Effects of astragaloside IV on glucocorticoid-induced avascular necrosis of the femoral head via regulating Akt-related pathways.

We investigated the role of astragaloside IV (AS-IV) in preventing glucocorticoid-induced avascular necrosis of the femoral head (ANFH) and the underlying molecular mechanisms. Network pharmacology was used to predict the molecular targets of AS-IV. Molecular dynamic simulations were performed to explore the binding mechanism and interaction mode between AS-IV and Akt. Rat models of glucocorticoid-induced ANFH with AS-IV intervention were established, and osteogenesis, angiogenesis, apoptosis and oxidative stress were evaluated before and after blocking the PI3K/Akt pathway with LY294002. The effects of glucocorticoid and AS-IV on bone marrow mesenchymal stem cells and human umbilical vein endothelial cells incubated with and without LY294002 were determined. Downregulated p-Akt expression could be detected in the femoral heads of glucocorticoid-induced ANFH patients and rats. AS-IV increased trabecular bone integrity and vessel density of the femoral head in the model rats. AS-IV increased Akt phosphorylation and upregulated osteogenesis-, angiogenesis-, apoptosis- and oxidative stress-related proteins and mRNA and downregulated Bax, cleaved caspase-3 and cytochrome c levels. AS-IV promoted human umbilical vein endothelial cell migration, proliferation and tube formation ability; bone marrow mesenchymal stem cell proliferation; and osteogenic differentiation under glucocorticoid influence. AS-IV inhibited apoptosis. LY294002 inhibited these effects. AS-IV prevented glucocorticoid-induced ANFH by promoting osteogenesis and angiogenesis via the Akt/Runx2 and Akt/HIF-1α/VEGF pathways, respectively, and suppressing apoptosis and oxidative stress via the Akt/Bad/Bcl-2 and Akt/Nrf2/HO-1 pathways, respectively.

The Ubiquitination of RIPK2 is Mediated by Peli3 and Negatively Regulates the Onset of Infectious Osteomyelitis.

Osteomyelitis is the infection and destruction of the bone. To date, there is no universal protocol for its treatment. Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) has been implicated in osteomyelitis development. However, the detailed mechanism remains unknown. Here, 6-8w wild-type or Pellino E3 Ubiquitin Protein Ligase Family Member 3 (Peli3)-deficient mice were injected with Staphylococcus aureus to induce osteomyelitis. RAW264.7 cells or bone marrow-derived macrophages isolated from mice were treated with lipopolysaccharide (LPS). Knocking down Peli3 in RAW264.7 cells increased the expression of inflammatory cytokines (interleukin-1ß, interleukin-6, and tumor necrosis factor-α) after LPS stimulation. Inflammation was also activated in S. aureus-induced Peli3-deficient mice. Moreover, S. aureus-infected Peli3-deficient mice also displayed more severe symptoms of osteomyelitis than S. aureus-infected wild-type mice. Moreover, Peli3 targets and degrades RIPK2 through K48-linked ubiquitination, and negatively modulates osteomyelitis by degrading RIPK2. Our data further expands the current understanding of RIPK2 in osteomyelitis, and suggests that RIPK2 might serve as a novel therapeutic target for treating osteomyelitis.

Merlin functions as a critical regulator in Staphylococcus aureus-induced osteomyelitis.

Merlin is known as a tumor suppressor, while its role in osteomyelitis remains unclear. This study aimed to investigate the role of Merlin in Staphylococcus aureus-induced osteomyelitis and its underlying mechanisms. S. aureus-induced osteomyelitis mouse model was established in Merlinfl/fl Lyz2cre/+ and Merlinfl/fl Lyz2+/+ mice. Bone marrow-derived macrophages (BMDMs) were isolated and stimulated by lipopolysaccharide (LPS). Bioassays, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and enzyme-linked immunosorbent assays, were conducted to determine the levels of target genes or proteins. Immunoprecipitation was applied to determine the interactions between proteins. DCAF1fl/fl mice were further crossed with Lyz2-Cre mice to establish myeloid cell conditional knockout mice (DCAF1fl/fl Lyz2cre/+ ). It was found that the level of Merlin was elevated in patients with osteomyelitis and S. aureus-infected BMDMs. Merlin deficiency in macrophages suppressed the production of inflammatory cytokines and ameliorated the symptoms of osteomyelitis induced by S. aureus. Merlin deficiency in macrophages also suppressed the production of proinflammatory cytokines in BMDMs induced by LPS. The inhibitory effects of Merlin deficiency on the inflammatory response were associated with DDB1-Cul4-associated factor 1 (DCAF1). In summary, Merlin deficiency ameliorates S. aureus-induced osteomyelitis through the regulation of DCAF1.

