Analysis of the Potential Angiogenic Mechanisms of BuShenHuoXue Decoction against Osteonecrosis of the Femoral Head Based on Network Pharmacology and Experimental Validation.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease with a high disability rate. The clinical effect of BuShenHuoXue decoction (BSHX) for ONFH is satisfactory. We aimed to elucidate the potential angiogenic mechanisms of BSHX in a rat femoral osteonecrosis model and bone marrow mesenchymal stem cells (BMSCs). METHODS: With in vivo experiments, we established the steroid-induced osteonecrosis of the femoral head (SONFH) model using Sprague-Dawley (SD) rats (8-week-old). The rats were randomly divided into five group of 12 rats each and given the corresponding interventions: control, model (gavaged with 0.9% saline), BSHX low-, medium- and high-dose groups (0.132 3, 0.264 6, and 0.529 2 g/mL BSHX solution by gavage). After 12 weeks, haematoxylin and eosin (H&E) staining was preformed to evaluate rat osteonecrosis. the expression of angiogenic factors (CD31, VEGFA, KDR, VWF) in rat femoral head was detected by immunohistochemistry, qPCR and western blotting. In cell experiment, BMSCs were isolated and cultured in the femoral bone marrow cavity of 4-week-old SD rats. BMSCs were randomly divided into eight groups and intervened with different doses of BSHX-containing serum and glucocorticoids: control group (CG); BSHX low-, medium-, and high-dose groups (CG + 0.661 5, 1.323, and 2.646 g/kg BSHX gavage rat serum); dexamethasone (Dex) group; and Dex + BSHX low-, medium-, and high-dose groups (Dex + 0.661 5, 1.323, and 2.646 g/kg BSHX gavaged rat serum), the effects of BSHX-containing serum on the angiogenic capacity of BMSCs were examined by qPCR and Western blotting. A co-culture system of rat aortic endothelial cells (RAOECs) and BMSCs was then established. Migration and angiogenesis of RAOECs were observed using angiogenesis and transwell assay. Identification of potential targets of BSHX against ONFH was obtained using network pharmacology. RESULTS: BSHX upregulated the expression of CD31, VEGFA, KDR, and VWF in rat femoral head samples and BMSCs (p < 0.05, vs. control group or model group). Different concentrations of BSHX-containing serum significantly ameliorated the inhibition of CD31, VEGFA, KDR and VWF expression by high concentrations of Dex. BSHX-containing serum-induced BMSCs promoted the migration and angiogenesis of RAOECs, reversed to some extent the adverse effect of Dex on microangiogenesis in RAOECs, and increased the number of microangiogenic vessels. Furthermore, we identified VEGFA, COL1A1, COL3A1, and SPP1 as important targets of BSHX against ONFH. CONCLUSION: BSHX upregulated the expression of angiogenic factors in the femoral head tissue of ONFH model rats and promoted the angiogenic capacity of rat RAOECs and BMSCs. This study provides an important basis for the use of BSHX for ONFH prevention and treatment.

Exploring the potential mechanism of Taohong Siwu decoction in the treatment of avascular necrosis of the femoral head based on network pharmacology and molecular docking.

Based on network pharmacology and molecular docking, this study seeks to investigate the mechanism of Taohong Siwu decoction (THSWD) in the treatment of avascular necrosis of the femoral head (AVNFH). The Traditional Chinese Medicine Systems Pharmacology database was used in this investigation to obtain the active ingredients and related targets for each pharmaceutical constituent in THSWD. To find disease-related targets, the terms "avascular necrosis of the femoral head," "necrosis of the femoral head," "steroid-induced necrosis of the femoral head," "osteonecrosis," and "avascular necrosis of the bone" were searched in the databases DisGeNET, GeneCards, Comparative Toxicogenomics Database, and MalaCards. Following the identification of the overlap targets of THSWD and AVNFH, enrichment analysis using gene ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, and WikiPathways was conducted. The "THSWD-drug-active compound-intersection gene-hub gene-AVNFH" network and protein-protein interaction network were built using Cytoscape 3.9.1 and string, and CytoHubba was used to screen hub genes. The binding activities of hub gene targets and key components were confirmed by molecular docking. 152 prospective therapeutic gene targets were found in the bioinformatics study of ONFH treated with THSWD, including 38 major gene targets and 10 hub gene targets. The enrichment analysis of 38 key therapeutic targets showed that the biological process of gene ontology analysis mainly involved cytokine-mediated signaling pathway, angiogenesis, cellular response to reactive oxygen species, death-inducing signaling complex. The Kyoto Encyclopedia of Genes and Genomes signaling pathway mainly involves TNF signaling pathway, IL-17 signaling pathway, and the Recactome pathway mainly involves Signaling by Interleukins, Apoptosis, and Intrinsic Pathway for Apoptosis. WikiPathways signaling pathway mainly involves TNF-related weak inducer of apoptosis signaling pathway, IL-18 signaling pathway. According to the findings of enrichment analysis, THSWD cured AVNFH by regulating angiogenesis, cellular hypoxia, inflammation, senescence, apoptosis, cytokines, and cellular proliferation through the aforementioned targets and signaling pathways. The primary component of THSWD exhibits a strong binding force with the key protein of AVNFH. This study sheds new light on the biological mechanism of THSWD in treating AVNFH by revealing the multi-component, multi-target, and multi-pathway features and molecular docking mechanism of THSWD.

