Elsevier

Nutrition

Volume 33, January 2017, Pages 1-13
Nutrition

Basic nutritional investigation
Inhibition of cartilage degradation and suppression of PGE2 and MMPs expression by pomegranate fruit extract in a model of posttraumatic osteoarthritis

https://doi.org/10.1016/j.nut.2016.08.004Get rights and content

Highlights

  • The present study examined the effect of oral consumption of pomegranate fruit extract (PFE) on the disease progression in rabbits with surgically induced osteoarthritis.

  • Significant inhibition in histologic scores, matrix metalloproteinase-13, and prostaglandin E2 are reported.

  • PFE-derived compounds may have potential value in the management of osteoarthritis.

Abstract

Objective

Osteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA.

Methods

OA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 β, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB was studied in IL-1 β-stimulated rabbit articular chondrocytes.

Results

Safranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly reduced IL-1 β induced IL-6 and MMPs expression in rabbit articular chondrocytes. These effects were also mimicked using MMP-13, MAPK, and NF-κB inhibitors in IL-1 β-stimulated rabbit chondrocytes. In an in vitro activity assay, PFE blocked the activity of MMP-13. Like MAPK and NF-κB inhibitors, PFE was also effective in inhibiting IL-1 β-induced PGE2 production in rabbit chondrocytes. PFE also reversed the inhibitory effect of IL-1β on COL2A1 mRNA and protein expression in IL-1 β-stimulated rabbit chondrocytes.

Conclusion

The present data highlight the chondroprotective effects of PFE oral consumption in a model of posttraumatic OA and suggest that PFE-derived compounds may have potential value in the management of OA.

Introduction

Osteoarthritis (OA) is the most common form of arthritis worldwide and a major cause of morbidity, physical limitation, disability, and socioeconomic burden [1], [2], [3]. Although cartilage matrix breakdown is the major characteristic of OA, the joint pathology also involves synovial membrane and subchondral bone changes [4]. OA is believed to be a consequence of mechanical and biochemical events that result in an imbalance between the synthesis and degradation of articular cartilage matrix consisting of proteoglycans (PGs), collagens (types II, IX, XI, and X, and others), and water [4], [5], [6]. Evidence now firmly supports the view that inflammation not only contributes to the development of symptoms but also plays a role in the progression of structural damage including cartilage degradation [7], [8], [9]. Excessive production of cartilage degrading enzymes such as the aggrecanases (ADAMTS-4 and -5) and matrix metalloproteinases (MMPs), which are the key player in degradation of aggrecan (ACAN) and collagen type II (COL2A1) has been shown in OA joints [10], [11], [12]. Studies have correlated increased chondrocyte apoptosis with OA severity in humans and animal models [13], [14], [15]. Moreover, cell death positively correlates with matrix degradation and calcification [16], [17]. In this regard, increased caspase-3 expression has been found to correlate with reduced cell density in human OA cartilage [18], whereas inhibition of apoptosis using caspase inhibitors reduced the severity of cartilage lesions in experimental OA [19]. Currently, goals of OA management include controlling pain, maintaining and improving the range of movement and stability of affected joints, and limiting functional impairment [20], [21]. Because of the association of severe adverse events with the use of nonsteroidal antiinflammatory drugs (NSAIDs), at least one-third of older adults use some form of complementary and alternative medicine for pain [22], [23].

Pomegranate (Punicagranatum L., Punicaceae) is an ancient fruit that has not changed much throughout human history. The fruit itself is a rich source of two types of polyphenolic compounds: anthocyanins (such as delphinidin, cyanidin, and pelargonidin), which give the fruit and juice its red color, and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic, and ellagic acid), which account for 92% of the antioxidant activity of the whole fruit [24], [25]. Studies have reported that the antioxidant capacity of pomegranate juice is three times that of the popular antioxidant-containing beverages such as red wine and green tea, presumably due to the presence of hydrolyzable tannins in the rind, along with anthocyanins and ellagic acid derivatives [25], [26], [27]. In a comparative analysis, anthocyanins from pomegranate were found to possess higher antioxidant activity than vitamin E (α-tocopherol), ascorbic acid and 3-carotene [26]. Pomegranate extract has been shown to protect from NSAID- and ethanol-induced gastric ulceration [28]. The whole-fruit extract is widely used in several traditional medicinal systems for the treatment of inflammation and pain in arthritis and other diseases [22]. The family of phytochemicals found in pomegranate fruit act together with greater potency than any single constituent alone [29]. Previously we have shown that a standardized pomegranate fruit extract (PFE) inhibit the production of MMPs via blocking the activation of p38-mitogen-activated protein kinase (MAPK) and the transcription factor nuclear factor (NF)-κB in OA chondrocytes [30]. Other studies found that bioavailable metabolites of PFE inhibited cyclooxygenase (COX)-2 activity in OA chondrocytes [31], and that consumption of PFE suppressed the inflammation and joint destruction in collagen-induced arthritis (CIA) mouse model [32]. also It has also been demonstrated that pretreatment of OA chondrocytes with PFE inhibited the interleukin (IL)-1 β-induced activation of the upstream kinase MKK3, resulting in the inhibition of p38 α-MAPK isoform and the activation and DNA-binding activity of the transcription factor RUNX-2 [33]. In the present study we examined the cartilage and chondroprotective effects of oral consumption of PFE in vivo using the rabbit model of posttraumatic OA in which the disease is induced through anterior cruciate ligament transection (ACLT) [34], [35]. Results from the present study demonstrated that in rabbits that orally consumed PFE, there was less cartilage damage and fewer apoptotic chondrocytes in the joints with surgically induced OA. Expression of IL-6, MMPs and prostaglandin (PG)E2 was also reduced, whereas the expression of ACAN and COL2A1 was high in the OA joints of rabbits fed PFE. Inhibition of IL-1 β-induced IL-6 and MMPs expression, MMP-13 activity and PGE2 production with an increase in COL2A1 level was observed by PFE pretreatment in rabbit articular chondrocytes. These data suggests that consumption of PFE may be chondroprotective and may be used as an adjunct therapy for the management of OA.

