scholarly journals The Protective Effect of Cordycepin On Alcohol-Induced Osteonecrosis of the Femoral Head

2017 ◽  
Vol 42 (6) ◽  
pp. 2391-2403 ◽  
Author(s):  
Yi-Xuan Chen ◽  
Dao-Yu Zhu ◽  
Zheng-Liang Xu ◽  
Jun-Hui Yin ◽  
Xiao-Wei Yu ◽  
...  
Keyword(s):  
Femoral Head ◽  
Protective Effect ◽  
Immunohistochemical Staining ◽  
Critical Role ◽  
Micro Ct ◽  
Bone Mesenchymal Stem Cells ◽  
Western Blots ◽  
Alizarin Red ◽  
Alp Activity

Background: Alcohol abuse is known to be a leading risk factor for atraumatic osteonecrosis of the femoral head (ONFH), in which the suppression of osteogenesis plays a critical role. Cordycepin benefits bone metabolism; however, there has been no study to determine its effect on osteonecrosis. Methods: Human bone mesenchymal stem cells (hBMSCs) were identified by multi-lineage differentiation. Alkaline phosphatase (ALP) activity, RT-PCR, western blots, immunofluorescent assay and Alizarin red staining of BMSCs were evaluated. A rat model of alcohol-induced ONFH was established to investigate the protective role of cordycepin against ethanol. Hematoxylin & eosin (H&E) staining and micro-computerized tomography (micro-CT) were performed to observe ONFH. Apoptosis was assessed by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemical staining was carried out to detect OCN and COL1. Results: Ethanol significantly suppressed ALP activity, decreased gene expression of OCN and BMP2, lowered levels of RUNX2 protein, and reduced immunofluorescence staining of OCN and COL1 and calcium formation of hBMSCs. However, these inhibitory effects were attenuated by cordycepin co-treatment at concentrations of 1 and 10 µg/mL Moreover, it was revealed that the osteo-protective effect of cordycepin was associated with modulation of the Wnt/β-catenin pathway. In vivo, by micro-CT, TUNEL and immunohistochemical staining of OCN and COL1, we found that cordycepin administration prevented alcohol-induced ONFH. Conclusion: Cordycepin treatment to enhance osteogenesis may be considered a potential therapeutic approach to prevent the development of alcohol-induced ONFH.

scholarly journals Loss of Signal Transducer and Activator of Transcription 3 Impaired the Osteogenesis of Mesenchymal Progenitor Cells in Vivo and in Vitro

2021 ◽  
Author(s):  
ZIJING HUANG ◽  
Jingyi Feng ◽  
Xin Feng ◽  
Laiting Chan ◽  
Jiarui Lu ◽  
...  
Keyword(s):  
Critical Role ◽  
Micro Ct ◽  
Signal Transducer ◽  
Alizarin Red ◽  
Mesenchymal Progenitors ◽  
Osteogenic Markers ◽  
Osteogenic Induction ◽  
Transcription 3

Abstract Background: Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors.Methods: Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and Micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers.Results: Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions: Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.

scholarly journals Loss of signal transducer and activator of transcription 3 impaired the osteogenesis of mesenchymal progenitor cells in vivo and in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zijing Huang ◽  
Jingyi Feng ◽  
Xin Feng ◽  
Laiting Chan ◽  
Jiarui Lu ◽  
...  
Keyword(s):  
Critical Role ◽  
Micro Ct ◽  
Signal Transducer ◽  
Alizarin Red ◽  
Mesenchymal Progenitors ◽  
Osteogenic Markers ◽  
Osteogenic Induction ◽  
Transcription 3

Abstract Background Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors. Methods Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers. Results Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.

scholarly journals Loss of signal transducer and activator of transcription 3 impaired the osteogenesis of mesenchymal progenitor cells in vivo and in vitro

2021 ◽  
Author(s):  
ZIJING HUANG ◽  
Jingyi Feng ◽  
Xin Feng ◽  
Laiting Chan ◽  
Jiarui Lu ◽  
...  
Keyword(s):  
Critical Role ◽  
Micro Ct ◽  
Signal Transducer ◽  
Alizarin Red ◽  
Mesenchymal Progenitors ◽  
Osteogenic Markers ◽  
Osteogenic Induction ◽  
Transcription 3

