Huogu I formula prevents steroid-induced osteonecrosis in rats by down-regulating PPARγ expression and activating Wnt/LRP5/β-catenin signaling
Authors: Xiangying Kong
Weiheng Chen
Na Lin
Daobing Liu
Yini Jiang
Ying Xu
Journal: Journal of Traditional Chinese Medicine (2014)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Huogu I formula; lipid metabolism; steroid-induced osteonecrosis of the femoral head; PPARgamma; Wnt signaling pathways

Abstract:  Objective: To investigate the effects of Huogu I formula on regulation of lipid metabolism in steroid-induced osteonecrosis of the femoral head (SONFH) rats and verify our hypothesis that Huogu I formula regulates lipid metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPARgamma) expression and activating Wnt signaling pathways. Methods: Eighty-five rats were divided into four groups: control, model, Huogu 15 g/kg and Huogu 30 g/kg. Six weeks later, animals were anaesthetized, femora was dissected for histopathological examination of the osteonecrotic changes and repair processes, micro computed tomography (Micro-CT)-based micro-angiography was performed to assess vascularization. Serum lipid levels were detected by haematological examination. The expressions of PPARy, Wnt3a, low density lipoprotein receptor-related protein 5 (LRP5) and beta-catenin were evaluated by immunohistochemistry, Western blot and quantitative real-time polymerase chain reaction analyses. Results: The incidence of osteonecrosis, ratio of empty lacuna, adipose tissue area and adipocyte perimeter in the bone marrow were dramatically lower in the Huogu I formula treatment groups. By micro-CT quantification, Huogu I formula treatment dose-dependently increased vessel volume, vessel surface, percentage of vessel volume and vessel thickness of the femoral heads of SONFH rats. Levels of serum lipid in Huogu 15 g/kg and Huogu 30 g/kg groups reduced significantly. Huogu I formula treatment could suppress the expression of PPARy and increase the expressions of Wnt3a, LRP5 and beta-catenin at both protein and mRNA levels. Conclusion: The results of our present study highlight the lipid-lowering potential of Huogu I formula, and provide further evidence of the involvement of the PPARgamma inhibition and Wnt/LRP5/ beta-catenin signaling activation in the effects of Huogu I formula. 

Retracted: No

PubPeer comments:
 Fig 2,  Huogu u2160 formula administration enhances femoral head neovascularization A: representative images of micro-CT reconstructed 3-D microangiography of proximal femur from control; B: model group; C: Huogu 15 g kg group ;D: Huogu 30 g kg group; E: statistical analysis on the differences of vessel volume; F: percentage of vessel volume; G: vessel surface; H: vessel thickness. ![file] Figure 2 from [Jiang et al (2014)]  Pravastatin administration enhances femoral head neovascularization. (a) Representative images of micro-CT reconstructed 3-D microangiography of proximal femur from control, model, Prava 10 mg kg, and Prava 20 mg kg groups. (b) Statistical analysis on the differences of vessel volume, vessel surface, percentage of vessel volume, and vessel thickness in the femoral heads of steroid-induced ONFH rats in different groups . ![file] The Control and Model images are the same, which seems reasonable if the data were all collected in one experiment and then separated for publication. However, the femoral-vasculature reconstruction for Prava 20 g kg has surprisingly much in common with that for Huogo-1 15 g kg. , 
 #1 There is much else to ponder about Fig 2. ![file] , 
 Fig 3 A-D.  Huogu u2160 formula administration suppresses PPARu03b3 expression in the femoral heads of SONFH rats Control and model group received no treatment. Huogu 15 g kg and Huogu 30 g kg groups respectively received Huogu u2160 formula dissolved in distilled water by oral gavage at dosages of 15 and 30 gu00b7kg-1 u00b7d-1 for 6 weeks. The protein expression levels of PPARu03b3 in the femoral heads of SONFH rats in control (A), model (B), Huogu 15 g kg group (C) and Huogu 30 g kg groups (D) were respectively detected by immunohistochemistry (u00d740). The curious glitch in 3A first caught my attention. ![file] It is possible, though, that 3B contains most easter-eggs. ![file] , 
 Figure 1  Huogu u2160 formula administration enhances osteogenesis Histological features of the normal bone in normal rats and the osteonecrotic bone in SONFH rats with or without Huogu u2160formula administration for six weeks (u00d720). A: histological features of the control group... ![file] 1(B). Model group. ![file] , 
 Figure 4A-C:  Huogu u2160 formula administration activates Wnt3a LRP5 u03b2-catenin signaling pathway in SONFH rats Protein expression levels of Wnt3a (A, D), low density lipoprotein receptor-related protein 5 (LRP5) (B,E), and u03b2-catenin (C, F) in the femoral heads of SONFH rats with or without Huogu u2160 formula administration were detected by Western blot, 1 for control, 2 for model, 3 for Huogu 15 g kg group and 4 for Huogu 30 g kg group . Here compared with (below) Figure 5 (b) from [Jiang et al (2014)] :  Pravastatin administration activates Wnt3a LRP5 b-catenin RUNX2 signaling pathway in steroid-induced ONFH rats. **Wnt3a, LRP5, b-catenin**, and RUNX2 expression at both mRNA and protein levels in the femoral heads of steroid-induced ONFH rats with or without pravastatin administration were detected by ...Western blot (b). ![file] The same GAPDH loading-control bands seem to be applied in both figures, despite the different lanes. , 
 #4 Fig 1D appears to have been stitched together from leopard-print fabric. ![file] , 
 More easter-eggs in Fig1A, red circles ![file] , 
 #7 OK, try Fig 3C. ![file] , 
 #8 Please check the green areas. ![file] , 
 More duplications in this figure, green areas. ![file] , 
 #1 ![] ![] A third sighting: Fig 4A from [Jiang et al (2014)] . ![] ,