Apigenin accelerates lipopolysaccharide induced apoptosis in mesenchymal stem ceils through suppressing vitamin D receptor expression

Background Transplantation of mensenchymal stem cells （MSCs） has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extremely harsh microenvironment such as excessive inflammation. Apigenin （API） has been reported to protect cells against inflammatory damage and cell death by exhibiting anti-inflammatory and anti-oxidative capacity. Here we investigated the modulatory effects of API in lipopolysaccharide （LPS）-mediated inflammation and apoptosis of MSCs, and further defined the underlying mechanism. Methods Effects of different concentrations of API （0, 5, 10, 20, 40 and 80 IJmol/L） for 24 hours, and LPS （0, 0.5 and 5.0 pg/ml） for 6 hours and 24 hours on MSCs viability were assayed by MTT. Based on this, MSCs were pretreated with different concentrations of API （0-40μmol/L） at the indicated times （6, 12 and 24 hours） followed by exposure to 5μg/ml LPS for 24 hours. MTT, phase-contrast microscopy, annexinV/propidium iodide （PI） double stain flow cytometry （FCM） and Hoechst staining were applied to explore the effects of API on MSCs induced by 5 μg/ml LPS for 24 hours. In addition, reverse-transcription polymerase chain reaction （RT-PCR） was applied to detect the mRNA expression of pro-inflammatory factors including cyclooxygenase-2 （COX-2）, inducible nitric oxide synthase （iNOS）, nuclear factor-kappa B （NF-KB）, pro-apoptotic gene caspase-3, Bad, and anti-apoptotic gene Bcl-2. Moreover, AutoDock software was used to imitate the docking score of API and vitamin D receptor （VDR）. In parallel, Western blotting and RT-PCR were used to investigate protein and mRNA expression of VDR. Results MSCs stimulated with LPS 5 IJg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API （0-40μmol/L） had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-KB at different time points in MSCs induced by LPS, except for API at the concentration of 5 μmol/L Results from phase-contrast microscopy, MTT, Hoechst staining and AnnexinV/PI double stain FCM demonstrated that with the increasing concentrations of API and extension of administrating time, significant morphological changes of MSCs occurred, viability of cells was strongly inhibited, and meanwhile, apoptosis of LPS-administrated MSCs was exacerbated, compared with LPS individual group. In addition, API promoted caspase-3, Bad mRNA expression and inhibited Bc