Puerarin Activates P38 And ERK1/2 Signaling Pathways And Promotes MC3T3-E1 Cell Proliferation

Objective:Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and degraded microstructure of bone,resulting in increased brittleness of the bone and prone to fracture.More common in elderly animals and post-sterilized animals.With the development of society,more and more pet owners will choose to perform sterilization for their pets.After the animals are sterilized,the level of estrogen in the body will decrease significantly,and the decrease in estrogen may cause osteoporosis.However,long-term estrogen supplementation will increase the risk of animals suffering from breast cancer and symmetrical hair loss.Therefore,the researchers sought to find a safe estrogen substitute with less side effects.Puerarin is a phytoestrogen,and the treatment effect of osteoporosis is similar to that of estrogen,but without the side effects of estrogen.This study intends to study the effect of puerarin on the proliferation of MC3T3-E1 osteoblasts at the cellular and molecular levels,and observe the dose-effect relationship and time-effect relationship.After further inhibiting the activities of P38 and ERK1/2,the main members of the MAPK signaling pathway in MC3T3-E1 osteoblasts,the expression of various markers during the colonization of downstream proteins and MC3T3-E1 cells was observed and compared to clarify the effect of puerarin on MC3T3-E1 The cell's mechanism of action and clarify the signaling pathways involved.Methods and results:Add different concentrations(0mol/L,10^-7mol/L,10^-6mol/L,10^-5mol/L,10^-4mol/L,10^-3mol/L)to MC3T3-E1 cell culture medium The puerarin was collected after 24 hours of culture,and the cell proliferation ability was detected by CCK-8.The activity of ALP in each group was detected by pNPP method.The results showed that puerarin at a concentration of 10^-6mol/L proliferated MC3T3-E1 cells.And ALP's vitality is the most obvious.Therefore,puerarin with a concentration of 10^-6mol/L was used for subsequent experiments.MC3T3-E1 cells were treated with puerarin at a concentration of 10^-6mol/L and cultured for 24h,36h,48h and 60h,respectively.The CCK-8 method was used to detect cell proliferation.The results showed that the cells were in the best condition after 48 hours of culture.Therefore,the cell culture time for subsequent experiments is 48h.The experiment was divided into blank control group,puerarin group,puerarin+P38 inhibitor group?PUE+SB203580?and puerarin+ERK1/2 inhibitor group?PUE+SCH772984?,and acted on MC3T3-E1 cells and cultured After 48h,the cell proliferation ability,ALP activity and type I collagen secretion level of each group were detected by CCK-8,pNPP and ELISA methods respectively,and the expression levels of P-P38 and P-ERK1/2 protein were detected by Western Blotting.The results showed that compared with the blank control group,the expression levels of P-P38and P-ERK1/2 protein in puerarin group were significantly increased.Puerarin can promote the proliferation of MC3T3-E1 cells and secrete ALP and type I collagen.Compared with the puerarin group,the puerarin+P38 inhibitor group can significantly inhibit the P-P38 protein expression level,and the puerarin+ERK1/2 inhibitor group can significantly inhibit the P-ERK1/2 protein expression level.Adding P38 and ERK1/2 signaling pathway inhibitors can significantly inhibit the proliferation of MC3T3-E1 cells and secrete ALP and type I collagen.Conclusion:Puerarin can promote the proliferation of MC3T3-E1 cells by activating P38 and ERK1/2 signaling pathways.Puerarin can promote the secretion of ALP and type I collagen by MC3T3-E1 cells by activating P38 and ERK1/2 signaling pathways,thereby promoting MC3T3-E1 Cell Differentiation.