[Characteristic changes of blood stasis syndrome in rat model of steroid-induced femoral head necrosis based on the combination of disease, syndrome, and symptom].

The approach combining disease, syndrome, and symptom was employed to investigate the characteristic changes of blood stasis syndrome in a rat model of steroid-induced osteonecrosis of the femoral head(SONFH) during disease onset and progression. Seventy-two male SD rats were randomized into a healthy control group and a model group. The rat model of SONFH was established by injection of lipopolysaccharide(LPS) in the tail vein at a dose of 20 µg·kg~(-1)·d~(-1) on days 1 and 2 and gluteal intramuscular injection of methylprednisolone sodium succinate(MPS) at a dose of 40 mg·kg~(-1)·d~(-1) on days 3-5, while the healthy control group received an equal volume of saline. The mechanical pain test, tongue color RGB technique, gait detection, open field test, and inclined plane test were employed to assess hip pain, tongue color, limping, joint activity, and lower limb strength, respectively, at different time points within 21 weeks of modeling. At weeks 2, 4, 8, 12, 16, and 21 after modeling, histopathological changes of the femoral head were observed by hematoxylin-eosin(HE) staining and micro-CT scanning; four coagulation items were measured by rotational thromboelastometry; and enzyme-linked immunosorbent assay(ELISA) was employed to determine the levels of six blood lipids, vascular endothelial growth factor(VEGF), endothelin-1(ET-1), nitric oxide(NO), tissue-type plasminogen activator(t-PA), plasminogen activator inhibitor factor-1(PAI-1), bone gla protein(BGP), alkaline phosphatase(ALP), receptor activator of nuclear factor-κB(RANKL), osteoprotegerin(OPG), and tartrate-resistant acid phosphatase 5b(TRAP5b) in the serum, as well as the levels of 6-keto-prostaglandin 1α(6-keto-PGF1α) and thromboxane B2(TXB2) in the plasma. The results demonstrated that the pathological alterations in the SONFH rats were severer over time. The bone trabecular area ratio, adipocyte number, empty lacuna rate, bone mineral density(BMD), bone volume/tissue volume(BV/TV), trabecular thickness(Tb.Th), trabecular number(Tb.N), bone surface area/bone volume(BS/BV), and trabecular separation(Tb.Sp) all significantly increased or decreased over the modeling time after week 4. Compared with the healthy control group, the mechanical pain threshold, gait swing speed, stride, standing time, and walking cycle of SONFH rats changed significantly within 21 weeks after modeling, with the greatest difference observed 12 weeks after modeling. The time spent in the central zone, rearing score, and maximum tilt angle in the open field test of SONFH rats also changed significantly over the modeling time. Compared with the healthy control group, the R, G, and B values of the tongue color of the model rats decreased significantly, with the greatest difference observed 11 weeks after modeling. The levels of total cholesterol(TC), total triglycerides(TG), low-density lipoprotein-cholesterol(LDL-C), and apoprotein B(ApoB) in the SONFH rats changed significantly 4 and 8 weeks after modeling. The levels of VEGF, ET-1, NO, t-PA, PAI-1, 6-keto-PGF1α, TXB2, four coagulation items, and TXB2/6-keto-PGF1α ratio in the serum of SONFH rats changed significantly 4-16 weeks after modeling, with the greatest differences observed 12 weeks after modeling. The levels of BGP, TRAP5b, RANKL, OPG, and RANKL/OPG ratio in the serum of SONFH rats changed significantly 8-21 weeks after modeling. During the entire onset and progression of SONFH in rats, the blood stasis syndrome characteristics such as hyperalgesia, tongue color darkening, gait abnormalities, platelet, vascular, and coagulation dysfunctions were observed, which gradually worsened and then gradually alleviated in the disease course(2-21 weeks), with the most notable differences occurred around 12 weeks after modeling.

Efficacy and safety of traditional Chinese medicine in the treatment of osteonecrosis of the femoral head.

