LPS Mediate The Osteoclastogenesis-related Cytokines And Mechanism Involved

ObjectiveTo investigate the possible mechanism of the expression of osteoclastogenesis-related cytokines induced by LPS, including interleukin-1alpha(IL-1α), interleukin-1beta(IL-1β), interleukin-6(IL-6), interleukin-10(IL-1-), macrophage colony-stimulating factor(MCSF) and tumor necrosis factor-alpha(TNF-α).Methods(1) The cell was induced by LPS with the concentration of 100 ng/ml and then stained with TRAP Kit when multinucleated cells was observed under the inverted fluorescence microscope after 24 h.(2) Protein expression of Ca SR was detected by Western blot and immunofluorescence technique under laser scanning confocal microscope.(3) The NPS2143 was used to pre-culture mouse macrophage cell line and the optimal dose of the NPS2143 was determined by the methods of CCK-8 and flow cytometry on condition that cell survival rate was not affected. On this basis, the experiment was divided into 4 groups: a) control group: RAW264.7 was cultured in complete culture medium without any treatment; b) LPS induced group: RAW264.7 cells were cultured with the complete culture medium containing 100 ng/ml LPS;c) NPS2143 group:RAW264.7 was cultured in complete culture medium with 3 μM NPS2143; d)LPS+NPS2143 group: RAW264.7 was cultured with the complete culture medium with3 μ M NPS2143 + 100 ng/ml LPS. For group b) and d), RAW264.7 cells were pretreated with 3 μM NPS2143 for 6 h, then cultured with fresh medium containing 3μM NPS2143 plus 100 ng/ml LPS or not. These groups were continued to stimulate for6 h. The level of the m RNA expression of IL-α, IL-1β, IL-6, IL-10, MCSF and TNF-αwas evaluated by q RT-PCR. The protein production level in supernatant of the culture medium was measured by liquid chip assay.Results(1) The cell fused into a multinucleated cell after induced by 100 ng/ml LPS for 24 h.The osteoclast-like cells were positive for the TRAP staining.(2) The fluorescence could be observed with the laser scanning confocal microscope and Western blot, confirming that the expression of Ca SR on the surface of the cell line.(3) Meanwhile, the 3 μM NPS2143 was chosen as the most optimal dose of antagonist by observing the cell viability by CCK-8 and the apoptosis rate by flow cytometry(FCM). The gene expression level of IL-1α、IL-6、MCSF and TNF-α was unregulated,while IL-1β and IL-10 were much lower compared with the control group( P <0.05).The measurements of liquichip was similar to that from m RNA level in the change of IL-6、IL-10 and TNF-α(P<0.05). On the other hand, no significance in the secretion of IL-1α, IL-1β or MCSF protein production was observed in the supernatant.ConclusionWith RAW264.7 cell line, the change of cytokine expression and protein secretion could be partly explained by the activation of calcium sensing receptor. The specific antagonist of Ca SR NPS2143 promote the expression of IL-6 and TNF-α and inhibit the expression of IL-10 on both gene and protein level. Ca SR may be a critical mediator of modify the LPS stimulated inflammatory reaction.