Abstract Background Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors. Methods Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and Micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers. Results Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.

scholarly journals Elucidation on Predominant Pathways Involved in the Differentiation and Mineralization of Odontoblast-Like Cells by Selective Blockade of Mitogen-Activated Protein Kinases

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Jia Tang ◽  
Takashi Saito
Keyword(s):  
Cell Differentiation ◽  
Signaling Pathway ◽  
Critical Role ◽  
Mapk Signaling ◽  
Significant Inhibition ◽  
Mitogen Activated Protein Kinase ◽  
Alizarin Red ◽  
Selective Blockade ◽  
Alp Activity ◽  
Mitogen Activated Protein

Aim. To analyze the effect of three mitogen-activated protein kinase (MAPK) inhibitors, namely, SB202190 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (ERK inhibitor) in Dex-stimulated MDPC-23 cell differentiation and mineralization. Methods. Experiment was divided into five groups, control (cells without Dex and inhibitors treatment), Dex (cells with Dex treatment but without inhibitors), Dex + SB202190, Dex + SP600125, and Dex + PD98059. Cell differentiation was assessed by alkaline phosphatase (ALP) activity assay and real time RT-PCR. Cell mineralization was investigated by alizarin red staining. Results. Exposure to SB202190 (20 μM) significantly decreased the mineral deposition in Dex-treated cells as demonstrated by alizarin red staining. Treatment of SP600125 (20 μM) attenuated the mineralization as well, albeit at a lower degree as compared to SB202190 (20 μM). Similarly, SB202190 (20 μM) completely abrogated the ALP activity stimulated by Dex at six days in culture, while no changes were observed with regard to ALP activity in SP600125 (20 μM) and PD98059 (20 μM) treated cells. The upregulation of bone sialoprotein (BSP), ALP, and osteopontin (OPN) in Dex challenged cells was completely inhibited by SB202190. Conclusion. Blockade of p38-MAPK signaling pathway resulted in significant inhibition of ALP activity, mineralization, and downregulation of osteogenic markers. The data implicated that p38 signaling pathway plays a critical role in the regulation of MDPC-23 cells differentiation and mineralization.

scholarly journals Silencing of Long Non-Coding RNA NONHSAT009968 Ameliorates the Staphylococcal Protein A-Inhibited Osteogenic Differentiation in Human Bone Mesenchymal Stem Cells

2016 ◽  
Vol 39 (4) ◽  
pp. 1347-1359 ◽  
Author(s):  
Yi Cui ◽  
Sheng Lu ◽  
Hongbo Tan ◽  
Jun Li ◽  
Min Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Activity Detection ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Inflammatory Microenvironment ◽  
Alp Activity ◽  
Spa Treatment

Background/Aims: Osteomyelitis is defined as an inflammation of the bones and bone marrow. The inflammatory microenvironment attenuates the osteogenic differentiation capacity of stem cells and inhibits osteoblast-mediated bone formation, leading to net bone loss. However, the whole expression profile, function and side effect of long non-coding RNAs (lncRNAs) on osteogenic differentiation of stem cells in an inflammatory microenvironment of osteomyelitis are not known. Methods: In the present study, human bone mesenchymal stem cells (hBMSCs) were treated with different concentrations of Staphylococcal protein A (SpA) to trigger an inflammatory microenvironment in vitro to partly duplicate the inflammatory microenvironment of osteomyelitis, which was confirmed using ELISA for detecting the inflammatory cytokines. The complete expression profiles of lncRNAs and mRNA during osteogenic differentiation of hBMSCs in an inflammatory microenvironment triggered by SpA were analyzed using a lncRNA microarray. LncRNA expression levels were verified by quantitative reverse transcription PCR analysis (qRT-PCR). The expression of NONHSAT009968 in hB-MSCs was silenced by infection with lentivirus expressing NONHSAT009968-shRNA. The expression of Runx2, OCN, OPN, COL1A1, and alkaline phosphatase (ALP) activity was detected by western blot. Alizarin red staining and ALP activity detection were carried out. Results: The results of ELISA showed that SpA treatment induced secretion of inflammatory cytokines IL-1A, IL-6, and TNFA. The results of alizarin red staining and ALP detection showed that SpA treatment suppressed the osteogenic differentiation of hBMSCs. A total of 2033 lncRNAs were found with aberrant expression in SpA-treated hBMSCs compared to controls. Among these lncRNAs, 641 were down-regulated and 1392 were up-regulated. Based on the results of qRT-PCR, lncRNA NONHSAT009968 was chosen for further investigation. The results of alizarin red staining, ALP activity detection, and western blot detection of Runx2, OCN, OPN, COL1A1, and ALP indicated that NONHSAT009968 silencing ameliorates SpA-inhibited osteogenic differentiation in hBMSCs. Conclusion: Our present study provides a basis for future analyses of the role of lncRNAs in osteoblastic differentiation in an inflammatory environment triggered by SpA, and lncRNA NONHSAT009968 might be a new target for promoting osteoblast formation.