BACKGROUND: Hip joint-preserving treatment options for osteonecrosis of the femoral head (ONFH) have been a research hotspot in recent years. The combination of Chinese and Western medicine has been used in clinical practice to treat early- and mid-stage ONFH. However, there is still a lack of high-quality evidence to verify the effectiveness and safety of this approach. OBJECTIVE: To systematically evaluate the clinical efficacy and safety of the combination of traditional Chinese medicine (TCM) with Western medicine in the treatment of early- and mid-stage ONFH. METHODS: Multiple electronic databases were searched to identify the randomized controlled trials (RCTs) examining the use of TCM in the treatment of ONFH. Based on the inclusion and exclusion criteria, eligible studies were selected, and the quality of the studies was evaluated using the risk of bias assessment tool recommended by the Cochrane system Evaluator manual 5.1.0. The meta-analysis of the included data was performed using Review Manager 5.4.1 software and Stata 17.0 software. RESULTS: A total of 47 RCTs involving 3266 subjects were included in the meta-analysis. The results are observed: (1) Harris score: TCM + Western medicine versus Western medicine (SMD = 1.25, 95% Cl: 1.02 to 1.48, P < 0.00001), TCM + physiotherapy versus physiotherapy (SMD = 2.26, 95% Cl: 1.42 to 3.10, P < 0.00001), and TCM + hip preservation surgery versus hip preservation surgery (SMD = 1.28, 95% Cl: 1.03 to 1.53, P < 0.00001); (2) Visual analogue scale score: TCM + Western medicine versus Western medicine (SMD = -3.99, 95% Cl: -7.41 to -0.57, P = 0.02), TCM + physiotherapy versus physiotherapy (SMD = -0.99, 95% Cl: -1.44 to -0.54, P < 0.0001), and TCM + hip preservation surgery versus hip preservation surgery (SMD = -1.08, 95% Cl: -1.75 to -0.40, P = 0.002); (3) Imaging improvement: TCM + physiotherapy versus physiotherapy (RR = 1.42, 95% Cl: 1.15 to 1.76, P = 0.001) and TCM + hip preservation surgery versus hip preservation surgery (RR = 1.21, 95% Cl: 1.11 to 1.31, P < 0.0001); and (4) Occurrence of adverse reaction: TCM + Western medicine versus Western medicine (RR = 0.73, 95% Cl: 0.28 to 1.92, P = 0.53), TCM + physiotherapy versus physiotherapy (RR = 0.46, 95% Cl: 0.03 to 7.33, P = 0.58), and TCM + hip preservation surgery versus hip preservation surgery (RR = 1.11, 95% Cl: 0.36 to 3.45, P = 0.86). CONCLUSION: TCM combined with Western medicine is an effective and safe approach for the treatment of ONFH. However, due to the low quality and quantity of the included studies, additional large-scale, high-quality studies are required to verify the above conclusions. SYSTEMATIC REVIEW REGISTRATION: https://www.crd.york.ac.uk/prospero/#recordDetails , CRD42023392030.

Exogenous melatonin ameliorates steroid-induced osteonecrosis of the femoral head by modulating ferroptosis through GDF15-mediated signaling.

BACKGROUND: Ferroptosis is an iron-related form of programmed cell death. Accumulating evidence has identified the pathogenic role of ferroptosis in multiple orthopedic disorders. However, the relationship between ferroptosis and SONFH is still unclear. In addition, despite being a common disease in orthopedics, there is still no effective treatment for SONFH. Therefore, clarifying the pathogenic mechanism of SONFH and investigating pharmacologic inhibitors from approved clinical drugs for SONFH is an effective strategy for clinical translation. Melatonin (MT), an endocrine hormone that has become a popular dietary supplement because of its excellent antioxidation, was supplemented from an external source to treat glucocorticoid-induced damage in this study. METHODS: Methylprednisolone, a commonly used glucocorticoid in the clinic, was selected to simulate glucocorticoid-induced injury in the current study. Ferroptosis was observed through the detection of ferroptosis-associated genes, lipid peroxidation and mitochondrial function. Bioinformatics analysis was performed to explore the mechanism of SONFH. In addition, a melatonin receptor antagonist and shGDF15 were applied to block the therapeutic effect of MT to further confirm the mechanism. Finally, cell experiments and the SONFH rat model were used to detect the therapeutic effects of MT. RESULTS: MT alleviated bone loss in SONFH rats by maintaining BMSC activity through suppression of ferroptosis. The results are further verified by the melatonin MT2 receptor antagonist that can block the therapeutic effects of MT. In addition, bioinformatic analysis and subsequent experiments confirmed that growth differentiation factor 15 (GDF15), a stress response cytokine, was downregulated in the process of SONFH. On the contrary, MT treatment increased the expression of GDF15 in bone marrow mesenchymal stem cells. Lastly, rescue experiments performed with shGDF15 confirmed that GDF15 plays a key role in the therapeutic effects of melatonin. CONCLUSIONS: We proposed that MT attenuated SONFH by inhibiting ferroptosis through the regulation of GDF15, and supplementation with exogenous MT might be a promising method for the treatment of SONFH.

Age is Not the Most Important Factor in the Prognosis of Femoral Neck Fracture: An Analysis of Long-Term Clinical Follow-Up.

Objective: The prognosis of femoral neck fractures is affected by factors including age and type of fracture. This study aimed to explore the associations among postsurgical outcomes of internal fixation for femoral neck fracture (healing rate, necrosis rate, and joint function score) and age and type of fracture. Methods: We retrospectively analyzed 297 cases of femoral neck fracture treated with internal fixation between February 2008 and October 2018. The postoperative femoral neck nonunion rate (a measure of healing) and femoral head necrosis rate were determined by x-ray and computed tomography. The Harris hip score (a measure of joint function and pain) was calculated. The effects of age and fracture type on these factors were analyzed. Results: There was no significant difference in the rate of femoral head necrosis and postoperative joint function scores among the different age groups. There was a significant difference in the postoperative rate of femoral head necrosis by Garden (P = .001) and Pauwels (P = .01) fracture types. No significant differences were noted for the Harris hip score for fractures characterized by the Pauwels classification (P = .09). However, the Harris hip scores differed significantly among groups for fractures categorized by the Garden classification (P = .001). Conclusions: Fracture type but not age is closely related to femoral head necrosis and Harris hip score after internal fixation of femoral neck fractures.

