| Objective:①To compare the impairing effects of continuous static gas pressure with that of IL-1βon annulus fibrosus cells cultured in alginate gel.②To investigate the protective effect of Astragalus Membranaceus on proliferation, apoptosis and cytoplasmic microfilament of rabbit annulus fibrosus cells which impaired by static gas pressure.Methods: Rabbit annulus fibrosus cells were cultured in alginate gel.①The cells were divided into 5 groups: group A as normal control, stimulated with 10ng·mL-1 IL-1βfor 24h in group B, with 10ng·mL-1 IL-1βfor 48h in group C, pressed with 1MPa static gas pressure for 24h in group D, and pressed with 1MPa static gas pressure for 48h in group E. Cell proliferation was detected with CCK-8 assay, while the apoptosis rate was detected with flow cytometry stained with Annexin V- PI. The expression of collagen I and II, MMP-3 and TIMP-1 was detected by RT-PCR.②The cells were divided into 5 group and impaired by 1Mpa static gas pressure, at the same time, the Astragalus Membranaceus injection of the does 10,50,100,500,1000μg·mL-1 was added to each group. After 48 hours, the cells proliferation were evaluated by CCK-8 method, and the cells were stained with Annexin V- PI to evaluate apoptosis using flow cytometry. meanwhile, each group cells were cultured in absence of gas pressure condintion in monolayer for 24h, then, the structure of cytoplasmic filaments was revealed by actin-tracker green.Results: Comparing the impairing effects of continuous static gas pressure with IL-1βwe found:①CCK-8 assay and flow cytometry showed that both IL-1βstimulation and static pressure could inhibit cell proliferation and increase apoptosis rate. The inhibition and increase was more obvious in group D and E.②RT-PCR showed that the expression of collagen I, II was significantly decreased in group B, C, D and E, especially collagen II expression in group E (declined about 75% as compared with group A, p<0.01). The expression of MMP-3 was increased in group B, C, D and E, especially in group D and E. The expression of TIMP-1 was decreased in group B and C, while increased in group D and E. The effect of Astragalus Membranaceus on impaired annulus fibrosus cells:①CCK-8 displays: Compared with control group, the Astragalus Membranaceus injection can promote the proliferation of annulus fibrosus cells, to improve the vitality of cells, and the significiant effect does is 50μg·mL-1 (p<0.01).②A nnexin V- PI staining reveals: Compared with control group, the does of 50μg·mL-1 Astragalus Membranaceus injection can significant reduce the apoptosis of annulus fibrosus cells(p<0.01). However, when the concentration increased to 1000μg·mL-1, the apoptosis rate decreased, but no statistical significance(p>0.05).③Actin staining showed that Astragalus Membranaceus injection can alleviating the destruction of microfilament network structure of annulus fibrosus cells. when dispelling the injuring factors, the annulus fibrosus cells displayed a relatively strong ability to recover.Conclusion:①Static pressure can induced similar impairing effects to that of IL-1βon rabbit annulus fibrosus cells. Static pressure can be a routine method for establishment of annulus fibrosus cell impairing model.②Astragalus Membranaceus injection can protect impaired annulus fibrosus cells, promoting proliferation and reducing apoptosis in vitro, which mechanism may be through the protection of microfilament network structure. Then, it has the potential for annulus fibrosus protection. |
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