Anti-inflammtion Drug Of Chiral Heterocyclic Compounds For The Osteogenic Differention Of Mesenchymal Stem Cells

OBJECTIVE:Bone defects caused by tumor,trauma and other reasons have been troubling many orthopedics and stomatologists,but we can not ignore the impact of inflammation and infection on the body.Excessive acute inflammatory response and the transformation of untreated acute inflammatory response into long-term chronic inflammation can stimulate osteoclast formation and impair the osteogenic differentiation function of stem cells,resulting in the imbalance between bone formation and bone resorption,and resulting in poor bone healing finally.Recently,the collaborator of our project developed a new class of synthetic compounds with chiral heterocyclic structure,with good anti-inflammatory activity and low toxicity.Therefore,this study explored a new method for the regeneration of inflammatory bone defects by adding chiral heterocyclic compounds to reduce the effect of inflammatory factors on bone marrow mesenchymal stem cells（BMSCs）and restore the osteogenic differentiation function of stem cells.Methods:We used lipopolysaccharide derived from Escherichia coli on BMSCs in vitro.Different concentrations of LPS(0,0.5μg·m L^-1,1μg·m L^-1,2μg·m L^-1,5μg·m L^-1,10μg·m L^-1)were added into the culture medium of BMSCs for different time（1 day,3 days,5 days,7 days）.Cell Counting Kit-8（CCK-8）was used to detect the effects of LPS on cell viability.According to CCK-8 results,different concentrations of LPS(0,0.5μg·m L^-1,1μg·m L^-1,2μg·m L^-1)were added into BMSCs culture medium,and cultured for different time（1 day,2 days,3 days）.The expression levels of TNF-ɑ,IL-1βand IL-6 were detected by RT-PCR to verify the effects of different concentrations of LPS and different duration of LPS on BMSCs.CCK-8,ELASA and RT-RCR were used to select the compound with best anti-inflammatory effects from five chiral heterocyclic compounds.The compound were treated with inflammatory BMSC and cultured for 7 days,14 days,21 days,respectively.The expression levels of RUNX2,ALP,OCN and COL1A1 were detected by RT-PCR,ALP activity was detected by alkaline phosphatase staining assay,calcium nodules were detected by alizarin red assay,and protein formation was detected by immunofluorescence assay.It aims to verify the ability of chiral heterocyclic compounds to promote the osteogenic differentiation of BMSCs in inflammatory microenvironment.RESULTS:The experimental results showed that the concentration of LPS higher than 1μg·ml^-1 could inhibite the activity of BMSCs.The higher the LPS concentration was,the more obvious the inhibition effect was.And the cell activity decreased further with the increase of LPS duration.Compared with BMSCs under normal condition,the expression level of inflammatory genes in BMSCs increased after LPS treatment.With the increase of LPS treatment time,the higher the LPS concentration,the more obvious the expression level of inflammatory genes.Compared with normal BMSCs,the expression level of osteogenic genes,the number of calcium nodules and alkaline phosphatase staining of BMSCs in inflammatory state decreased significantly.In addition,CCK-8 and RT-PCR results showed that compound C had the best anti-inflammatory activity.When 10μmol·L^-1 compound C was used on inflammatory BMSCs,the expression levels of inflammatory genes and proteins were significantly decreased,the expression levels of osteogenic genes and proteins were increased,and the staining area of alizarin red and alkaline phosphatase was increased compared with that of inflammatory BMSCs without adding compound C.Conclusion:The inflammatory model of BMSCs was successfully constructed by LPS.LPS at the concentration of 1μg·m L^-1 or below had no significant effect on the activity of BMSCs.However,with the increase of LPS concentration,the activity of BMSCs cells was inhibited.With the increase of LPS treatment time,the inhibitory effect increased,and the osteogenic differentiation function of BMSCs was impaired.Chiral heterocyclic compound C has good anti-inflammatory activity,which can inhibit the inflammatory cytokines and restore the osteogenic differentiation function of stem cells.Therefore,this study provides a new treatment method for inflammatory bone defect through the ability of anti-inflammtion and osteogenic differentiation from chiral heterocyclic compounds.