cartilage degradation and improved pain relief. The reduction in MMPs observed in this study could partially be explained by (a) the effect of treatment materials in reducing the precipitating pro-inflammatory cytokines and/or (b) the activity of treatment materials by directly suppressing expression of these matrix degrading enzymes.
Urine C-terminal telopeptide of type II collagen (uCTX-II) has been by far the most studied and frequently referred biomarker of cartilage degradation that could be used for the purpose of diagnosis, determining the severity of disease or extent of disease progression, prognosis, and monitoring efficacy of treatment [60
]. In clinical studies, high levels of CTX-II are a good predictor of increased risk of joint destruction [61
]. Degradation and loss of articular cartilage are fundamental features of collagen-induced arthritis whereby increased CTX-II level directly correlated with the time course of paw swelling and arthritis severity. Our results were in accord with previous reports [35
]. In the current study, substantiating the effects on paw swelling, paw thickness, arthritis severity, pro-inflammatory cytokines, and matrix degrading enzymes, rats treated with UP1306 showed significantly reduced levels of uCTX-II. These findings indicate that cartilage protection activity is one of the primary functions of UP1306 suggesting its usage in management of arthritic conditions.
Together with symptoms and biomarkers, histopathological analyses of articular cartilage, synovial membrane, and subchondral bone have been used to evaluate arthritis disease progression or to measure outcome of therapeutic interventions [63
]. In the current study, significant improvements in maintenance of the articular structural integrity of rats treated with UP1306 and Methotrexate were observed. These effects were demonstrated in the histopathology data as exhibited by limited loss, degeneration, or necrosis of chondrocytes, smoother articular cartilage surface, deeper and uniform stain of intracellular matrix, and close to normal contour of the subchondral bone. The changes in magnitude of histopathological severity scores for cartilage degradation, bone damage, inflammation, reactivity, and matrix integrity were computed and were found that UP1306 treatment resulted in 86.8%, 71.8%, 58.6%, 70.6%, and 87.1% inhibitions, respectively, when compared to vehicle-treated CIA rats.
Previously, active constituents of UP1306 (i.e., catechins, prenylated flavonoids, and stilbenes) have showed to possess activities suggestive of their benefits in OA management. These include (i) the inhibition of the activities of cyclooxygenase-2 (COX-2), lipoxygenase (5-LOX), platelet phospholipase A2, and pro-inflammatory cytokines, such as TNF-α, ILs 1, 2, 6, 8, and 12 [64
] as a result of catechin; (ii) inhibition of inflammation activities [66
]; (iii) suppression effect of T-cell migration and inflammation induction [67
]; (iv) inhibition of nitric oxide (NO), inducible NO synthase expression, prostaglandin E2 production, and activation of NF-κB [68
]; (v) inhibition of pro-inflammatory mediators, such as IL-1β, IL-6, and COX-2 [69
]; and (vi) activation of total antioxidant ability [69
] as a result of prenylated flavonoids and stilbenes from M. alba root bark extract.