IKKe in osteoclast inhibits the progression of methylprednisolone-induced osteonecrosis.

Previous studies have described that NF-κB signaling mediated by NFκB-inducing kinase (NIK) plays a critical role of the differentiation of osteoclasts. We aim to explore the role of IKKe in methylprednisolone -induced osteonecrosis of the femoral head (ONFH). Methylprednisolone-induced ONFH mice model was successfully established, and subjected to micro computed tomography to detect the femoral head image of the mice. Bone marrow cells from experimental mice were collected and cultured. qPCR and immunoblot were performed to examine the possible signal pathways of IKKe involvement, and osteoclast-related gene expressions in IKKe+/+ and IKKe-/- cells in vitro and in vivo were examined. It was found that the levels of IKKe decreased in ONFH patients, and IKKe interacted with NIK in the NF-κB signal pathway to suppress osteoclasts via inhibiting the transcription of NIK. Furthermore, IKKe knockout promoted the osteoclastogenesis in mice model. Finally, IKKe knockout suppressed methylprednisolone-induced ONFH and pro-inflammatory responses in mice model. Our findings show a mechanism of IKKe inhibition of the progression of methylprednisolone-induced ONFH via the NIK/NF-κB pathway.

Astragaloside IV ameliorates steroid-induced osteonecrosis of the femoral head by repolarizing the phenotype of pro-inflammatory macrophages.

Osteonecrosis of the femoral head (ON-FH) is a common complication of steroid use. Pro-inflammatory macrophages play a crucial role in the apoptosis of osteocytes. The objective of the study was to evaluate a plant extract astragaloside IV (AS-IV) in treating ON-FN. Bone-marrow-derived macrophages (BMDMs) were treated with lipopolysaccharides (LPS), IFN-γ or IL-4 to induce M1 and M2-like phenotypes. Quantitative real-time PCR and Western blot were used to examine M1 and M2 phenotypic markers. Flow cytometry was used to analyze MHC II, CD206, F4/80, and CD11b levels and cell apoptosis. Glucocorticoid was used to induce ON-FN in mice. TNF-α and IL-1ß levels in femoral head were determined using enzyme-linked immunosorbent assay. AS-IV repolarized macrophages from M1 to M2 phenotypes. Culture medium from AS-IV treated M1 macrophages induced less cell apoptosis osteocytes compared to that from untreated M1 macrophages. In ON-FH mice, the ratio of M1 macrophages was decreased in the femoral head by AS-IV, concomitant with a decrease in TNF-α and IL-1ß levels. AS-IV is effective in alleviating ON-FH through its effects in repolarizing macrophages from M1-like phenotype to M2-like phenotype, promoting survival of osteocytes, reducing arthritic symptoms, and decreasing inflammatory cytokines.

Tumor necrosis factor-α promotes Staphylococcus aureus-induced osteomyelitis through downregulating endothelial nitric oxide synthase.

BACKGROUND: Infections of Staphylococcus aureus (S. aureus) often result in osteomyelitis, which is the acute or chronic infections of the bone marrow or bones. TNF-α is long recognized as a key factor contributing to the pathogenesis of osteomyelitis, but little is known about the underlying molecular mechanism. METHODS: Expression levels of TNF-α, and several candidate genes, including endothelial nitric oxide synthase (eNOS), known to be downregulated by TNF-α were analysed in MC3T3-E1 cells with S. aureus infection and osteomyelitis patient blood. MicroRNA(miR)-129-5p was predicted and experimentally verified to target eNOS. Alizarin red sulfate (ARS) and alkaline phosphatase (ALP) staining assays were conducted on MC3T3-E1 cells with S. aureus infection to assess the role of TNF-α/miR-129-5p/eNOS on mineralization defect. RESULTS: TNF-α and miR-129-5p were upregulated while eNOS was downregulated in MC3T3-E1 cells with S. aureus infection and osteomyelitis patients, showing inversely correlated expression profiles. MiR-129-5p directly binds to the 3'-UTR of eNOS mRNA to suppress eNOS expression in MC3T3-E1 cells. TNF-α blocker inhibited miR-129-5p and elevated eNOS expression, likely contributing to rescued mineralization defect in S. aureus-infected MC3T3-E1 cells. During S. aureus infection, upregulated TNF-α increases endogenous miR-129-5p expression, which in turn inhibits eNOS, contributing to osteomyelitis. CONCLUSION: Our study thereby proposes a novel signalling cascade involving TNF-α/miR-129-5p/eNOS in the pathogenesis of osteomyelitis, which may also serve as therapeutic targets.