Dried root of Rehmannia glutinosa extracts prevents steroid-induced avascular necrosis of femoral head by activating the wingless-type (Wnt)/ß-catenin signal pathway.

Steroid-induced avascular necrosis of femoral head (SANFH) is one of the most common complications caused by long-term or excessive clinical use of glucocorticoids. This study aimed to investigate the effects of dried root of Rehmannia glutinosa extracts (DRGE) in SANFH. First, SANFH rat model was established by dexamethasone (Dex). Tissue change and proportion of empty lacunae were detected by hematoxylin and eosin staining. Protein levels were detected by western bloting analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess apoptosis of femoral head tissue. Cell viability and apoptosis of MC3T3-E1 cells were assessed by Cell Counting Kit-8 assay and flow cytometry. ALP activity and cell mineralization were detected by ALP staining assay and Alizarin red staining. The findings showed that DRGE improved tissue damage, inhibited apoptosis, and promoted osteogenesis in SANFH rats. In vitro, DRGE increased cell viability, inhibited cell apoptosis, promoted osteoblast differentiation, reduced the levels of p-GSK-3ß/GSK-3ß, but increased the levels of ß-catenin in cells treated with Dex. Furthermore, DKK-1, an inhibitor of the wingless-type (Wnt)/ß-catenin signaling pathway, reversed the effect of DRGE on cell apoptosis and ALP activity in cells treated with Dex. In conclusion, DRGE prevents SANFH by activating the Wnt/ß-catenin signaling pathway, indicating that DRGE may be a hopeful choice drug to prevent and treat patients with SANFH.

Suppression of apoptosis in osteocytes, the potential way of natural medicine in the treatment of osteonecrosis of the femoral head.

OBJECTIVES: In the field of orthopedics, osteonecrosis of the femoral head (ONFH) is a common and refractory condition sometimes known as "immortal cancer" due to its complicated etiology, difficult treatment, and high disability rate. This paper's main goal is to examine the most recent literature on the pro-apoptotic effects of traditional Chinese medicine TCM monomers or compounds on osteocytes and to provide a summary of the potential signal routes. METHODS: The last ten years' worth of literature on ONFH as well as the anti-ONFH effects of aqueous extracts and monomers from traditional Chinese medicine were compiled. CONCLUSIONS: When all the relevant signal pathways are considered, the key apoptotic routes include those mediated by the mitochondrial pathway, the MAPK signaling pathway, the PI3K/Akt signaling pathway, the Wnt/-catenin signaling pathway, the HIF-1 signaling network, etc. As a result, we anticipate that this study will shed light on the value of TCM and its constituent parts for treating ONFH by inducing apoptosis in osteocytes and offer some guidance for the future development of innovative medications as anti-ONFH medications in clinical settings.

Analysis of the potential biological mechanisms of Danyu Gukang Pill against osteonecrosis of the femoral head based on network pharmacology.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is still a challenge for orthopedists worldwide and can lead to disability if patients are not treated effectively. Danyu Gukang Pill (DGP), a traditional Chinese medicine (TCM) formulation, is recognized to be effective against ONFH. Nevertheless, its molecular mechanisms remain to be clarified. METHODS: The active ingredients of DGP were collected from the online databases according to oral bioavailability (OB) and drug-likeness (DL). The potential targets of DGP were retrieved from the TCMSP database, while the potential targets of ONFH were obtained from the GeneCards and NCBI databases. The functions and signaling pathways of the common targets of DGP and ONFH were enriched by GO and KEGG analyses. Subsequently, molecular docking and in vitro cell experiments were performed to further validate our findings. RESULTS: In total, 244 active ingredients of DGP and their corresponding 317 targets were obtained, and 40 ONFH-related targets were predicted. Afterwards, 19 common targets of DGP and ONFH were obtained and used as potential targets for the treatment of ONFH. Finally, combined with network pharmacology analysis, molecular docking and in vitro cell experiments, our study first demonstrated that the treatment effect of DGP on ONFH might be closely related to the two targets, HIF1A (HIF-1α) and VEGFA, and the HIF-1 signaling pathway. CONCLUSIONS: This study is the first to investigate the molecular mechanisms of DGP in the treatment of ONFH based on network pharmacology. The results showed that DGP might up-regulate the expression of HIF-1α and VEGFA by participating in the HIF-1 signaling pathway, thus playing an anti-ONFH role.

Tongluo Shenggu capsule promotes angiogenesis to ameliorate glucocorticoid-induced femoral head necrosis via upregulating VEGF signaling pathway.