Section snippets

ACLT surgery to induce OA in rabbits

The model used to study the effects of PFE consumption on the development of OA was the rabbit ACLT model as it produces a reliable and reproducible degradation of articular cartilage [36]. All animal studies were approved by the IACUC of the Case Western Reserve University (CWRU), Cleveland, Ohio (protocol no. 2009-0209) and the IACUC of the Northeast Ohio Medical University (Protocol #13-002). Animal surgeries and sacrifice were carried out using the facilities of the Animal Resource Center

Effect of PFE administration on macroscopic and histologic parameters

Postmortem inspection of rabbit knees revealed that ACLT was complete in all rabbits included in the study. In the right femorotibial joints subjected to sham surgery, the articular cartilage macroscopic grades were normal or scarcely perceptible India ink uptake and no cartilage erosions were observed in any of the joint compartments (Supplementary Fig. 1), whereas the induced OA lesions (erosion of articular cartilage) were focal in joints in which ACLT was performed. In animals that were fed

Discussion

The present study was carried out to determine the effect of oral administration of PFE on cartilage degeneration in a rabbit model of posttraumatic OA. In this model, oral administration of PFE was markedly effective against joint destruction. The histologic findings were informative with respect to the effects of PFE on OA cartilage structural changes. In fact, rabbits in both groups that were fed PFE exhibited a significant decrease in the markers of cartilage matrix damage, such as loss of

References (54)

  • M. Shukla et al.

    Consumption of hydrolyzable tannins-rich pomegranate extract suppresses inflammation and joint damage in rheumatoid arthritis

    Nutrition

    (2008)
  • N. Badlani et al.

    The protective effect of OP-1 on articular cartilage in the development of osteoarthritis

    Osteoarthritis Cartilage

    (2008)
  • S. Laverty et al.

    The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the rabbit

    Osteoarthritis Cartilage

    (2010)
  • B.C. Sondergaard et al.

    Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation

    Osteoarthritis Cartilage

    (2006)
  • J.P. Pelletier et al.

    The inhibition of subchondral bone resorption in the early phase of experimental dog osteoarthritis by licofelone is associated with a reduction in the synthesis of MMP-13 and cathepsin K

    Bone

    (2004)
  • N.P. Seeram et al.

    Bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate (Punica granatum L.) juice

    Clin Chim Acta

    (2004)
  • N.P. Seeram et al.

    Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours

    J Nutr

    (2006)
  • M. Cross et al.

    The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study

    Ann Rheum Dis

    (2014)
  • R.C. Lawrence et al.

    Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part II

    Arthritis Rheum

    (2008)
  • M.W. Lark et al.

    Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints

    J Clin Invest

    (1997)
  • R.C. Billinghurst et al.

    Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase

    Arthritis Rheum

    (2000)
  • J. Samuels et al.

    Osteoarthritis: a tale of three tissues

    Bull NYU Hosp Jt Dis

    (2008)
  • M. Kapoor et al.

    Role of proinflammatory cytokines in the pathophysiology of osteoarthritis

    Nat Rev Rheumatol

    (2011)
  • M. Wang et al.

    MMP13 is a critical target gene during the progression of osteoarthritis

    Arthritis Res Ther

    (2013)
  • P.S. Burrage et al.

    Matrix metalloproteinases: role in arthritis

    Front Biosci

    (2006)
  • F.J. Blanco et al.

    Chondrocyte apoptosis induced by nitric oxide

    Am J Pathol

    (1995)
  • H.A. Kim et al.

    Apoptotic chondrocyte death in human osteoarthritis

    J Rheumatol

    (2000)
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    This work was supported in part by grants from National Institute of Health grants (RO1 AT-003267; RO1-AT-005520, R21-AT504615) and funds from the Northeast Ohio Medical University. TMH conceived the study. NA and TMH were responsible for its design and coordination of the data acquisition and analyses, interpretation of the data, manuscript preparation, and statistical analyses. NA performed the ACLT surgeries. NMK set up the rabbit chondrocyte cultures, performed the MMPs activity assay and enzyme-linked immunosorbent assay (ELISA), data acquisition and interpretation. OA performed the PGE2 ELISA and data acquisition and interpretation. All authors approved the final version of the manuscript. The authors have no conflicts of interest to declare.

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