Hydrostatic Pressure Of Apigenin On Apoptosis And Matrix Metabolism In The Human Nucleus Pulposus

BackgroundAs a result of lumbar disc degeneration, lumbar disc herniation and spinal stenosis become common diseases in orthopedics department. With the advent of an aging society in China, lumbar degenerative diseases pose a threat to the health of the people and has serious impact on the life quality of patients. With the popularity of mechanical and computer office, more and more people work in a sitting position, which led to the lumbar disc herniation, and the disease tends to younger. So lumbar intervertebral disc degeneration has become the hot research issue.Disc degeneration has become one of the problems plagued orthopedic surgeon, and its unlikely to reverse in case of degeneration. Current treatment methods are conservative treatment and surgical treatment. To some extent, operation can alleviate the clinical symptoms, but can't block the process of disc degeneration. Spinal surgery with Internal fixation will lead to changes in the structure, speed up the adjacent segment disc degeneration, while giving patients a serious psychological and economic burden. Therefore, how to prevent or delay the process of disc degeneration become the focus of research.ObjectiveDiscuss the effect Apigenin on human nucleus pulposus cells in vitro hydrostatic pressure environment, including functional status, apoptosis and matrix metalloproteinase MMP-3 and its inhibitor TIMP-1.And to demonstrate the possibilities that Apigenin as a nitric oxide synthase(NOS) inhibitors in delaying the process of disc degeneration and reveal part mechanism of action. To seek some new ideas that Chinese medicine for the prevention and treatment of degenerative disc disease.Methods1. Human Nucleus Pulposus Cells (HNPC) are provided by U.S ScienCell Research Laboratories. In this study, we use Laboratory culture, proliferation and passage. The passages 1-7 were observed, proliferation activity was detected and the main extracellular matrix expression (proteoglycans, collagen-Ⅰ, collagen-Ⅱ) were analysis. Select the appropriate passage for hydrostatic pressure and drug intervention.2. Research the effect of Apigenin on the NO output and drug dose-dependent of human nucleus pulposus cells which were monolayer cultured in vitro. Classic nitric oxide donor drugs SNAP and nitric oxide synthase inhibitor L-NMMA were used to compare with Apigenin for the Comparative Analysis. Discussion the feasibility of using Apigenin as a nitric oxide synthase (NOS) inhibitor.3. Build a convenient and safe hydrostatic pressure system which was designed by Oxford University Physiology Institute and Space Development Medical Research Center in Japan. We improved the system in order to realize hydrostatic pressure on cells that were Monolayer cultured in vitro. Analysis the differences of Nucleus Pulposus Cells in morphology, survival rate and functional status under different hydrostatic pressure. From the result, we demonstrate the feasibility of the hydrostatic pressure system and identify the value and loading-time of hydrostatic pressure in the next step.4. Integrated intervention on human nucleus pulposus cells with hydrostatic pressure system and Apigenin. Were detected nucleus pulposus cells apoptosis rate, MMP-3 and TIMP-1 production volume and collagen-Ⅱexpression. The experimental results were analyzed statistically. Analysis the effect of Apigenin on nucleus pulposus cells including apoptosis, balance expression of MMP-3 and TIMP-1 and extracellular matrix in different hydrostatic pressure environment. Discuss the feasibility of using traditional Chinese medicine Apigenin for delaying the process of disc degeneration and reveal part mechanism of action.Results1. Normal human nucleus pulposus cells can be monolayer cultured for good growth state in vitro. The nucleus pulposus cells before passage 4th are in normal state:having good morphology and proliferative ability, as well as stabile phenotypic expression. With the increase of passage will appear "dedifferentiation" phenomenon:the passage 5th decreased cell proliferation, and the expression of collagen-Ⅰcan be detected which indicates cell aging, so is not suitable for seed cells;2. Apigenin and L-NMMA have similar effect in inhibiting NO production of nucleus pulposus cells. Apigenin can significantly decreased NO production of nucleus pulposus cells when concentration is 12.5μmol/L.When L-NMMA concentration is 200μmol/L can also decrease NO output which is similar to Apigenin. However, the drug concentration of Apigenin was significantly lower than L-NMMA. SNAP drug can significantly increase NO production of nucleus pulposus cells when concentration is 500μmol/L.3. The constructed hydrostatic pressure system can provide a wide range of stress levels to meet the needs of a variety of experimental studies and has good biocompatibility. When nucleus pulposus cells were under 0.3Mpa hydrostatic pressure sustained for 120min, they have better morphology, survival rate is relatively high, strong expression of extracellular matrix. When under 3Mpa hydrostatic pressure for 120min, the nucleus pulposus cells survival rate was reduced, the expression of extracellular matrix was significantly less in the function inhibition state.4. Apigenin as a NOS inhibitors can impact nucleus pulposus cells'function status, apoptosis rate and the metabolism of matrix metalloproteinase.And the effect is different in different hydrostatic pressure environment:under 0.3Mpa hydrostatic pressure, Apigenin can reduce the degradation of extracellular matrix PG and collagen-Ⅱby regulating the balance between matrix metalloproteinases MMP-3 and TIMP-1.Improve nucleus pulposus cells inhibition status due to the high concentration of NO. In 3Mpa hydrostatic pressure, Apigenin have a role in reducing the apoptosis rate of nucleus pulposus cells and reduce the excessive breakdown of extracellular matrix.ConclusionsApigenin is a safe and effective Chinese medicine like NOS synthase inhibitor, can reduce the amount of NO production by nucleus pulposus cells. Apigenin can reduce the degradation of extracellular matrix PG and collagen-Ⅱby regulating the balance between matrix metalloproteinases MMP-3 and TIMP-1 and improve nucleus pulposus cells function inhibition status due to the high concentration of NO, reducing the apoptosis rate of nucleus pulposus in high hydrostatic pressure. Overall, Apigenin play a protective role on the intervertebral disc nucleus pulposus cells and extracellular matrix.