scholarly journals Melatonin attenuates vascular calcification by activating mitochondrial fusion and mitophagy via an AMPK/OPA1 signaling pathway

2019 ◽  
Author(s):  
chenweiren chen ◽  
jia qi yang ◽  
fang liu ◽  
xue qin shen ◽  
yuan sha
Keyword(s):  
Vascular Calcification ◽  
Mitochondrial Fusion ◽  
Calcium Deposition ◽  
Protective Effects ◽  
Melatonin Treatment ◽  
Western Blots ◽  
Alizarin Red ◽  
Mitofusin 2 ◽  
Alp Activity ◽  
Compound C

Abstract Background: Mitochondrial fusion/mitophagy play a role in cardiovascular calcification. Melatonin has been shown to protect against cardiovascular disease. This study sought to explore whether melatonin attenuates vascular calcification by regulating mitochondrial fusion/mitophagy via an AMP activated protein kinase/ Optic atrophy 1 (AMPK/OPA1) signaling pathway.Methods: The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualised by Alizarin red staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2), mitofusin 2 (Mfn2), mito-light chain 3 II (LC3II) and cleaved caspase3. Results: Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase3 were found to be down-regulated and Mfn2 or mito-LC3II was found to be enhanced in response to melatonin, together with a decrease in mitochondrial superoxide levels. Melatonin also maintained mitochondrial function and promoted mitochondrial fusion/mitophagy via OPA1 pathway. But OPA1 deletion abolished the protective effects of melatonin on VSMC calcification. Melatonin treatment significantly increased the p-AMPK and OPA1 protein expression. Treatment with compound C ablated the benefit observed with melatonin treatment. Conclusions: Melatonin protects VSMC against calcification by promoting mitochondrial fusion/mitophagy via AMPK/OPA1 pathway.

Pluripotent Unrestricted Somatic Stem Cells (USSC) from Cord Blood Generate Hepatocytes in Vivo, but In Vitro Convert into Immature Endodermal Precursors: The Critical Role of the Biological Niche.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3596-3596
Author(s):  
Sandra Sensken ◽  
Juergen Enczmann ◽  
Thorsten Trapp ◽  
Esmail Zanjani ◽  
Peter Wernet ◽  
...  
Keyword(s):  
Exocrine Pancreas ◽  
Critical Role ◽  
Oncostatin M ◽  
Liver Development ◽  
Pancreas Development ◽  
Western Blots ◽  
Fetal Sheep ◽  
Pancreatic Development