BACKGROUND: Tongluo Shenggu Capsule (TLSGC) is a product of Traditional Chinese patent medicine that has been effective in glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) clinically for many years. It is made from water extracts of a well-used herbal and dietary supplement-pigeon pea leaves. Nevertheless, the material basis and pharmacological mechanisms of TLSGC ameliorating GIONFH needed to be better defined. PURPOSE: To investigate the material basis and pharmacological mechanisms of TLSGC to ameliorate GIONFH. METHODS: The chemical compositions in TLSGC were characterized using the LC-MS system. Based on integrating the relevant targets of TLSGC in MedChem Studio software and GIONFH-related genes in our previous work, a "drug targets-disease genes" interaction network was constructed. The candidate targets of TLSGC ameliorating GIONFH were filtrated by topological characteristic parameters and further experimental validated based on methylprednisolone-induced rat model and dexamethasone-inhibited human umbilical vein endothelial cells (HUVECs). RESULTS: A total of 33 chemical compositions were characterized in TLSGC. Based on these compositions and GIONFH-related genes, 122 hub genes were selected according to topological parameters calculation. Biological functions were mainly enriched in four over-expressed modules of vascular damage, inflammation and apoptosis, bone metabolism and energy metabolism. The hub genes had the maximum enrichment degree in the VEGF-VEGFR2-PKC-Raf1-MEK-ERK signaling axis of the VEGF pathway. Experimentally, the therapeutic effects of TLSGC against GIONFH in rats were proved by micro-CT and pathological examination. Then, the protective effects of TLSGC on vascular damage were determined using angiography, CD31 immunohistochemistry, vascular function indicators in vivo, aortic ring test ex vivo, and the HUVECs activities in vitro including migration, invasion and tube formation. Mechanically, TLSGC effectively suppressed the downregulation of VEGF and VEGFR2 and their downstream targets, including Raf-1, PKC, p-MEK, and p-ERK proteins both in vivo and in vitro. CONCLUSION: TLSGC could promote angiogenesis by upregulating the VEGF-VEGFR2-PKC-Raf-1-MEK-ERK signaling axis, thereby exerting an apparent curative effect on GIONFH.

Network-Based Pharmacology and Bioinformatics Study on the Mechanism of Action of Gujiansan in the Treatment of Steroid-Induced Avascular Necrosis of the Femoral Head.

Objective: To investigate the main pharmacological basis and mechanism of action of Gujiansan in the treatment of steroid-induced avascular necrosis of the femoral head (SANFH). Methods: The active constituents and targets of Gujiansan were screened by using TCMSP and other databases, and relevant disease targets were obtained by analyzing the microarray of SANFH in the GEO database. The intersection of the two was taken to obtain the potential targets of Gujiansan for the treatment of SANFH, and key active constituents were screened with the "active constituent-target" network constructed by the Cytoscape software; then, the STRING database was used to construct the protein interaction network to screen the key targets. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of key targets were performed by the DAVID database, and the relationship between the "key active constituent-key target-key signaling pathway" was explored. Finally, the molecular docking between key active constituents and key targets was verified. In addition, qPCR detection technology was used to evaluate the preventive and therapeutic effects of key active constituents of Gujiansan in a rat osteoblast model of SANFH to verify the possible mechanism of the effect of Gujiansan in the treatment of SANFH. Results: (1) 106 active constituents and 55 targets were obtained for the treatment of SANFH. (2) Quercetin, luteolin, kaempferol, cryptotanshinone, and naringenin were the key active constituents for the treatment of SANFH. (3) IL1B, STAT3, CAT, PTGS2, and MAPK3 were the key targets for the treatment of SANFH. (4) IL1B, STAT3, CAT, PTGS2, MAPK3, and HMOX1 are key targets in the protein interaction network. (5) DAVID enrichment analysis mainly covers the regulation of DNA-binding transcription factor activity, positive regulation of cytokine production, and response to oxidative stress and other biological processes, involving IL-17, AGE-RAGE, C-type lectin receptor, and other signaling pathways. (6) Gujiansan is a multitarget and multisignaling pathway for the treatment of SANFH. (7) Good binding activity exists between key active constituents and key targets. Conclusion: This study analyzes the potential mechanism of action of Gujiansan in the treatment of SANFH with network pharmacology, which can provide a reference for the further study of its pharmacological basis and targets.

Tongue Manifestation in Patients with Osteonecrosis of the Femoral Head: A Cross-sectional Study.

OBJECTIVE: Although tongue manifestation is a vital component of Traditional Chinese Medicine (TCM), relevant research on patients with osteonecrosis of the femoral head (ONFH) is still lacking. This study will explore the characteristic tongue manifestation of ONFH patients to inform future research and clinical practice. METHODS: This is a cross-sectional study. All ONFH patients meeting criteria and their clinical data were collected from the online China osteonecrosis of the femoral head database (CONFHD) since it was created. Organized tongue manifestations of eligible patients through the tongue manifestation acquisition instrument, including tongue shape, tongue color, tongue coating thickness, tongue coating color and tongue coating moisture. We used descriptive analysis for the general information while systematic clustering analysis for the better summary of tongue characteristics. RESULTS: A total of 375 ONFH patients were included with an average age of 46.3 years. Most patients appeared with enlarged tongue body (54.4%), and the proportions of pale and red tongue (62.4%) were higher than others. Tongue coating were mainly showed as thick (64.5%), white (57.6%) and moist (79.7%). Comparison of tongue shape between different causes of ONFH had a significant statistically difference (P = 0.000). Tongue manifestations could be cluster analyzed into three categories which were matched into four TCM syndromes. CONCLUSIONS: The tongue manifestation of ONFH patients has a significant change both in tongue body and coating, and different features may be related to the ONFH pathology. This study provides new and valuable tongue informations for a preliminary screening of ONFH patients.

