Effect Of Aspirin On Osteogenic Differentiation Of BMSCs In Postmenopausal Osteoporosis

Objectives:The pathogenesis of postmenopausal osteoporosis(PMOP)is unclear,and studies have shown that aspirin has a definite effect on the treatment of PMOP.Osteoporosis and normal human bone mesenchymal stem cells(BMSCs)were used as the research object to investigate the effects of different doses of aspirin on osteogenic differentiation of BMSCs.This study sought the molecular mechanism of aspirin intervention for osteogenic differentiation,and explored the relationship between autophagy and osteogenic differentiation of BMSCs.We elucidated the molecular mechanism of aspirin in the treatment of PMOP and provided a new theoretical basis for the clinical prevention and treatment of PMOP.Methods:1.This study used clinical comparative analysis of experimental research methods to collect 45 inpatients in our hospital.According to the inclusion and exclusion criteria,20 patients were excluded.A total of 10 subjects with normal bone density and 15 patients with PMOP were included.All patients underwent joint replacement or intramedullary nailing.The intramedullary red bone marrow was collected and subcultured by density gradient centrifugation and adherent method.The purity of BMSCs was identified by immunofluorescence staining.2.BMSCs were intervened in different doses of aspirin during osteogenic induction.Alkaline phosphatase-calcium-cobalt staining and alkaline phosphatase assay,alizarin red staining and MTT were used to detect osteocalcification and cell proliferation.3.According to the different doses of aspirin on the osteogenic differentiation of BMSCs,the BMSCs in the osteoporosis group were selected before and after the induction eontrol group,the best dose group of aspirin and the highest dose group,and the total RNA was extracted,Transcriptome sequencing analysis and comparison of differential genes.4.Cell homogenate lysis in the pre-induction group,the post-induction control group,the aspirin optimal dose group,and the highest dose group.The protein of autophagy-related such as EEA-1,LAMPI,Atg7,Beclinl,P62 and LC-3B were detected concentration by SDS-polyacrylamide gel(SDS-PAGE)electrophoresis.In order to explore the molecular mechanism of the effect of aspirin on osteogenic differentiation of BMSCs.Results:There were no significant differences in age,fracture site,injury to surgery days,and BMI between the two groups included in the study(P>0.05).In the operation,BMSCs obtained from the femur red bone marrow of the two groups were isolated and cultured.The cells expressing CD 105-positive and CD45-negative cells by imnunofluorescence staining accounted for more than 95%of the total number of cells,which can meet the requirements of subsequent experiments.2.In the osteogenic differentiation of BMSCs,8 different doses of aspirin were added to intervene BMSCs.Alkaline phosphatase calcium and cobalt staining and alkaline phosphatase assay showed that the alkaline phosphatase content of BMSCs was higher when the aspirin dose was between 25 μM and 75 μM.The results of alizarin red staining showed that the aspirin dose was 5μM to 50μM,and more calcium crystals were formed for osteogenic induction.High doses of aspirin significantly inhibited the process of osteogenic differentiation.There was no significant difference in the proliferation rate of BMSCs between different doses of aspirin.3.In the analysis of RNA-seq sequencing results,the gene expression differences before induction,50 μM and 1000 μM were 4698,248 and 4069,respectively,compared with the 0 μM group.The GO enrichment of differential genes between groups was mainly concentrated in pathways such as biological regulation,cytoplasmic and protein binding.The KEGG enrichment of differential genes between the groups was mainly concentrated in the P13K-Akt signaling pathway,cancer pathway,cytokine-cytokine receptor interaction and calcium signaling.4.In the results of protein electrophoresis,the levels of autophagy in BMSCs were significantly increased after osteogenic induction.Under the intervention of 50 μM aspirin,the expression of LAMP1(P=0.0019 vs 0 μM)and Atg7(P=0.0074 vs 0 μM)was decreased,and the expression of LC3B-Ⅱ/LC3B-I(=0.9971 vs 0 μM)was not different.High doses of aspirin have a significant inhibitory effect on autophagy.Conclusions:1.Low dose of aspirin can promote osteogenic differentiation of BMSCs.2.High-dose aspirin has a significant inhibitory effect on osteogenic differentiation of BMSCs and is associated with inhibition of autophagy.3.Different doses of aspirin had no effect on the eell proliferation rate during differentiation of BMSCs.4.Increased autophagy is involved in the osteogenic differentiation of BMSCs,accompanied by changes in the expression of multiple gene pathways.