Abstract
Purpose: In Persian traditional medicine, application of Mummy material has been advised since hundred years ago for treatment of different diseases as bone fracture, cutaneous wounds and joint inflammation. Regarding to the claim of indigenous people for application of this material in the treatment of joint inflammation, the present study was designed to evaluate whether Mummy can revoke the inflammatory responses in chondrocytes stimulated with interleukin 1-β (IL-1β).Methods: Isolated chondrocytes at the second passage were plated in 50 ml conical tubes at density of 1x106 for pellet culture or were plated in T75 culture flasks as monolayer. Cells in both groups were treated as control (receiving serum free culture medium), negative control (receiving IL-1β (10ng/ml for 24 hr)) and IL-1β pre-stimulated cells which treated with Mummy at concentrations of 500 and 1000µg/ml for 72hrs. After 72 hrs, to evaluate whether Mummy can revoke the inflammatory response in chondrocytes, cell in different groups were prepared for investigation of gene expression profile of collagen II, Cox-2, MMP-13, C-Rel and P65 using real-time RT-PCR.Results: Treatment of chondrocytes with IL-1β (10ng/ml) resulted in a significant increase in expression level of Cox-2, MMP-13, C-Rel and P65 in pellet culture system, while treatment of IL-1β-stimulated choncrocytes with Mummy at both concentrations of 500 and 1000µg/ml inhibited the expression level of above mentioned genes. Compared to the pellet culture, Mummy did not affect expression level of genes in monolayer condition.Conclusion: The obtained data from this investigation revealed that Mummy can be used as a potent factor for inhibiting the inflammatory responses induced by IL-1β in chondrocytes probably through inhibition of NF-қB subunits activation.
Highlights
Articular cartilage is basically composed of unique cell type named chondrocytes which are trapped in a delicately organized extracellular matrix (ECM).[1,2] Despite of low division activity, chondrocytes are responsible for maintaining the homeostasis in cartilage tissue through regulation of ECM components synthesis and catabolism.[3]
Well-preserved cartilage samples were transferred to the culture lab, after washing three times with phosphate buffer saline (PBS)(Cat No: P4417, Sigma-Aldrich, Germany) containing 1% penicillin/ Streptomycin (P/S)(Cat No:ATRA-010, ATOCEL, Hungary), samples were cut into 1x1 mm thickness pieces using sterile scalpel for isolation of chondrocytes, obtained pieces were incubated in pronase 1%(Cat No:10165921001,Roche, Germany) for 60 minutes, further digestion was performed by incubation in collagenase enzyme (Cat No:11088807001,Roche, Germany) at concentration of 0.2% in shaking water bath for 3-4 hours
To understand if Mummy can revoke the effects of interleukin 1-β (IL-1β) on chondrocytes, cells were stimulated with IL-1β (24 hr) and treated with Mummy at above mentioned concentrations
Summary
Articular cartilage is basically composed of unique cell type named chondrocytes which are trapped in a delicately organized extracellular matrix (ECM).[1,2] Despite of low division activity, chondrocytes are responsible for maintaining the homeostasis in cartilage tissue through regulation of ECM components synthesis and catabolism.[3]. Osteoarthritis (OA) which is the most common debilitating and chronic disorder of synovial joints, is characterized by progressive loss of articular cartilage and involvement of other joint tissues including subchondral bone and synovium.[5,6] At molecular level, over production of pro-inflommatory cytokines such as IL-1β and TNF-α by stimulated synoviocytes and chondrocytes has a crucial function in osteoarthritis pathogenesis.[7,8] Elevated IL-1β levels upregulates the production of degrading enzymes such as matrix metallopreteinases (MMPs) and downregulates the ECM molecules synthesis by chondrocytes which results in degradation of articular cartilage.[7,9] local synthesis and release of IL-1β induces the production of inflammatory pain mediators such as Cox-2 and inducible nitric oxide synthase (iNOS) which leads to clinical symptoms of OA.[10,11] The biological effects of pro-inflammatory cytokines on chondrocytes such as increased level of Cox-2 and MMPs, are mainly excreted by activation of transcription
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