Ginkgo biloba L. extract prevents steroid-induced necrosis of the femoral head by rescuing apoptosis and dysfunction in vascular endothelial cells via the PI3K/AKT/eNOS pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. extract (EGb) is one of the world's most extensively used herbal medicines. Due to the diverse pharmacological properties of EGb, it has been used in the treatment of neurological illnesses, as well as cardiovascular and cerebrovascular ailments. However, the effect and pharmacological mechanism of EGb on steroid-induced necrosis of the femoral head (SINFH) are still unclear. AIM OF THE STUDY: SINFH remains a challenging problem in orthopedics. Previous investigations have shown that EGb has the potential to reduce the occurrence of SINFH. The goal was to determine the effect and mechanism of EGb in preventing SINFH by inhibiting apoptosis and improving vascular endothelial cells (VECs) functions. MATERIALS AND METHODS: CCK-8, nitric oxide (NO) production and flow cytometry were used to determine the cell apoptosis and function. The scratch and angiogenesis tests assessed migration and tube formation. Western blot analysis detected the expressions of apoptosis-related proteins and PI3K/AKT/eNOS pathway-related proteins. Apoptosis and angiogenesis were also detected treated with the inhibitors. A mouse model of SINFH was established. Paraffin section was used to determine the necrotic pathology and apoptosis. Vessels in the femoral heads were assessed by immunofluorescence staining. RESULTS: When stimulated by methylprednisolone (MPS), cell viability, NO generation and tube formation were decreased, the apoptotic rate increased. Simultaneously, MPS decreased the expression levels of p-PI3K, p-AKT, and p-eNOS. EGb increased the expression levels of these proteins, restrained apoptosis, and restored cell functions. The addition of the inhibitors decreased anti-apoptotic effect and angiogenesis. In addition, when compared to the model mice, there were fewer empty lacunae and normal trabecular arrangement after taking different doses of EGb. The protective effect was also confirmed by the vascular quantitative analysis in vivo. CONCLUSION: This study established that EGb increased endothelial cell activity and inhibited apoptosis and function loss induced by MPS, elucidating the effect and molecular mechanism of EGb on early SINFH.

Study on the Mechanism of Treating Femoral Head Necrosis with Drynariae Rhizoma Based on Network Pharmacology.

Through the network pharmacology thought, the action target of the active ingredients of Drynariae Rhizoma was predicted, and the mapping was combined with the related targets of ONFH, and the key nodes of interaction were identified for enrichment analysis, so as to comprehensively explore the pharmacological mechanism of Drynariae Rhizoma against ONFH. The main active ingredients of Drynariae Rhizoma were screened based on pharmacokinetic characteristics in pharmacokinetic database and analysis platform of TCM system (TCMSP). We used the organic small molecule bioactivity database (PubChem) and Swiss target prediction database to predict related targets based on 2D or 3D structural similarity and then mined the known ONFH therapeutic targets through the Human Mendelian Genetic Database (OMIM) and Pubmed texts. Combined with the predicted targets, String database was imported to construct the OP target interaction network diagram of bone fracture therapy. CytoNCA software was used to topology the key nodes of interaction according to relevant node parameters, and String was imported again to construct the protein interaction network diagram. Finally, biological functions and metabolic pathways of key nodes were analyzed through DAVID database. It was revealed that Drynariae Rhizoma may regulate stem cells, osteoblasts, osteoclasts, and immune cells through multiple pathways, including proliferation, differentiation, immunity, and oxidative stress. Conclusion: Pharmacological studies based on network indicate that Drynariae Rhizoma may participate in the regulation of several major signaling pathways through direct or indirect action targets and affect the proliferation and differentiation of multiple types of cells, thus playing an anti-ONFH role, which provides a scientific basis for explaining the material basis and mechanism of its anti- ONFH.

Revealing the mechanisms of Weishi Huogu I capsules used for treating osteonecrosis of the femoral head based on systems pharmacology with one mechanism validated with in vitro experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Weishi Huogu I (WH I) capsules, developed through traditional Chinese medicine, have been used to treat clinical osteonecrosis of the femoral head (ONFH) for decades. However, the mechanisms have not been systematically studied. AIM OF THE STUDY: In this study, the mechanisms of WH I capsules used in treating ONFH were examined through a systems pharmacology strategy, and one mechanism was validated with in vitro experiments. MATERIALS AND METHODS: WH I capsules compounds were identified by screening databases; then, a database of the potential active compounds was constructed after absorption, distribution, metabolism and excretion (ADME) evaluation. The compounds were identified through a systematic approach in which the probability of an interaction of every candidate compound with each corresponding target in the DrugBank database was calculated. Gene Ontology (GO) and pathway enrichment analyses of the targets was performed with the Metascape and KEGG DISEASE databases. Then, a compound-target network (C-T) and target-pathway network (T-P) of WH I capsule components were constructed, and network characteristics and related information were used for systematically identifying WH I capsule multicomponent-target interactions. Furthermore, the effects of WH I capsule compounds identified through the systematic pharmacology analysis of the osteogenic transformation of human umbilical mesenchymal stem cells (HUMSCs) were validated in vitro. RESULTS: In total, 152 potentially important compounds and 176 associated targets were identified. Twenty-two crucial GO biological process (BP) or pathways were related to ONFH, mainly in regulatory modules regulating blood circulation, modulating growth, and affecting pathological processes closely related to ONFH. Furthermore, the GO enrichment analysis showed that corydine, isorhamnetin, and bicuculline were enriched in "RUNX2 regulates osteoblast differentiation", significantly increased alkaline phosphatase activity and calcium deposition and upregulated runt-related transcription factor 2 mRNA and protein expression and osteocalcin mRNA expression in HUMSCs, suggesting that these compounds promoted the mesenchymal stem cell (MSC) osteogenic transformation. CONCLUSIONS: The study showed that the pharmacological mechanisms of WH I capsule attenuation of ONFH mainly involve three therapeutic modules: blood circulation, modulating growth, and regulating pathological processes. The crosstalk between GOBPs/pathways may constitute the basis of the synergistic effects of the compounds in WH I capsules in attenuating ONFH. One of the pharmacological mechanisms in the WH I capsule effect on ONFH involves enhancement of the osteogenic transformation of MSCs, as validated in experiments performed in vitro; however, more mechanisms should be validated in further studies.

