Study On Effects And Mechanism Of Ferulic Acid On Osteoblast By Regulating ENaC

Objective:To investigate the effects and mechanism of Ferulic acid (FA) on osteogenic function of Ob in vitro, in order to understand that what important role FA plays in metabolism bone disease, and provide a theoretical basis for the use of activating blood and resolving stasis in the prevention and treatment of osteoporosis(OP).Methods: The effective concentrations of FA were screened from 0.001-1000 mg.L^-1, and then select the final concentration of FA 7.8, 31.2, 125, 500 mg.L^-1 for the experiment.Primary osteoblasts were isolated from the SD neonatal rat's skull and cultured in DMEM containing 10% heat-inactivated FBS and antibiotics (100μg/mL streptomycin, 100 U/mL penicillin).The third generation of cells, the density of 1×10⁴/mL, was used in the experiment. Adherent cells (at 80–90% confluency) were trypsinized and seeded into 96 well plates at a density of 1×10⁴/mL. In some experiments, the medium was replaced with a fresh mixture supplemented with 5 mMβ-glycerophosphate and 50μg/mL ascorbate (i.e., osteogenic- promoting medium) 24 h after plating. Medium and inducing agents were changed every 3–4 days. The proliferation of Ob was analyzed by CCK-8 assay. Cell cycle was examined by flow cytometry. Alkaline phosphatase(ALP) was detected by alkaline phosphatase kit. Calcified nodules was observed using alizarin red staining. Elisa kit was used to measure the cGMP levels from Ob extracts. The mRNA synthesis of various Ob specific genes and cGMP/PKG/ENaC signaling related genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). Si RNA PKGâ…¡was transfected into cells following the Lipofectamine protocol.Results:(1)pharmacodynamic evaluation: FA markedly increased Ob proliferation in dose of 0.01 - 100 mg.L^-1,at 1000 mg.L^-1,it inhibited the proliferation,and alkaline phosphatase (ALP) activity was elevated by FA in of dose 1 - 1000 mg.L^-1.However,the selected concentration of FA (7.8, 31.2, 125, 500 mg.L^-1),compared with control group, four concentration groups all stimulated the proliferation of Ob(P<0.05),only 500 mg.L^-1 concentration group had significantly difference compared with other groups(P<0.01); The ALP activity was elvated in 4 doses, there were significant differences from each other except comparing between 31.2 and 125 mg.L^-1 groups. Cell cycle tested by flocytometry showed that 4 doses of FA only increased the proportion of G2/M phase, and did not influence the other part vs control with no dose dependent effect.FA (7.8, 31.2, 125 mg.L^-1) also increased calcified nodule formation, in which the 500 mg.L^-1 group didn't promote the formation of calcified nodules; Stimulation with FA increased the ALP,Osteopontin,Collagen-â… mRNA expression .(2) mechanism research: FA at concentration from 7.8 to 500 mg.L^-1 could increase the cGMP levels in OB(P<0.01),there was a dose-dependent increase among 7.8, 31.2, 125 mg.L^-1 groups but not in 125 and 500 mg.L^-1 groups. RT-PCR results showed that FA from 7.8 to 500 mg.L^-1 could promote PKGâ…¡mRNA expression, which is the downstream molecule of the cGMP signaling pathway(P<0.05), but have no significant effect on PKGⅠα,PKGⅠβmRNA expression. Moreover, FA induced expression of ENaCα, ENaCγmRNA in Ob, and the expression of ENaCαmRNA was reduced when PKGâ…¡small interfering RNA was introduced. However, the siRNA PKGâ…¡with FA, did not completely down-regulate ENaCαmRNA expression, which was higher than siRNA PKGâ…¡alone(P<0.01) and lower than FA alone(P<0.05). Conclusion: FA could enhance proliferation, differentiation and mineralization of Ob in vitro, and promote the expression of osteogenic genes. The mechanism may be partly related to cGMP- PKGâ…¡-ENaC signaling pathway, and partly related to FA or cGMP directly regulating ENaC.