The Effect Of Ica On The Proliferation, Secretion And Mapks Pathway Of Articular Cartilage Cells

Articular cartilage diseases has a harmful effect on animal and human health, caused great economic losses to animal production, became a common problem was concerned by medical field, scientific community and society. The occurs of it because a variety of pathogenic factors invade the body cause cartilage tissue damaged, but due to the special anatomical structure of the joint, the prevention and treatment of the disease become difficult. As one of the main monomer in the epimedium, ICA can activate osteoblast MAPK signaling pathway to promote osteoblast proliferation, differentiation, and inhibition the apoptosis to prevent and treat arthritis, but the function and mechanism of ICA effect on articular cartilage cell is not clear. The experiment used a variety of research methods of cell culture techniques, histology, immunohistochemistry, flow cytometry technology and so on to mainly explored the ICA effect on proliferation, differentiation, apoptosis, NO/NOS, MMP-13of chondrocytes. While used Western-blot technology to researching the regulation mechanism of ICA impact on the chondrocytes MAPK signaling pathway, to provide theoretical support of ICA for clinical application of ICA to prevent and treat chondrocytes diseases of animal.Chondrocytes was isolated by Ⅱ type collagnase digestion from healthy swine articular cartilage, established the efficient training methods of porcine articular cartilage cells in vitro. Then used light microscopy observation of cell morphology, HE staining, Ⅱ type collagen immuneohistochemical and electron microscopic observation of cell ultrastructure methods to identificating the cultured cells is the articular catilage cells, found that cell grew, shape, density of cultivated chondrocytes by enzyme digestion method was well can be used for this test work.MTT assay was applied to detecting chondrocytes proliferation under the action of ICA, the results was final concentrationlng/mL,10ng/mL,20ng/mL,30ng/mL,40ng/mL ICA promoting chondrocytes proliferation. But with the increase of time (Id、3d、5d、7d), the effect of different concentration of ICA promoting to chondrocytes proliferation was different:Low, medium concentration of ICA on promoting chondrocytes proliferation was more and more significant (p<0.05), and the high concentration of ICA promoting fuction was decline.10ng/mLICA had better promoting effect on chondrocytes proliferation, this experiment adopted the final concentration10ng/mL ICA as the optimal concentration act on articular cartilage cells were studied.Flow cytometry results showed that cultured chondrocytes with10ng/mLICA1d can blocked G1phase of cell cycle (p<0.05), but cell cycle had no significant change when add ICA3d、5d and7d (p>0.05). However,10ng/mL ICA had no significant effect on apoptosis of chondrocytes (p>0.05), so ICA was beneficial to the growth and proliferation of articular cartilage cells.Results of using colorimetry detecting the secretion of NO/NOS showed that the expression of NO/NOS significantly reduce (p<0.05) when cultivated chondrocytes with10ng/mL ICA5d; while elisa detected the secretion of MMP-13founded that MMP-13reduce (p<0.05) when cultured chondrocytes with final concentration10ng/mL ICA3d,5d,7d. The results suggested that ICA can obviously regulate the secretion of various factor of articular cartilage cells.Cultured chondrocytes with10ng/mLICA in the experimental group Id,3d,5d,7d founded that phosphorylation level of ERK1/2had no significant difference after ICA effect5min,25min,1.5h (p>0.05), while the phosphorylation level of the experimental group was significantly higher than the control group (p<0.05). These data demonstrated that days of cultivating chondrocytes with ICAld-7d, the phosphorylation of ERK1/2has no significant change, ICA can activate ERKl/2within a short time (5min) and maintain ERK1/2activated1.5h at the least were also observed. In addition, ICA activate p38MAPK to the maximum of activation after ICA effect25min, slower than ERK1/2and holding time is shorter. Thus ICA can activate MAPK in a short time and maintain the activation of it, while activate p38MAPK slower than ERK1/2.In conclusion, using enzyme digestion can get good cultivated model of articular cartilage cells in vitro cell culture. Final concentration of10ng/mLICA has a good function on promoting proliferation of chondrocytes. ICA can activate MAPKs pathway in a short time and maintain the activation of it, at the same time ICA can reduce the secretion of NO/NOS, MMP-13and cell proliferation through MAPKs pathway, so these results indicated ICA can improve articular cartilage cells.