Circ_0058792 regulates osteogenic differentiation through miR-181a-5p/Smad7 axis in steroid-induced osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) caused by steroids is a severe orthopedic disorder resulting from the use of high-dose steroid drugs, characterized by structural changes in the bone, joint dysfunction, and femoral head collapse. CircRNAs and miRNAs have increasingly been suggested to play pivotal roles in osteogenic differentiation and osteogenesis. Significant upregulation of circ_0058792 was observed in patients with steroid-induced ONFH. Bioinformatic analysis showed that circ_0058792 might act as a sponge for miR-181a-5p. This study further investigated the mechanisms underlying the role of circ_0058792 and miR-181a-5p in osteogenic differentiation in methylprednisolone-induced ONFH rats and MC3T3-E1 cells. The results showed a notable decrease in the serum of miR-181a-5p in methylprednisolone-induced ONFH rats. Silencing of circ_0058792 using siRNAs and overexpression of miR-181a-5p significantly increased alkaline phosphatase activity and matrix mineralization capacity. Additionally, markers for osteogenic differentiation were significantly upregulated in miR-181a-5p-transfected cells. However, overexpression of circ_0058792 and the addition of the miR-181a-5p inhibitor reversed this increase. Smad7 was identified to be miR-181a-5p's direct target and circ_0058792 was confirmed to be miR-181a-5p's competing endogenous RNA (ceRNA). Upregulation of miR-181a-5p promotes phosphorylation of Smad2 and Smad3. Furthermore, circ_0058792 and miR-181a-5p had opposing effects on Smad7 expression. Collectively, these findings indicate that circ_0058792 regulates osteogenic differentiation by sponging miR-181a-5p via the TGF-ß/Smad7 pathway. These findings elucidated the functions of circ_0058792 and miR-181a-5p in the regulation of steroid-induced ONFH. Our findings also indicated that circ_0058792 and miR-181a-5p are possible diagnostic markers and therapeutic targets for treating steroid-induced ONFH.

Treatment of Femoral Head Osteonecrosis with Ozone Therapy: Pilot Trial of a New Therapeutic Approach.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a progressive and painful disorder due to impaired blood supply to the femoral head, yet little is known about the effect of ozone therapy in femoral head necrosis. OBJECTIVES: We aimed to evaluate the clinical and radiographic outcomes of ozone therapy in the treatment of ONFH. STUDY DESIGN: Nonrandomized clinical trial. SETTINGS: The study was conducted in a single-center, academic institution. METHODS: A total of 71 patients (107 hip joints) with Association Research Circulation Osseous (ARCO) stage-I, II, III, and IV ONFH were included and assigned to undergo either intraarticular O2-O3 mixture hip injections with ozonated autohemotherapy (ozone therapy group, n = 39, 58 hip joints) or protected weight bearing (control group, n = 32, 49 hip joints). The primary outcomes included the Visual Analog Scale (VAS) for pain intensity and Harris Hip Score (HHS) for hip function. The secondary outcomes included bone marrow edema examination, and conversion to total hip arthroplasty (THA). RESULTS: Ozone therapy effectively improves VAS for pain intensity and HHS during the follow-up period compared to the control group. Ozone therapy showed a significant resolution of bone marrow edema of the femoral head compared to the control group (P < 0.001). Thirteen of the 49 hips (26.53%) in the control group underwent THA, whereas only 6 hips (10.34%) in the ozone therapy group required THA during a 30-month follow-up (P = 0.041). The cumulative analysis revealed a low rate of conversion to THA in the ozone therapy group (logrank test; P = 0.022). LIMITATIONS: The study is limited by a single treatment protocol in addition to the lack of a randomized design. CONCLUSIONS: Ozone therapy was associated with significant pain relief, improvement in hip function, and bone marrow edema resolution that may delay the need for THA in patients affected by ONFH.Institutional Review Board (IRB) approval number: HK2018-10-28.Clinical trials registration number: ChiCTR1900023449.

Lycium barbarum polysaccharide protects against osteonecrosis of femoral head via regulating Runx2 expression.

