mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway.

PURPOSE: To study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin. METHODS: We isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone. RESULTS: Up-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin. CONCLUSION: miR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

CCL2 Knockdown Attenuates Inflammatory Response After Spinal Cord Injury Through the PI3K/Akt Signaling Pathway: Bioinformatics Analysis and Experimental Validation.

Spinal cord injury (SCI) is a common clinical problem in orthopedics with a lack of effective treatments and drug targets. In the present study, we performed bioinformatic analysis of SCI datasets GSE464 and GSE45006 in the Gene Expression Omnibus (GEO) public database and experimentally validated CCL2 expression in an animal model of SCI. This was followed by stimulation of PC-12 cells using hydrogen peroxide to construct a cellular model of SCI. CCL2 expression was knocked down using small interfering RNA (si-CCL2), and PI3K signaling pathway inhibitors and activators were used to validate and observe the changes in downstream inflammation. Through data mining, we found that the inflammatory chemokine CCL2 and PI3K/Akt signaling pathways after SCI expression were significantly increased, and after peroxide stimulation of PC-12 cells with CCL2 knockdown, their downstream cellular inflammatory factor levels were decreased. The PI3K/Akt signaling pathway was blocked by PI3K inhibitors, and the downstream inflammatory response was suppressed. In contrast, when PI3K activators were used, the inflammatory response was enhanced, indicating that the CCL2-PI3K/Akt signaling pathway plays a key role in the regulation of the inflammatory response. This study revealed that the inflammatory chemokine CCL2 can regulate the inflammatory response of PC-12 cells through the PI3K/Akt signaling pathway, and blocking the expression of the inflammatory chemokine CCL2 may be a promising strategy for the treatment of secondary injury after SCI.

Identification of the CCL2 PI3K/Akt axis involved in autophagy and apoptosis after spinal cord injury.

Spinal cord injury (SCI) is a devastating neurological disease with no cure that usually results in irreversible loss of sensory and voluntary motor functions below the injury site. We conducted an in-depth bioinformatics analysis combining the gene expression omnibus spinal cord injury database and the autophagy database and found that the expression of the autophagy gene CCL2 was significantly upregulated and the PI3K/Akt/mTOR signaling pathway was activated after SCI. The results of the bioinformatics analysis were verified by constructing animal and cellular models of SCI. We then used small interfering RNA to inhibit the expression of CCL2 and PI3K to inhibit and activate the PI3K/Akt/mTOR signaling pathway; western blot, immunofluorescence, monodansylcadaverine, and cell flow techniques were used to detect the expression of key proteins involved in downstream autophagy and apoptosis. We found that when PI3K inhibitors were activated, apoptosis decreased, the levels of autophagy-positive proteins LC3-I/LC3-II and Bcl-1 increased, the levels of autophagy-negative protein P62 decreased, the levels of pro-apoptotic proteins Bax and caspase-3 decreased, the levels of the apoptosis-inhibiting protein Bcl-2 increased. In contrast, when a PI3K activator was used, autophagy was inhibited, and apoptosis was increased. This study revealed the effect of CCL2 on autophagy and apoptosis after SCI through the PI3K/Akt/mTOR signaling pathway. By blocking the expression of the autophagy-related gene CCL2, the autophagic protective response can be activated, and apoptosis can be inhibited, which may be a promising strategy for the treatment of SCI.

Identification of the Fosl1/AMPK/autophagy axis involved in apoptotic and inflammatory effects following spinal cord injury.

Strategies for reducing spinal cord injury (SCI) have become a research focus because an effective treatment of SCI is unavailable. The objective of this study was to explore the underlying mechanisms of Fosl1 following SCI. Based on the analysis of the Gene Expression Omnibus (GEO) database, Fosl1 was found to be highly enhanced in SCI. This result was confirmed in our animal model, and Fosl1 was found to be obviously expressed in neurons. Next, we treated PC-12 cells with H2O2 to mimic injured neurons and further verified that Fosl1 silencing upregulated AMPK expression, promoted autophagy and inhibited inflammation and apoptosis. Subsequently, a special inhibitor of AMPK was used to examine the role of AMPK, and we learned that the inhibition of AMPK suppressed autophagy and promoted inflammation and apoptosis following Fosl1 silencing. These changes completely reversed the beneficial effects of Fosl1 silencing on injured PC-12 cells. Moreover, treatment with an AMPK activator resulted in effects that were opposite those of the inhibitor. Finally, rats were injected intrathecally with si-Fosl1 to detect its role in vivo. The results showed that si-Fosl1 improved neurological function and decreased apoptosis and inflammation at 14 d postoperation, and the activator further benefited the rats of si-Fosl1 treatment. In conclusion, Fosl1 inhibits autophagy and promotes inflammation and apoptosis through the AMPK signaling pathway following SCI in vivo and in vitro.