Involvement of mechanosensitive ion channels in the effects of mechanical stretch induces osteogenic differentiation in mouse bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) can be induced to process osteogenic differentiation with appropriate mechanical and/or chemical stimuli. The present study described the successful culture of murine BMSCs under mechanical strain. BMSCs were subjected to 0%, 3%, 8%, 13%, and 18% cyclic tensile strain at 0.5 Hz for 8 hr/day for 3 days. The expression of osteogenic markers and mechanosensitive ion channels was evaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The expression of alkaline phosphatase (ALP) and matrix mineralization were evaluated with histochemical staining. To investigate the effects of mechanosensitive ion channel expression on cyclic tensile strain-induced osteogenic differentiation, the expression of osteogenic markers was evaluated with real-time RT-PCR in the cells without mechanosensitive ion channel expression. This study revealed a significant augment in osteogenic marker in BMSC strained at 8% compared to other treatments; therefore, an 8% strain was used for further investigations. The ALP expression and matrix mineralization were enhanced in osteogenic induced BMSCs subjected to 8% strain after 7 and 14 days, respectively. Under the same conditions, the osteogenic marker and mechanosensitive ion channel expression were significantly promoted. However, the loss function of mechanosensitive ion channels resulted in the inhibition of osteogenic marker expression. This study demonstrated that strain alone can successfully induce osteogenic differentiation in BMSCs and the expression of mechanosensitive ion channels was involved in the process. The current findings suggest that mechanical stretch could function as efficient stimuli to induce the osteogenic differentiation of BMSCs via the activation of mechanosensitive ion channels.

MicroRNA-23b-3p participates in steroid-induced osteonecrosis of the femoral head by suppressing ZNF667 expression.

BACKGROUND: Clinical treatment with high-dose of steroid hormone causes steroid-induced osteonecrosis of the femoral head (SONFH), whereas the internal regulation mechanism remains elusive. Numerous studies have reported that microRNAs participated in the development of SONFH through modulating gene expression. The aim of the current study was to clarify the function of microRNA-23b-3p (miR-23b-3p) and ZNF667 in SONFH. EXPERIMENTAL DESIGN: Bioinformatics prediction and luciferase reporter system were utilized to confirm the target relation between miR-23b-3p and ZNF667. To examine the function of miR-23b-3p in vivo, rat SONFH models were established by specific inducers. The morphological changes, plasma viscosity, blood lipid, and inflammatory cytokines were measure by corresponding experiments. RESULTS: MiR-23b-3p and ZNF667 was negatively correlated in SONFH patient tissues, miR-23b-3p was down-regulated, while ZNF667 was up-regulated. MiR-23b-3p targeted ZNF667, the expression level of ZNF667 was suppressed by miR-23b-3p activation whereas strengthened by miR-23b-3p inhibition. SONHF rats with overexpressed miR-23b-3p displayed alleviated symptoms, including reduced plasma viscosity, declined blood lipids, decreased levels of pro-inflammatory cytokines and improved bone integrality. Moreover, elevation of ZNF667 reversed the repression of SONFH induced by miR-23b-3p overexpression. CONCLUSIONS: We found that miR-23b-3p played a protective role in SONFH by targeting ZNF667, which provided a novel reference for SONFH prevention and therapy.

Open reduction through a posterior incision in the surgical treatment of shoulder posterior dislocation associated with proximal humeral fractures.