Abstract As shown recently by our group (Koegler et al. 2004; J. Exp. Med.200: 19:1–13) transplantation of USSC in a non-injury model, the preimmune fetal sheep, resulted in more than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion. Here we examined whether we are able to trigger USSC in vitro into the an endodermal differentiation pathway applying protocols described for both embryonal as well as adult stem cell. The following matrix/growth factors/organic substances were applied: Neurobasal medium/supplement B27/bFGF with and without nicotinamid, on fibronectin, laminin or matrigel for differentiation into the pancreatic development as well as HGF, NGF, bFGF and HGF, FGF4 with or without Oncostatin M to induce liver development. 15 different USSC lines were differentiated for 1, 2, 3, 4 and 6 weeks. Primers were designed with the strategy to define stages of endodermal development on the basis of the embryonic cell development from mouse and human. Therefore the following markers were established: GATA 4, HNF-1, HNF3ß and HNF 4a to define the embryonic and visceral (extra-embryonic) endoderm, a common precursor phenotype for both liver/exocrine pancreas development. To further assess differentiation into liver cells, a-1 antitrypsin, a-fetoprotein, albumin, HGF, Cyp2B6, Cyp3A4, Gys2 and PDX-1, PAX4, ISL-1, NKx6.1, NeuroD, insulin to determine differentiation into the pancreatic development as well as epithelial markers as CK8, CK18, CK19 were analyzed by RT-PCR and subsequent hybridization using gene-specific oligonucleotide probes. USSC were tested negativ for the majority of the markers, only HGF and cytokeratin markers CK8/18 and CK19 were tested positiv. In vitro differentiation shows that USSC never expressed Neuro D, HNF1, HNF3b, PDX-1, PAX4, insulin and a-fetoprotein, but do express depending on the culture conditions common endodermal precursor markers HNF4a, GATA4 (but not HNF1 or HNF3ß) as well as albumin, Cyp2B6, Cyp3A4, Gys2 (liver development) and Nkx6.1 and ISL-1 (pancreatic development. It is very interesting to note that ISL-1, required for the formation of the dorsal mesenchym and essential for the dorsal exocrine pancreas development is strong expressed, although all other markers as PDX-1 and PAX4 were always tested negative. We have never observed the expression of a-fetoprotein, which might be explained by the kinetic of this factor (Young-Yang, Nat.Cell Biology 2004). In summary, the results show that under the vitro conditions applied, only an endodermal precursor phenotype could be established. Therefore at present co-cultivation experiments with injured hepatocytes from sheep/rat/human are performed to mimic the biological niche in vitro as shown by Young-Yang et al. Western blots with an human albumin specific antibody (moAb, clone HAS-11, Sigma) revealed that this approach resulted in an strong expression of albumin in USSC. Moreover, this co-cultivation model is useful for the identification and characterization of factors who are responsible for the biological niche in vivo.

scholarly journals Protective Effects of Total Saponins of Panax Notoginseng on Steroid-Induced Avascular Necrosis of the Femoral Head In Vivo and In Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Heng-feng Yuan ◽  
Jian-feng Pan ◽  
Shuo Li ◽  
Chang-an Guo ◽  
Shu-hao Liu ◽  
...  
Keyword(s):  
Femoral Head ◽  
Protective Effect ◽  
Avascular Necrosis ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Panax Notoginseng ◽  
Protective Effects ◽  
Model Group

This research was designed to investigate the protective effects of TSPN on steroid-induced avascular necrosis of the femoral head (ANFH) and the likely mechanisms of those effects. As an in vivo study, TSPN was shown to be protective against steroid-induced ANFH due to the upregulation of VEGF-A. Furthermore, TSPN attenuated the apoptosis of osteocytes and reduced the expression of Caspase-3 relative to the model group. As an in vitro study, TSPN exerted a concentration-dependent protective effect against apoptosis in MC3T3-E1 cells. Moreover, TSPN (at a dose of 100 μg/mL) significantly reversed the dexamethasone-induced augmentation of Caspase-3 expression and activity. Therefore, our study demonstrated that TSPN had a protective effect against steroid-induced ANFH that was related to the upregulation of VEGF-A and the inhibition of apoptosis and Caspase-3 activation.

scholarly journals Lysophosphatidic Acid Analogue rather than Lysophosphatidic Acid Promoted the Bone Formation In Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Zi-Li Yu ◽  
Bin-Fang Jiao ◽  
Zu-Bing Li
Keyword(s):  
Bone Formation ◽  
Lysophosphatidic Acid ◽  
Histological Analysis ◽  
Alizarin Red S ◽  
Porous Scaffolds ◽  
Micro Ct ◽  
Micro Computed Tomography ◽  
Alizarin Red ◽  
Lipid Molecule

