title: A novel semisynthesized small molecule icaritin reduces incidence of steroid-associated osteonecrosis with inhibition of both thrombosis and lipid-deposition in a dose-dependent manner.
abstract: BACKGROUND
  Intravascular-thrombosis and extravascular-lipid-deposition are the two key pathogenic events considered to interrupt intraosseous blood supply during steroid-associated osteonecrosis (ON) development. However, there are no reported candidate agents capable of simultaneously targeting these two key pathogenic events. The authors' published experimental studies have shown that Epimedium-derived flavonoids possess an anti-ON effect. Further, the authors have recently identified a small molecule Icaritin as an intestinal metabolite of Epimedium-derived flavonoids.
  
  
  OBJECTIVE
  The present study was to evaluate the prevention effect of the available semisynthesized small molecule Icaritin on steroid-associated ON development in a rabbit model.
  
  
  METHODS
  After receiving an established inductive protocol for inducing steroid-associated ON, eighty-four male 28-week-old New-Zealand white rabbits were divided into the following three daily oral administration groups, including low dose Icaritin group (L-ICT; n=28; 5 mg x kg(-1) x day(-1)), high dose Icaritin group (H-ICT; n=28; 10 mg x kg(-1) x day(-1)), and control vehicle group (CON; n=28). Before and after induction, dynamic contrast-enhanced MRI was performed on proximal femur for intra-osseous perfusion function index. Meanwhile, blood samples were examined for coagulation, fibrinolysis, lipid-transportation, endothelium injury, oxidative stress, and hepatocyte injury index, while marrow samples were quantified for adipogenic potential index of mesenchymal stem cell by in vitro culture and proliferator-activated receptor-gamma (PPARgamma) protein expression by western blot. At baseline, week 1 and 2 post-induction, 4, 8 and 16 rabbits in each group were sacrificed, respectively. After sacrifice, femora were dissected for micro-CT-based micro-angiography, followed by histological examination of ON lesion, intravascular thrombosis, extravascular fat-cell and vascular endothelial growth factor (VEGF) localized expression.
  
  
  RESULTS
  The ON incidence in the L-ICT and H-ICT groups was both significantly lower than that in the CON group (p<0.05 for both). The ON incidence in the H-ICT group was significantly lower than that in the L-ICT group (p<0.05). A significant decrease in the vascularization index and a significant increase in the permeability index seen in the CON group was attenuated in the L-ICT group and almost prevented in the H-ICT group at week 1 post-induction. Reduced perfusion to vessel-like structural units was more rarely found in the H-ICT group than in the L-ICT group. Regarding intravascular thrombosis, a significant increase in the thrombotic vessel count, endothelium injury index, coagulation index, and a significant decrease in both the fibrinolysis and oxidative stress index in the CON group were attenuated in the L-ICT group and prevented in the H-ICT group. For extravascular lipid-deposition, a significant increase in the fat cell area fraction, adipogenic potential index, PPARgamma expression and lipid-transportation index in the CON group was attenuated in the L-ICT group and prevented in the H-ICT group. Increased immunoreactivity of VEGF in the CON group was attenuated in the L-ICT group and prevented in the H-ICT group. Regarding safety, the hepatocyte injury index did not show significant change from baseline in any group.
  
  
  CONCLUSIONS
  Icaritin, a novel semisynthesized small molecule with osteoprotective potential, exerts dose-dependent effect on reducing incidence of steroid-associated ON with inhibition of both intravascular thrombosis and extravascular lipid-deposition. Suppression of the up-regulated PPARgamma expression for extravascular adipogenesis of mesenchymal stem cells and protection from activated oxidative stress for intravascular endothelium injury were found to be involved in the underlying mechanisms.
authors.affiliation: Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, the Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR.
authors.forename: Ge
authors.initials: G
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Qin
authors.forename: Hui
authors.initials: H
authors.lastname: Sheng
authors.forename: Xin-Luan
authors.initials: XL
authors.lastname: Wang
authors.forename: Yi-Xiang
authors.initials: YX
authors.lastname: Wang
authors.forename: David Ka-Wai
authors.initials: DK
authors.lastname: Yeung
authors.forename: James F
authors.initials: JF
authors.lastname: Griffith
authors.forename: Xin-Sheng
authors.initials: XS
authors.lastname: Yao
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xie
authors.forename: Zi-Rong
authors.initials: ZR
authors.lastname: Li
authors.forename: Kwong-Man
authors.initials: KM
authors.lastname: Lee
authors.forename: Kwok-Sui
authors.initials: KS
authors.lastname: Leung
mesh_terms: D000792:Angiography; D000818:Animals; D001808:Blood Vessels / Q000187:drug effects / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000008:administration & dosage* / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D050356:Lipid Metabolism / Q000187:drug effects; D008279:Magnetic Resonance Imaging; D008297:Male; D010020:Osteonecrosis / Q000188:drug therapy / Q000209:etiology* / Q000473:pathology / Q000517:prevention & control*; D010316:Particle Size; D010477:Perfusion; D011817:Rabbits; D013256:Steroids / Q000009:adverse effects*; D013927:Thrombosis / Q000188:drug therapy* / Q000517:prevention & control; D016896:Treatment Outcome; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D055114:X-Ray Microtomography
chemical_list: D005419:Flavonoids; D013256:Steroids; D042461:Vascular Endothelial Growth Factor A; C499403:icaritin