Therapeutic effect of neohesperidin on TNF-α-stimulated human rheumatoid arthritis fibroblast-like synoviocytes.

During the pathogensis of rheumatoid arthritis (RA), activated RA fibroblast-like synoviocytes (RA-FLSs) combines similar proliferative features as tumor and inflammatory features as osteoarthritis, which eventually leads to joint erosion. Therefore, it is imperative to research and develop new compounds, which can effectively inhibit abnormal activation of RA-FLSs and retard RA progression. Neohesperidin (Neo) is a major active component of flavonoid compounds with anti-inflammation and anti-oxidant properties. In this study, the anti-inflammation, anti-migration, anti-invasion, anti-oxidant and apoptosis-induced effects of Neo on RA-FLSs were explored to investigate the underlying mechanism. The results suggested that Neo decreased the levels of interleukin IL-1ß, IL-6, IL-8, TNF-α, MMP-3, MMP-9 and MMP-13 in FLSs. Moreover, Neo blocked the activation of the MAPK signaling pathway. Furthermore, treatment with Neo induced the apoptosis of FLSs, and inhibited the migration of FLSs. It was also found that Neo reduced the accumulation of reactive oxygen species (ROS) induced by TNF-α. Taken together, our results highlighted that Neo may act as a potential and promising therapeutic drug for the management of RA.

Utilizing benchmarked dataset and gene regulatory network to investigate hub genes in postmenopausal osteoporosis.

OBJECTIVE: The objective of this paper was to investigate hub genes of postmenopausal osteoporosis (PO) utilizing benchmarked dataset and gene regulatory network (GRN). MATERIALS AND METHODS: To achieve this goal, the first step was to benchmark the dataset downloaded from the ArrayExpress database by adding local noise and global noise. Second, differentially expressed genes (DEGs) between PO and normal controls were identified using the Linear Models for Microarray Data package based on benchmarked dataset. Third, five kinds of GRN inference methods, which comprised Zscore, GeneNet, context likelihood of relatedness (CLR) algorithm, Partial Correlation coefficient with Information Theory (PCIT), and GEne Network Inference with Ensemble of trees (Genie3), were described and evaluated by receiver operating characteristic (ROC) and precision and recall (PR) curves. Finally, GRN constructed according to the method with best performance was implemented to conduct topological centrality (closeness) for the purpose of investigate hub genes of PO. RESULTS: A total of 236 DEGs were obtained based on benchmarked dataset of 20,554 genes. By assessing Zscore, GeneNet, CLR, PCIT, and Genie3 on the basis of ROC and PR curves, Genie3 had a clear advantage than others and was applied to construct the GRN which was composed of 236 nodes and 27,730 edges. Closeness centrality analysis of GRN was carried out, and we identified 14 hub genes (such as TTN, ACTA1, and MYBPC1) for PO. CONCLUSION: In conclusion, we have identified 14 hub genes (such as TN, ACTA1, and MYBPC1) based on benchmarked dataset and GRN. These genes might be potential biomarkers and give insights for diagnose and treatment of PO.

Evodiamine attenuates adjuvant-induced arthritis in rats by inhibiting synovial inflammation and restoring the Th17/Treg balance.

OBJECTIVES: Evodiamine (Evo) possesses strong anti-inflammatory activity. In this study, we determine the antiarthritic effect of Evo. METHODS: Evo was administered to rats with adjuvant-induced arthritis (AA). We evaluated arthritis symptoms & histopathological changes and measured inflammatory cell infiltration, pro-inflammatory cytokine production and Th17 & Treg percentages in arthritic rats. KEY FINDINGS: Evo significantly improved the clinical signs of AA in rats, including decreases in paw swelling, the polyarthritis index and the number of swollen paw joints. Based on the histopathological analysis, Evo improved synovial inflammation and bone injury by inhibiting inflammatory cell infiltration, synoviocyte proliferation, pannus formation and cartilage erosion. Furthermore, the numbers of synovial CD3+ or CD68+ inflammatory cells were reduced, and the elevated levels of tumour necrosis factor-α, interleukin-1ß (IL-1ß) and IL-6 were restored to control levels by the Evo treatment. In addition, Evo therapy regulated the abnormal differentiation of Treg and Th17 cells, decreasing IL-17 production and increasing IL-10 levels. Finally, Evo inhibited Stat3 phosphorylation and induced Stat5 phosphorylation in rats with AA. CONCLUSIONS: Based on our results, Evo is a promising antiarthritic agent, potentially due to its inhibitory effect on synovial inflammation and regulatory effects on Treg and Th17 differentiation.

