| Osteoarthritis (OA) is the most common joint disorder and a major contributor to disability in the elderly. It is generally viewed as a degenerative disorder involving cartilage degradation. Typically, the degenerative changes are mostly caused by unbalance of the MMPs and TPIMs, accompanied by a local inflammatory component that accelerates the collagen and agrrecan destruction in the cartilage tissues. Genistein is one kind of plant flavonoids which are found to attenuate inflammation response through their inhibition of important regulatory enzymes. These anti-inflammatory properties of flavonoids provide the rationale for investigating the role of isoflavones in conditions such as OA.Objective: In the present study, we developed an appropriate model for osteoarthritis research with lipopolysaccharide (LPS)-treated cultured chondrocytes from New Zealand rabbit articular cartilage. We investigated the effect of genistein on nitric oxide (NO) or interleukin-1 beta (IL-1β) production in response to increasing doses of genistein in LPS-treated cultured rabbit chondrocytes.Methods: Part 1: One month old New Zealand rabbits were chosen in this study. Articular cartilage tissue from the limbs was cut out under sterile conditions after sacrifice. Plenty of chondrocytes were obtained by using mechanical separation and enzyme digestion method. Cells were cultured in DMEM high glucose-based culture medium containing 10% fetal bovin serum. Chondrocytes were identified by morphologic and immunochemistry methods. Normal cultured group and LPS-treated group were set. Cell viablity was measured by MTT test, the inflammatory cytokines NO and IL-1βwas determined with biochemical methods or RT-PCR.Part 2: We further investigated the protective effect of genistein on LPS-treated chondrocytes. Chondrocytes were cultured in 96-well plates and set into different groups as bellow: normal control, LPS-treated alone, LPS-treated supplemrnted with estradiol, LPS-treated supplemented with genistein in doses ranging from 0.1μg/ml~25.0μg/ml. Cell viability assay were carried out by MTT test. Total RNA was extracted from the lysed cell fraction, which was used for RT-PCR assay to determine the expression levels of iNOS, IL-1β, MMP-1, type II collagen, and p53.Results:Part 1: Treatment with LPS affected articular chondrocytes viability negatively, with higher levels of inflammatory cytokines NO and IL-1βproduction. An appropriate research model was successfully developed through this LPS-treated articular chondrocyte culture.Part 2: Cell viability in LPS-treated group was significantly lower than that of control group, with upregulated IL-1β, iNOS, MMP-1, p53 mRNA expression levels. Combination of LPS and genistein in doses of 1.0μg/ml~15.0μg/ml did not alter cell viability significantly compared to normal control group. Higher levels of Genistein even showed inhibitory effect for cell viability. Results from the RT-PCR showed that genistein at any chosen doses can prevent LPS-induced increase of inflammatory mediators IL-1β, iNOS, MMP-1, and p53. Type II collagen expression levels were not affected significantly within different groups.Conclusion: LPS treatment affected chondrocyte viability negatively, by increasing proinflammatory mediator production and/or inducing apoptosis in cultured articular chondrocytes. The LPS-treated cultured chondrocytes can be used as an appropriate model for cartilage disease research. Genistein showed no significant effect of reversing LPS induced chondrocyes viability. However at lower levels, similarly to estradiol, genistein suppressed proinflammatory mediator IL-1β, iNOS, MMP-1 and p53 mRNA expression, which may be the machanism of genistein asserting it's protective effects in OA prevention.
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