Study On The Effect Of Ferulic Acid On Osteogenic Ability Of MC3T3-E1 Cells Under High Glucose Environment And Related Mechanism

BackgroundDiabetes is a common endocrine disease,hyperglycemia in diabetic patients leads to a series of bone destruction diseases.The functional damage of osteoblasts plays an important role in the occurrence of such bone destruction-related diseases,so finding effective drugs to protect osteoblasts has become a research focus for the treatment of these diseases.But currently,there are very limited therapeutic drugs for treatment.Ferulic acid is a natural ingredient exists in some Chinese herbs such as Angelica sinensis and Chuanxiong.It has been confirmed by previous studies that it has anti-inflammatory,antioxidant and antisepsis biological effects,but its effect on the osteogenic ability of osteoblasts has not been studied yet.ObjectiveThe purpose of this study was to investigate the effect of ferulic acid on the osteogenic function of mouse pre-osteoblasts(MC3T3-E1 cells)under high glucose environment,and to explore the possible mechanism it may involve.MethodsMC3T3-E1 cells were cultured in the high glucose medium containing different concentrations of ferulic acid,we used the CCK-8 experiment to detect cell proliferation viability to screen most appropriate drug intervention condition(denoted as FA group).Under these interference condition,the reactive oxygen species(ROS)content was detected by flow cytometry to reflect the level of oxidative stress.The expression levels of interleukin-1(IL-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and advanced glycation end products(AGEs)were detected by ELISA kit to reflect the level of cellular inflammation.Alkaline phosphatase(ALP)assay kit was used to determine the activity of ALP to reflect the function of osteogenic differentiation.Alizarin red staining was used to determine the formation of mineralized nodules to reflect the mineralization function of cells.Finally,after intervened by signaling pathway specific inhibitor LY294002,the expression of key osteogenic genes such as Runt-associated transcription factor 2(Runx2),osteocalcin(OCN)and osteopontin(OPN)was detected by qPCR,the expression of osteogenic related proteins OCN、OPN、Runx2 and key proteins in PI3K/Akt signaling pathway such as p-PI3K,p-Akt,PI3K,Akt was detected by Western blot.ResultsIn the ferulic acid concentration screening experiment,CCK-8 results showed that 0.1-10 μM ferulic acid can alleviate the suppression of MC3T3-E1 cells proliferation viability under high glucose environment(P<0.05),this promoting effect of ferulic acid at the concentration of 1 μM was more obvious.Moreover,1μM ferulic acid could restrain the level of oxidative stress and cellular inflammation,decrease the expression of ROS,AGEs,inflammatory cytokines IL-1,IL-6 and TNFa in the high glucose condition(P<0.01).In addition,it can improve the ALP activity,mineralized nodules formation and the key osteogenic genes OCN\OPN\Runx2 mRNA expression(P<0.05).In the FA group(1 μM),the inhibitory effect of high glucose environment on key pathway proteins p-Akt and p-PI3K was relieved(P<0.01).However,in the inhibitor group,the expression levels of these proteins were decreased to a certain degree compared with the FA group(P<0.05).ConclusionAppropriate ferulic acid has protective effect on alleviating the damage of osteogenic function on MC3T3-E1 cells under glucose environment,which may play a positive role in promoting bone formation.In addition,the PI3K/Akt signaling pathway may be involved in the regulation on above process.