Lysophosphatidic acid (LPA), a bioactive lipid molecule, has recently emerged as physiological and pathophysiological regulator in skeletal biology. Here we evaluate the effects of LPA on bone formation in vivo in murine femoral critical defect model. Primary femoral osteoblasts were isolated and treated with osteogenic induction conditional media supplemented with 20 μM LPA or LPA analogue. Mineralized nodules were visualized by Alizarin Red S staining. Forty-five C57BL/6 mice underwent unilateral osteotomy. The femoral osteotomy gap was filled with porous scaffolds of degradable chitosan/beta-tricalcium phosphate containing PBS, LPA, or LPA analogue. 2, 5, and 10 weeks after surgery, mice were sacrificed and femurs were harvested and prepared for Micro-Computed Tomography (Micro-CT) and histological analysis. Alizarin Red S staining showed that LPA and LPA analogue significantly enhanced the mineral deposition in osteoblasts. Micro-CT 3D reconstruction images and HE staining revealed that significantly more newly formed bone in osteotomy was treated with LPA analogue when compared to control and LPA group, which was verified by histological analysis and biomechanical characterization testing. In summary, our study demonstrated that although LPA promotes mineralized matrix formation in vitro, the locally administrated LPA was not effective in promoting bone formation in vivo. And bone formation was enhanced by LPA analogue, administrated locally in vivo. LPA analogue was a potent stimulating factor for bone formation in vivo due to its excellent stability.

scholarly journals Icariin Protects against Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats

2018 ◽  
Vol 47 (2) ◽  
pp. 694-706 ◽  
Author(s):  
Zengfa Huang ◽  
Cheng Cheng ◽  
Beibei Cao ◽  
Jing Wang ◽  
Hui Wei ◽  
...  
Keyword(s):  
Femoral Head ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Adipogenic Differentiation ◽  
Ct Scanning ◽  
Control Group ◽  
Micro Ct ◽  
Alizarin Red ◽  
Immunohistochemical Analyses

Background/Aims: Glucocorticoid (GC)-related osteonecrosis of the femoral head (ONFH) is a common complication following administration of steroids to treat many diseases. Our previous study demonstrated that icariin (ICA) might have a beneficial effect on the bone marrow mesenchymal stem cells (BMSCs) of patients with steroid-associated osteonecrosis. In this study, we investigated the underlying mechanisms of ICA associated with the potential enhancement of osteogenesis and anti-adipogenesis in GC-related ONFH. Methods: In vitro cell proliferation was evaluated by CCK-8 assay. Alizarin red S and alkaline phosphatase (ALP) activity were used to measure osteogenic differentiation, while adipogenic differentiation was revealed by oil red O staining and TG content assay. The expression level of osteogenesis-associated genes and PPARγ was evaluated by RT-qPCR, western blotting and immunofluorescence. A total of 30 female SD rats were randomly separated into three groups: a control group, a methylprednisolone (MPS) group and a MPS + ICA group. Serum ALP and TG (triglyceride), micro-CT scanning, histological and immunohistochemical analyses were performed in the animal model. Results: In the in vitro study, ICA promoted proliferation, improved osteogenic differentiation and suppressed adipogenic differentiation of BMSCs treated with MPS. The group treated with MPS and 10-6 M ICA expressed higher levels of Runx2, ALP, bone morphogenetic protein (BMP) 2, and OC and lower expression of PPARγ than the MPS group. In the in vivo study, ICA prevented bone loss in a rat model of GC-related ONFH as shown by micro-CT scanning, histological and immunohistochemical analyses. Conclusions: ICA is an effective compound for promoting bone repair and preventing or delaying the progression of GC-associated ONFH in rats. This effect can be explained by its ability to improve the balance between adipogenesis and osteogenesis, indicating that ICA is an effective candidate for management of GC-associated ONFH.

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