Knockdown of SGK1 alleviates the IL-1ß-induced chondrocyte anabolic and catabolic imbalance by activating FoxO1-mediated autophagy in human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the progressive degeneration of articular cartilage with no effective treatment methods available. Cartilage degeneration is closely related to an anabolic and catabolic imbalance in chondrocytes, and accumulating evidence has revealed that autophagy is a crucial protective mechanism that maintains the balance of anabolic and catabolic activities. Therefore, studies aiming to identify additional genes that regulate autophagy as a promising therapeutic strategy for OA are needed. In this study, we analyzed the GSE113825 datasets from Gene Expression Omnibus and validated that serum- and glucocorticoid-regulated kinase 1 (SGK1) was upregulated in OA cartilage. Based on the results from loss-of-function studies, SGK1 silencing promoted the deposition of glycosaminoglycans in interleukin 1 beta (IL-1ß)-treated chondrocytes, and significantly alleviated IL-1ß-induced downregulation of Collagen II and Aggrecan, as well as the upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 5 and matrix metalloproteinase-13. Furthermore, SGK1 knockdown reversed the IL-1ß-induced chondrocyte anabolic and catabolic imbalance by activating autophagy. Moreover, SGK1 directly bound to forkhead box protein O1 (FoxO1) and increased its phosphorylation, which in turn resulted in its translocation from the nucleus. The decreased FoxO1 levels led to a decrease in LC3-I/LC3-II conversion and Beclin-1 levels, subsequently inhibiting autophagosome formation and increasing P62 levels, thus indicating a downregulation of autophagy. Taken together, we identified a critical role of SGK1 in the IL-1ß-induced chondrocyte anabolic and catabolic imbalance, which may represent a potential novel therapeutic target for OA.

Proximal humeral internal locking plate combined with a custom neutral-position shoulder and elbow sling for proximal humerus fractures: A randomized study.

OBJECTIVES: The aim of this study was to investigate the effectiveness of the proximal humeral internal locking system (PHILOS) plate combined with a custom neutral-position shoulder and elbow sling for proximal humerus fractures. METHODS: A total of 112 patients with proximal humerus fractures were assigned randomly into 2 groups. Group A (n = 56) was treated by open reduction and internal fixation (ORIF) with a PHILOS plate; group B (n = 56) was treated by ORIF with a PHILOS plate in combination with the use of a custom neutral-position shoulder and elbow sling for 30 days after surgery. The incidence of internal fixation failure, the Constant-Murley shoulder assessment, and Visual Analogue Scale (VAS) score were recorded and analyzed. RESULTS: Patients included were followed up for an average of 15 months (range, 6-24 months). No significant differences were observed in mean VAS scores and mean Constant-Murley shoulder assessment scores at 1-day preoperative and postoperative day 3 between groups A and B. However, mean VAS scores and mean Constant-Murley shoulder assessment in group B were significantly improved when compared with group A at postoperative day 30 and the final follow-up. No cases of postoperative infection, loss of reduction, PHILOS break, or vascular nerve injury occurred in either group. CONCLUSIONS: Proximal humerus fractures treated with the combination of the PHILOS and custom neutral-position shoulder and elbow sling for 30 days after operation was associated with a lower incidence of internal fixation failure. There was no increase in adverse events compared with open reduction and internal fixation with a PHILOS plate alone.

CTHRC1 mediates IL­1ß­induced apoptosis in chondrocytes via JNK1/2 signaling.

Osteoarthritis (OA), also known as degenerative joint disease or degenerative arthritis, is characterized by chondrocyte apoptosis. The aim of the present study was to investigate the effects of collagen triple helix repeat containing 1 (CTHRC1) and the c­Jun N­terminal kinase (JNK) 1/2 inhibitor SP600125 on rat chondrocytes cultured in vitro with interleukin (IL)­1ß. Chondrocytes were treated with different doses of IL­1ß and cell viability and CTHRC1 expression were assessed using Cell Counting Kit­8 and western blot assays, respectively. In separate experiments, chondrocytes were treated with CTHRC1­expressing constructs (pLVX­Puro­CTHRC1) and/or SP600125, or IL­1ß with either CTHRC1 short hairpin (sh)RNA constructs (shNRA­CTHRC1) or SP600125. The expression of CTHRC1, B­cell lymphoma (Bcl)­2, Bcl­2­associated X protein (Bax), cleaved caspase­3, poly ADP ribose polymerase (PARP)­1 and matrix metalloproteinase (MMP)­13 was measured using reverse transcription­quantitative polymerase chain reaction and western blotting assays. A Cell Counting Kit­8 assay was performed to examine cell viability. Annexin V/propidium iodide staining and flow cytometry assays were used to detect chondrocyte apoptosis. The expression of JNK1/2 and phosphorylated JNK1/2 was measured using western blotting. CTHRC1 was highly expressed in patients with OA compared with normal controls. IL­1ß treatment (5, 10 and 20 ng/ml) increased the protein expression of CTHRC1 in a dose­dependent manner and decreased the viability of chondrocytes in a time­dependent manner. pLVX­Puro­CTHRC1 mimics the effect of IL­1ß on chondrocyte apoptosis and JNK1/2 activity, and this is reversed by SP600125 treatment. However, transfection with shRNA­CTHRC1 or treatment with SP600125 inhibited IL­1ß­induced cell apoptosis and JNK1/2 activation. These results indicate that CTHRC1 downregulation may protect chondrocytes from IL­1ß­induced apoptosis by inactivating the JNK1/2 pathway.