BACKGROUND: We explored the surgical technique of reducing the humeral head and repairing the fractures through a combined approach in the treatment of this complex injury. METHODS: Six patients with posterior shoulder dislocations associated with proximal humerus fractures were enrolled in this study. The posteriorly dislocated head was first reduced through a shoulder posterior incision and the ruptured posterior capsular tissues were repaired simultaneously using a suture anchor. The fractures were then reduced and fixed with a PHILOS through a deltopectoral approach. The affected shoulders were immobilized in a neutral position for 6 weeks postoperatively with a customized orthosis and then permitted active shoulder exercises after removal of the orthosis. At the last visit, union of the fractures was evaluated. Degrees of anterior forward of the affected shoulder were recorded. Outcomes were evaluated according to UCLA and Constant criteria. RESULTS: Six patients were followed up for an average of 24.5 ± 7.4 (range 13-35) months. At the last visit, the mean degree of anterior forward was 171.7 ± 7.5 (range 160-180) degrees. An average of 32.9 ± 1.2 (range 31-34) points was obtained according to UCLA criteria, demonstrating excellent and good results in two and four cases, respectively. The mean Constant score was 87.3 ± 4.1 (range 83-92) points. CONCLUSIONS: The dislocated humeral head can be reduced through a posterior approach, while fractures can be reduced and fixed through a deltopectoral approach. This technique has the advantages of simplicity and its minimally invasive approach for reducing the dislocation.

Treatment of Transverse with or without Posterior Wall Fractures of Acetabulum Using a Modified Smith-Petersen Combined with Kocher-Langenbeck Approach.

BACKGROUND The aim of this study was to explore the surgical treatment of transverse with or without posterior wall fractures of the acetabulum. MATERIAL AND METHODS We surgically treated 21 consecutive cases of pure transverse (7 cases) and with posterior wall (14 cases) fractures of the acetabulum. The anterior column fractures were firstly reduced, temporarily fixed through a modified Smith-Petersen small incision, and finally fixed after the fixation of the posterior column and wall fractures, which were reduced and fixed through a Kocher-Langenbeck approach. The operative time, intra-operative blood loss, quality of reduction (Matta criteria), perioperative complications, osseous union, subsequent complications, and hip function evaluation were recorded. RESULTS The mean operative time was 198.1 min and the mean intra-operative blood loss was 938.1 ml. Anatomic reduction of the anterior column was obtained in 20 cases and was imperfect in 1 case. All posterior column and wall fractures were anatomically reduced. We followed up 18 cases for a mean duration of 16.3 (8-30) months. All the fractures achieved osseous union. The mean Harris score was 85.1 points, with an excellent result in 7 cases, good in 8, fair in 2, and poor in 1. According to modified Merle d' Aubigne and Postel score system, the results were excellent in 2 cases, good in 15, and poor in 1. Avascular necrosis of the femoral head occurred in 1 case, heterotopic ossification in 3 cases, and numbness of the anterolateral thigh in 6 cases. CONCLUSIONS For transverse with or without posterior wall fractures of the acetabulum, reduction and fixation of anterior and posterior column should be done in sequence, and a modified Smith-Petersen small incision might be a good choice in reduction and fixation of the anterior column because it possesses advantages of direct visualization and minimal invasion.

New strategy to rescue the inhibition of osteogenesis of human bone marrow-derived mesenchymal stem cells under oxidative stress: combination of vitamin C and graphene foams.

To rescue the oxidative stress induced inhibition of osteogenesis, vitamin C (VC) was chemically modified onto three-dimensional graphene foams (3D GFs), then their regulation on osteogenesis of human bone marrow-derived mesenchymal stem cells (BM-MSCs) was studied. Combined action of VC + GF significantly decreased H2O2-induced oxidative stress, and rescued H2O2-inhibited cell viability, differentiation and osteogenesis of BM-MSCs in vitro. Further studies revealed that Wnt pathway may be involved in this protection of osteogenesis. Furthermore, an in vivo mouse model of BM-MSCs transplantation showed that VC + GF remarkably rescued oxidative stress inhibited calcium content and bone formation. The combination of VC and GF exhibited more pronounced protective effects against oxidative stress induced inhibition of osteogenesis, compared to monotherapy of VC or GF. Our study proposed a new strategy in stem cell-based therapies for treating bone diseases.

[Operation via anterior approach in treating pelvic crescent fracture].

