Mechanism Of Icariin Promoting Osteoblast Proliferation By Activating ERK Phosphorylation Through Non-nuclear Effect

Objective:To detect the mechanism of Icariin(ICA)promoting osteoblasts proliferation by activating ERK phosphorylation through non-nuclear effect,to elucidate the mechanism of regulating ICA's non-nuclear effect promoting OB proliferation,and to e×plore the relationship between the two.It is e×pected to provide reference for the discovery of new therapeutic targets against osteoporosis in Icariin.Methods:OB was e×tracted from newborn rat skull by enzymatic digestion and cultured in vitro.OB was identified by alkaline phosphatase(ALP)staining and calcium nodule staining.ICA and BSA are coupled,and ICA-BSA coupling is used to interfere with OB.Osteoblasts were divided into group A(ICA intervention group),group B(ica-bsa intervention group),group C(ICI182780+icariin-bsa intervention group)and group D(PD98059+icariin-bsa intervention group).The effect of the drugs on the proliferation of osteoblasts was detected by tetramethylazo salt colorimetry(MTT)and cytometry.The phosphorylation of ERK in osteoblasts was detected by Western Blot.The effect of calcium ion concentration changes in cells was observed under a confocal fluorescence microscope.Results:ICA,BSA and ica-bsa solution were scanned by uv-visible absorption spectrum.The peak value of the absorbance curve of ica-bsa solution was significantly different from that of ICA and BSA,indicating that energy transfer occurred between molecules,that is,ICA and BSA reacted to form a coupling.The impenetrability of the coupling was proved by liquid mass spectrometry.Flow cytometry and MTT results indicated that ICA-BSA promoted osteoblasts to enter mitosis,so it promoted the proliferation of osteoblasts.This effect was inhibited by the estrogen receptor antagonist ICI 182780 and MEK1PD98059 pathway inhibitors,suggesting that ICA's nonnuclear action in osteoblasts is associated with ER and MAPK signaling pathways.Western Blot showed that the e×pression level of ERK was higher in the ICA-BSA group,while the samples treated wittj the specific inhibitor PD98059 of MEK1 and the specific estrogen receptor antagonist ICI 182780 in the two groups had a significant inhibitory effect on the e×pression level of ERK.The free calcium in osteoblasts was measured by confocal laser.Within 30 seconds after the ica-bsa drug intervention,the intracellular fluorescence intensity was significantly higher than that in other groups,indicating the presence of non-nuclear effects.Conclusion:Impenetrable ica-bsa activates rapid phosphorylation of ERK in osteoblasts,increases intracellular calcium content,and promotes proliferation of osteoblasts.However,the estrogen receptor antagonist ICI 182780 and the MEK1 specific inhibitor PD98059 may affect the effect of ICA-BSA on osteoblasts and have inhibitory effect.Therefore,it can be inferred that estrogen receptor mediates the non-nuclear action of Icariin,and MAPK signaling pathway plays a crucial role in this process.