Serum Levels of the Inflammatory Cytokines in Patients with Lumbar Radicular Pain Due to Disc Herniation.

STUDY DESIGN: Cohort study. PURPOSE: This study primarily aimed to evaluate the serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-4 in patients with lumbar radiculopathy 1 and 12 months after microdiscectomy. OVERVIEW OF LITERATURE: Lumbar radiculopathy is possibly caused by inflammatory changes in the nerve root. The intraneural application of pro-inflammatory cytokines induces behavioral signs associated with pain. Anti-inflammatory cytokine treatment effectively reduces hyperalgesia. METHODS: The role of TNF-α and IL-4 in long-lasting lumbar radiculopathy was addressed. A total of 262 patients were recruited from Anqing Hospital, Anhui Medical University. During inclusion at 1 and 12 months, serum concentrations of TNF-α and IL-4 were analyzed by enzyme-linked immunosorbent assay, and pain intensity was reported on a 0-10 cm visual analog scale (VAS). RESULTS: Sixty six patients had VAS <3 and 196 patients had VAS ≥3. Serum concentrations of pro-inflammatory TNF-α and anti-inflammatory IL-4 in patients with lumbar radiculopathy related to disc herniation were measured at 1- and 12-month follow-up. TNF-α decreased in both VAS groups with time. In contrast, IL-4 increased in both groups at 1 month and then decreased gradually until month 12. The changes in serum levels of TNF-α and IL-4 over time between the VAS ≥3 and VAS <3 groups were significantly different. CONCLUSIONS: Chronic lumbar radiculopathy may be associated with high level of pro-inflammatory substances, such as TNF-α, in serum after disc herniation, and elevated anti-inflammatory cytokine in patients with lumbar radiculopathy may indicate a favorable outcome.

Epidermal growth factor increases the expression of Nestin in rat reactive astrocytes through the Ras-Raf-ERK pathway.

Astrocytes undergo de-differentiation and become activated during a response to injury. Several studies have found that reactive astrocytes re-express markers, such as Nestin, which are normally expressed in neural stem cells. It was recently shown that the epidermal growth factor receptor (EGFR) is up-regulated in astrocytes after injury and promotes reactive astrocyte transformation. However, the signaling pathways involved in this process have not been elucidated. In the present study, we showed that Nestin was strongly expressed in reactive astrocytes. Furthermore, as shown by immunoblot analyses, epidermal growth factor (EGF) regulated Nestin expression through EGFR activation. Inhibition of the PLCγ, PI3K, ERK, p38, and JNK pathways did not affect Nestin expression in reactive astrocytes. However, treatment with a Raf-1 inhibitor inhibited Nestin expression in a concentration-dependent manner. Taken together, the signaling analyses revealed that EGF induced and regulated Nestin expression through activation of the Ras-Raf--ERK signaling pathway. This is the first study to show that Nestin expression is regulated by an extracellular signaling molecule in reactive astrocytes.

Surgical management of the fractures of axis body: indications and surgical strategy.