BACKGROUND: Osteonecrosis of femoral head (ONFH) is a pathological state caused by lack of blood supply in femoral head. This study aimed to explore the function of Lycium barbarum polysaccharide (LBP), an antioxidant agent extracted from L. barbarum, on ONFH. METHODS: Osteonecrosis rat model was generated using lipopolysaccharide (LPS) and methylprednisolone followed by examination of body weight, blood glucose, morphology, and BMSC osteoblast differentiation. The effect and underlying mechanism of LBP on the proliferation, apoptosis, and osteoblast differentiation of BMSC were determined with or without LPS or hypoxia treatment using CCK-8. Alizarin Red S staining, flow cytometry, and western blot, respectively. RESULT: LBP could protect against glucocorticoid-induced ONFH in rats, resulting in improved sparse trabecular bone, empty lacunae and bone cell coagulation. Moreover, LBP promoted the proliferation and osteoblast differentiation of bone mesenchymal-derived stem cells (BMSCs) in a dose-dependent manner. Furthermore, LBP enhanced osteoblast differentiation of BMSCs under hypoxia condition. Mechanistically, we found that LBP treatment enhanced Runx2 and ALP expression in BMSCs. LBP restored the expression of Runx2 and ALP under hypoxia, suggesting that LBP might be involved in regulating Runx2/ALP expression and contributed to osteoblast differentiation. Knockdown of Runx2 significantly inhibited BMSCs proliferation, while LBP treatment did not rescue the osteoblast differentiation ability of BMSCs with Runx2 knockdown. CONCLUSION: Our findings suggested that LBP protects against ONFH via regulating Runx2 expression, which could be utilized to treat patients suffering ONFH.

Combination of hyperbaric oxygen and core decompression therapies improve outcomes in the treatment of hip osteonecrosis.

INTRODUCTION: To investigate whether combined treatment of hyperbaric oxygen (HBO) and core decompression (CD) result with better outcomes and have an additional influence on health quality scores when compared with HBO alone. METHODS: 63 consecutive patients' 80 hips (43 male, 20 female, 17 bilateral), diagnosed with Stage II Osteonecrosis of the femoral head were included in our study. The mean age at presentation in the HBO and CD + HBO groups were 39.9 years and 39.2 years, respectively. The mean follow-up was 39.8 months (24-56 months) for HBO group and 43.1 months (24-58 months) for the CD + HBO group. Standard radiographs and MRI were performed initially and during controls. Clinical outcomes were assessed using the modified Harris Hip Score (HHS), the visual analogue score (VAS) and SF-36 life quality score. RESULTS: 52 hips (65%) were Ficat Stage IIa and 28 hips (35%) were IIb. Totally, 46 hips (30 hips IIa, 16 hips IIb) were in HBO alone group and 34 hips (22 hips IIa, 12 hips IIb) were in CD + HBO group. Both VAS and HHSs were improved in each group after treatment (p < 0.001). When both groups were compared, this improvement was more distinct and evident in CD + HBO combination group than HBO alone group (p < 0.001). The physical function and pain components of SF-36 survey were found to be different in between two groups (p < 0.005). DISCUSSION: HBO treatment decreases pain, increases functional scores for Ficat Stage II patients. Addition of HBO treatment to decompression of the femoral head improves the results better than HBO alone. In particular, reduction of pain is more prevalent for Stage IIa patients than IIb with combination of HBO and CD therapies.

A Network Pharmacology and Molecular Docking Strategy to Explore Potential Targets and Mechanisms Underlying the Effect of Curcumin on Osteonecrosis of the Femoral Head in Systemic Lupus Erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a refractory immune disease, which is often complicated with osteonecrosis of the femoral head (ONFH). Curcumin, the most active ingredient of Curcuma longa with a variety of biological activities, has wide effects on the body system. The study is aimed at exploring the potential therapeutic targets underlying the effect of curcumin on SLE-ONFH by utilizing a network pharmacology approach and molecular docking strategy. METHODS: Curcumin and its drug targets were identified using network analysis. First, the Swiss target prediction, GeneCards, and OMIM databases were mined for information relevant to the prediction of curcumin targets and SLE-ONFH-related targets. Second, the curcumin target gene, SLE-ONFH shared gene, and curcumin-SLE-ONFH target gene networks were created in Cytoscape software followed by collecting the candidate targets of each component by R software. Third, the targets and enriched pathways were examined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Eventually, a gene-pathway network was constructed and visualized by Cytoscape software; key potential central targets were verified and checked by molecular docking and literature review. RESULTS: 201 potential targets of curcumin and 170 related targets involved in SLE-ONFH were subjected to network analysis, and the 36 intersection targets indicated the potential targets of curcumin for the treatment of SLE-ONFH. Additionally, for getting more comprehensive and accurate candidate genes, the 36 potential targets were determined to be analyzed by network topology and 285 candidate genes were obtained finally. The top 20 biological processes, cellular components, and molecular functions were identified, when corrected by a P value ≤ 0.05. 20 related signaling pathways were identified by KEGG analysis, when corrected according to a Bonferroni P value ≤ 0.05. Molecular docking showed that the top three genes (TP53, IL6, VEGFA) have good binding force with curcumin; combined with literature review, some other genes such as TNF, CCND1, CASP3, and MMP9 were also identified. CONCLUSION: The present study explored the potential targets and signaling pathways of curcumin against SLE-ONFH, which could provide a better understanding of its effects in terms of regulating cell cycle, angiogenesis, immunosuppression, inflammation, and bone destruction.