title: Apigenin accelerates lipopolysaccharide induced apoptosis in mesenchymal stem cells through suppressing vitamin D receptor expression.
abstract: 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 µmol/L) for 24 hours, and LPS (0, 0.5 and 5.0 µg/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 - 40 µmol/L) at the indicated times (6, 12 and 24 hours) followed by exposure to 5 µg/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-κB), 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 µg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API (0 - 40 µmol/L) had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-κB 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 Bcl-2 mRNA expression in a time-dependent and concentration- dependent manner. Further study found that pro-apoptosis effect of API was related to suppress VDR expression.
  
  
  CONCLUSIONS
  API could inhibit the expression of inducible inflammatory factors, therefore exert the strong anti-inflammatory function. However, API could not protect MSC apoptosis induced by LPS but amplified the apoptosis. The apoptosis is related to Bad/Bcl-2 increasing and caspase-3 activation, which is mediated through suppressing VDR expression.
authors.affiliation: Institute of Orthopaedic Diseases, Jinan University & Department of Orthopaedics, First Affiliated Hospital of Jinan University, Guangzhou, Guangdong 510630, China.
authors.forename: Huan-Tian
authors.initials: HT
authors.lastname: Zhang
authors.forename: Zhen-Gang
authors.initials: ZG
authors.lastname: Zha
authors.forename: Jia-Hui
authors.initials: JH
authors.lastname: Cao
authors.forename: Zu-Jian
authors.initials: ZJ
authors.lastname: Liang
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.forename: Ming-Tao
authors.initials: MT
authors.lastname: He
authors.forename: Xiao
authors.initials: X
authors.lastname: Zang
authors.forename: Ping
authors.initials: P
authors.lastname: Yao
authors.forename: Jia-Qing
authors.initials: JQ
authors.lastname: Zhang
mesh_terms: D000818:Animals; D047310:Apigenin / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D008070:Lipopolysaccharides; D018167:Receptors, Calcitriol; D047310:Apigenin