OBJECTIVE: To evaluate the effectiveness of operation via anterior approach in treating pelvic crescent fracture. METHODS: Between June 2007 and December 2009, 18 patients with pelvic crescent fracture were treated, including 11 males and 7 females with an average age of 32 years (range, 19-52 years). The locations were the right in 8 cases and the left in 10 cases. Fractures were caused by traffic accident in 10 cases, by falling from height in 5 cases, and by crushing with heavy weights in 3 cases. All patients suffered lateral compression injuries of the pelvis. The mean time from injury to operation was 7.8 days (range, 3-22 days). The preoperative mean displacement of the posterior pelvic ring was 13.7 mm (range, 5-24 mm) according to the method described by Matta et al. The operation time, intraoperative blood loss, displacement correction of the posterior pelvic ring, fracture healing time, and Hannover pelvic score were recorded. RESULTS: The average operation time was 175 minutes (range, 110-230 minutes); the average intraoperative blood loss was 561.7 mL (range, 300-1,100 mL); the postoperative mean displacement of the posterior pelvic ring was 1.2 mm (range, 0-3 mm); and the mean displacement correction of the posterior pelvic ring was 12.6 mm (range, 4-23 mm). No intraoperative lumbosacral nervous injury occurred. Superficial wound infection occurred in 2 cases and was cured after 1 week of wound drainage and application of antibiotic. The others achieved healing of incision by first intention. Fifteen patients were followed up 16.1 months on average (range, 13-22 months). The X-ray films showed fracture healing in all patients. The fracture healing time was 3.6 months on average (range, 3-4 months). No patient had loss of reduction or failure of internal fixation. The clinical outcome was excellent in 10 cases (66.7%) and good in 5 cases (33.3%) according to Hannover pelvic score; social reintegration was complete in 13 cases (86.7%) and incomplete in 2 cases (13.3%). CONCLUSION: Operation via anterior approach is a good choice in the treatment of pelvic crescent fracture.

[Comparison of effectiveness between minimally invasive plating osteosynthesis and expandable intramedullary nailing technique in treatment of middle third humeral shaft fractures].

OBJECTIVE: To compare the effectiveness of two minimally invasive methods: minimally invasive plating osteosynthesis (MIPO) and expandable intramedullary nailing technique in treatment of middle third humeral shaft fractures. METHODS: The clinical data were retrospectively analyzed and compared from 33 cases with middle third humeral shaft fractures between May 2004 and December 2008. All the patients were divided into 2 groups: 14 patients were treated with MIPO technique (group A) and 19 with expandable intramedullary nailing technique (group B). In group A, there were 10 males and 4 females with an average age of 35 years (range, 21-51 years). The disease cause was traffic accident in 5 cases, tumbling in 6 cases, machine related trauma in 2 cases, crushed by a heavy object in 1 case. Six fractures were classified as AO type A, 6 as type B, and 2 as type C. The time from injury to operation was 3 to 11 days with an average of 5.9 days. In group B, there were 12 males and 7 females with an average age of 40 years (range, 19-68 years). The disease cause was traffic accident in 7 cases, tumbling in 8 cases, falling from height in 3 cases, crush injury in 1 case. Ten fractures were classified as AO type A, 8 as type B, and 1 as type C. The time from injury to operation was 2 to 6 days with an average of 4.2 days. There was no significant difference in general data between 2 groups (P > 0.05). RESULTS: The operation time was (104.6 +/- 25.8) minutes in group A and (85.0 +/- 35.7) minutes in group B, showing no significant difference (P > 0.05). Incision healed by first intention without iatrogenic radial nerve palsy in 2 groups. The patients were followed up 21.4 months on average (range, 12-37 months) in group A and 20.5 months on average (range, 22-35 months) in group B. The X-ray films showed bony healing in all patients. The fracture union time was (16.4 +/- 6.1) weeks in group A and (15.0 +/- 2.5) weeks in group B, showing no significant difference (P > 0.05). The University of California Los Angeles (UCLA) End-Result scores were 34.1 +/- 1.1 in group A and 31.8 +/- 2.6 in group B and the Mayo Elbow Performance scores were 100 in group A and 97.6 +/- 3.9 in group B; all showing significant differences (P < 0.05). CONCLUSION: Good clinical outcomes could be obtained when middle third humeral shaft fractures are treated by either MIPO or expandable intramedullary nailing techniques. However, MIPO technique could offer better shoulder and elbow functional results.