PURPOSE: The axis body fractures are relatively uncommon and have a variety of presentations. Surgical management to them has been only reported as case reports or included as a minor part of clinical management. The objective of this study is to summarize the indications for surgery and report the clinical outcome of surgical treatment based on different fracture patterns. METHODS: A retrospective analysis of 28 consecutive patients presenting with the axis body fractures was undertaken. The indications for surgical treatment were defined as: (1) fractures associated with instability of adjacent joints; (2) irreducible displaced superior articular facet fracture; (3) fractures resulting in spinal cord compression. The fractures were classified as sagittal, coronal, transverse and lateral mass fracture. One of the following surgical procedures was applied according to the fracture pattern: posterior C1-C2 pedicle screws fixation and fusion (I); posterior C1-C3 screws fixation and fusion (II); posterior osteosynthesis with C2 transpedicular half-thread lag screws (III). RESULTS: 13 patients were successfully managed operatively. Two transverse and two unilateral lateral mass fractures were treated with surgical procedure I, five sagittal fractures with II, four coronal fractures with III. Complications of malposition of screws and neurologic deficit did not occur during operation. Satisfactory reduction and bony union were demonstrated on postoperative radiographics. CONCLUSIONS: Conservative treatment is still advocated as primary management for most axis body fractures. But for patients with obvious adjacent joints instability or irreducible displaced superior articular facet fracture, surgical intervention based on the different fracture pattern is necessary.

Treatment of osteonecrosis of the femoral head with thorough debridement, bone grafting and bone-marrow mononuclear cells implantation.

Osteonecrosis of the femoral head (ONFH) is a disease with a wide-ranging etiology and poorly understood pathogenesis seen commonly in young patients. Various head-preserving procedures have been used to avert the need for total hip replacement. These include vascularized and non-vascularized bone grafting procedures, bone marrow containing osteogenic precursors implanted into the necrotic lesion. We use drills, curettes, broaches under image intensifier to perform a thorough debridement of all necrotic lesion, pack autogenous cortical and cancellous bone which were harvested from the ipsilateral iliac crest tightly into the femoral head, implant bone-marrow mononuclear cells containing mesenchymal stem cells into the necrotic lesion. The study included 15 patients (20 hips, 10 males, 5 females, mean age 35 years, range 23-58 years) with stage II-III ONFH according to the association research circulation osseous classification. The outcome was determined by changes in the Harris hip score (HHS), by progression in radiographic stages, and by the need for hip arthroplasty. The mean follow-up was 24 months (range 9-36 months). The mean HHS increased from 64 to 85 points. The overall clinical success rate is 80 %. There were no infection, femoral neck fracture or other complications. Thorough debridement, autogenous bone grafting and bone-marrow mononuclear cells implantation is an effective procedure in patient with small lesion, early-stage ONFH.

Chordin-like protein 1 promotes neuronal differentiation by inhibiting bone morphogenetic protein-4 in neural stem cells.

In the present study, the effects of chordin­like protein 1 (CHRDL1) overexpression, together with bone morphogenetic protein­4 (BMP­4) treatment, on the differentiation of rat spinal cord­derived neural stem cells (NSCs) was investigated. Adult rat spinal cord­derived NSCs were cultured in serum­free medium. The recombined eukaryotic expression vector pSecTag2/Hygro B­CHRDL1 was transfected into adult rat spinal cord­derived NSCs using a lipid­based transfection reagent and protein expression was assessed by western blot analysis. Differentiation of transfected NSCs following BMP­4 treatment was determined by immunocytochemistry. The percentage of microtubule­associated protein­2 (MAP­2)­positive cells in the BMP­4­treated (B) group was found to be significantly lower compared with that in the non­transfected control (N) group. The percentage of MAP­2­positive cells in the pSecTag2/Hygro B­CHRDL1­transfected, BMP­4­treated group was identified to be significantly higher compared with that in group B, however, no significant difference was observed between group N and the transfected, non­BMP­4­treated control group. The current study indicates that CHRDL1 protein antagonizes BMP­4 activity and induces spinal cord­derived NSCs to differentiate into neurons.

[Surgical treatment of the sacrum tumor].

OBJECTIVE: To discuss the surgical methods and effects in the treatment of sacrum tumor. METHODS: Fifteen patients of sacrum tumor included 12 males 3 females aged from 17 to 68 years old,mean 54.6 years. Ten cases were primary tumor and 5 were metastatic tumor. Five cases underwent anterior approach tumor extirpation, 3 posterior approach tumor extirpation and 7 posterior tumor extirpation with bone graft and internal fixation of a pedicle screw and rod system. Additionally, all cases were treated with radiotherapy or/and chemotherapy post-operatively according to the character of the tumor. RESULTS: Thirteen patients were followed-up for 4 months to 5 years. One patient had exacerbation accompanying dysfunction of urinary and feca after surgery, which relieved after four months of non-operative treatments. One chordoblastoma and 2 metastatic tumor died of recurrence and metastasis 1 to 2 years after operation, respectively. And in another case of giant cell tumor occurred the local recurrence 6 months after operation, who refused secondary surgical treatment. CONCLUSION: Individualized surgical treatment with conbination of radio therapy or/and chemotherapy will make good results for patients with sacrum tumor.