Effect Of Nourishing Kidney And Activating Blood On Interleukin-1Beta/MAPK Signal Pathway By Human Osteoarthritis Chondrocytes

Osteoarthritis (Osteoarthritis, OA) is the elderly joint disease, the main pathological changes is the excessive apoptosis of cartilage cells and extracellular matrix degradation. In the osteoarthritis pathological change process, cytokines play a key role, not only can promote cartilage cells secrete degradation enzyme, led to the imbalance of extracellular matrix, and accelerate the apoptosis of cartilage cells, interleukin-1in numerous cytokines is one of the most important degradation factors. Interleukin-1via MAPKs signaling pathways and nuclear factor kappa-B controls the matrix metalloproteinases (MMPs) is the main way of cartilage matrix degradation. It has now been confirmed that JNK, p38and ERK are the main MAPK family involved in the pathogenesis of OA. ERK1/2is main to promote proliferation, control the terminal differentiation of the cells, and JNK, p38is involved in the inflammatory response. Nuclear factor kappa-B combined with a variety of gene promoter or a specific enhanced site, to promote related gene transcription. One of the most typical nuclear factor kappa-B is p65and p50, formed two subunits dimers. In the OA, MAPKs signaling pathways and nuclear factor kappa-B mediated matrix metalloproteinases, the extracellular matrix is the key enzyme of degradation, can degrade almost all of the extracellular matrix, MMP-1, MMP-3, MMP-13is the main protease to accelerate the occurrence and development of OA. Therefore, adjusting IL-1mediated the stability of MAPKs signaling pathways and nuclear factor kappa-B, down-regulated the production of MMPs, delay the pathological changes of OA, will provide new targets for the prevention and treatment of OA.This topic in the academic ideas of the nourishing kidney and activating blood method in the treatment of OA, supported by the national natural science fund, cultured human OA chondrocytes in vitro as the research object, used nourishing kidney and activating blood drug-containing serum intervention, with the aid of CCK-8method, western blotting technique, etc. Detect related factors ERK, P-38, JNK, P-65of MAPKs signaling pathways and nuclear factor kappa-B regulated by IL-1beta, down-regulated the expression of MMP-3and13, explore the pathological mechanism of OA that it is intervented by nourishing kidney and activating blood drug, provides a new way of idea for the treatment of OA. Experiment1Objective:Compared with normal cartilage cells, to observe the expression of MAPKs and NF-kB signals in OA chondrocytes.Methods:Compared the expression of ERK, P38, JNK and P65transduction factor in normal and OA chondrocytes by using Western blot techniques.Results:The protein expression of ERK, P38, JNK and P65in OA chondrocytes obviously enhanced than in normal cartilage cells (P<0.05).Conclusion: MAPKs signaling pathways and NF-kB transduction factors are closely related with OA development.Experiment2Objective:To observe whether Niuxijianbu Particles can adjust MAPKs and NF-kB signal transduction factors in OA chondrocytes.Methods: OA chondrocytes is divided into5groups, OA chondrocytes group, OA chondrocytes+control serum group, OA chondrocytes+drug-containing serum low-dose group, the OA chondrocytes group+drug-containing serum middle-dose group, the OA chondrocytes+drug-containing serum high-dose group, after48h, using Western blot technique to detect the protein expression of ERK, P38, JNK, P65.Results:Niuxijianbu Particles can inhibit ERK, P38and P65, and the inhibition of P38presents dose dependent relationship (P<0.05); Niuxijianbu Particles have a promoting effect on JNK signal, significantly higher than the OA group (P<0.05).Conclusion:Niuxijianbu Particles can intervene in the MAPKs and NF-kB signal transduction pathway, with protective effect on cartilage.Experiment3Objective: To observe the expression of MAPKs and NF-kB signals induced by IL-1in OA chondrocytes.Methods:To investigate the influence of different IL-1concentrations (Ong/ml, lng/ml,10ng/ml,100ng/ml) on OA cartilage cell proliferation by CCK-8technique. To detect the protein expression of P38, JNK, P65, MMP-3, MMP-13in OA and IL-1group by Western blot technique.Results:IL-1could inhibit osteoarthritis cartilage cell proliferation in concentration-response and time-effect relationship from1ng/ml to100ng/ml, and OA cartilage cell growth inhibition is smooth by10ng/ml IL-1on48h; The expression of OA chondrocytes JNK, P38and P65, MMP-3, MMP-13protein significantly enhanced induced by IL-1, statistically significant (P<0.05).Conclusion:IL-1can significantly inhibit OA chondrocytes proliferation in lOng/ml dose, and induce the expression of MAPKs and NF-kB signals, as the best concentration.Experiment4Objective:to observe the effect of Niuxijianbu Particles on OA cartilage cell proliferation induced by IL-1.Methods:OA chondrocytes were randomly divided into6groups, OA chondrocytes group with10%fetal bovine serum, OA chondrocytes+control serum group+10ng/mlIL-1, OA chondrocytes+high-dose-decoction-containing serum group+10ng/mlIL-1, OA chondrocytes+middle-dose-decoction-containing serum group+10ng/mlIL-1, OA chondrocytes+low-dose-decoction-containing serum group+10ng/mllL-1.10ng/mlIL-1model group with10%fetal bovine serum. Then cell proliferation was detected by CCK-8assay after24h,48h and72h.Results: Serum containing Niuxijianbu Particles groups increased significantly Cartilage cell proliferation after48h compared with the24h.Conclusion: Niuxijianbu Particles can promote the OA cartilage cell proliferation, which may be one of the important mechanisms to protect cartilage cells. Experiment5Objective:To observe whether Niuxijianbu Particles can adjust the IL-1/MAPKs and NF-kB/MMPs signals pathway in OA chondrocytes.Methods:The chondrocytes were divided into OA chondrocyte group, OA chondrocyte+control serum group+10ng/mlIL-1β, OA chondrocyte+high-dose-decoction-containing serum group+10ng/mlIL-1β, OA chondrocyte+middle-dose-decocti on-containing serum group+10ng/mlIL-1β, OA chondrocyte+low-dose-decoction-containing serum group+10ng/mlIL-1β, OA model group with10%fetal bovine serum+10ng/mlIL-1β.Then each group of the protein expression was detected by Western blotting after48h.Results:Niuxijianbu Particles can significantly inhibited MMP-13, P38and P65induced by IL-1and with dose dependent relationship (P<0.05); But the inhibitory effect of MMP-3is weak; Improve JNK protein. Conclusion:Niuxijianbu Particles can adjust the expression of P38, P65protein, inhibit MMP-13, and then protect the cartilage cells, has a certain significance to delay the cartilage cells damage.