ABSTRACT
<p><b>OBJECTIVE</b>To explore and identify the method for IL-1beta induced New Zealand rabbit knee chondrocyte degeneration, thus providing experimental bases for Chinese medical research on osteoarthritis from in vitro cultured chondrocytes.</p><p><b>METHODS</b>Under aseptic conditions, bilateral knee joint cartilage was collected from 4-week old New Zealand rabbits. Chondrocytes were separated by type II collagenase digestion and mechanical blowing method. They were randomly divided into two groups when passaged to the 2nd generation, the normal control group (group Z) and the IL-1beta induced model group (group M). No intervention was given to those in group Z. 10% FBS culture media containing 10 ng/mL IL-1beta was added to group M. All cells were passaged to the 3rd generation. They were compared using morphological observation, toluidine blue staining, type II collagen immunohistochemical staining, and flow cytometry.</p><p><b>RESULTS</b>Under inverted microscope, the second and the 3rd generation chondrocytes' phenotype of group Z was stable with good proliferation. Most cells turned into fusiform and slabstone shaped. In group M, most cells turned into long spindle shape or irregular shape. Results of toluidine blue staining and immunohistochemistry showed that the positive expression of chondrocytes after staining in group Z was superior to that in group M. Results of flow cytometry showed that there was statistical difference in the apoptosis rate of the second generation chondrocytes between group M and group Z (P < 0.01).</p><p><b>CONCLUSION</b>It was obviously seen that chondrocytes in IL-1beta induced New Zealand rabbit knee chondrocyte model obviously degenerated, which could be used in related experimental researches on osteoarthritis.</p>
Subject(s)
Animals , Rabbits , Cartilage , Cell Biology , Cells, Cultured , Chondrocytes , Cell Biology , Interleukin-1beta , Pharmacology , Knee Joint , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 β (IL-1 β)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.</p><p><b>METHODS</b>Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 β stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 β, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction.</p><p><b>RESULTS</b>IL-1 β stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-α, MMP-3 and MMP-13. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.</p><p><b>CONCLUSION</b>ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.</p>