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To investigate the effectiveness of a Chinese patent medicine, Jintiange capsules with the main component of artificial tiger bone powder, combined with alfacalcidol on muscle strength and balance of the lower extremities in patients with primary osteoporosis.A randomized, double-blind, double-dummy, positive-controlled, multicenter clinical trial.A total of 400 patients diagnosed with primary osteoporosis or osteopenia were recruited and randomized into the Jintiange or control groups. During the 52-week treatment, the participants in the Jintiange group were treated with Jintiange capsules (1.2 ​g each time, 3 times per day) and calcium carbonate simulant, while those in the control group were treated with calcium carbonate (element calcium 0.3 ​g, twice a day) and a Jintiange capsule simulant. Alfacalcidol (0.25 ​μg/d) was applied in both groups. The timed up and go test (TUG), chair rising test (CRT), and tandem gait test (TGT) were performed to evaluate balance, muscle strength and fall risk of the participants.There were 154 participants in the Jintiange group, and 157 participants in the control group were included in the per-protocol set. Comparing the data at week 52 from those at baseline, the TUG time decreased from 9.60 ​± ​2.25 ​s to 8.53 ​± ​2.06 ​s (p ​ ​0.001) in the Jintiange group and decreased from 9.50 ​± ​1.91 ​s to 9.11 ​± ​1.95 ​s (p ​ ​0.001) in the control group; the CRT time decreased from 11.49 ​± ​4.05 ​s to 8.57 ​± ​2.13 ​s (p ​ ​0.001) and 11.17 ​± ​3.21 ​s to 9.74 ​± ​1.98 ​s (p ​ ​0.001) in the Jintiange and control groups, respectively; the number of correct steps in the TGT increased significantly in both the control (7.40 ​± ​1.27 vs. 7.69 ​± ​0.87, p ​ ​0.01) and Jintiange groups (7.21 ​± ​1.58 vs. 7.60 ​± ​1.12, p ​ ​0.001). At the end of the study, the TUG and CRT results in the Jintiange group were superior to those in the control group (all p value ​ ​0.05), while no obvious difference was found in the TGT between the two groups. At week 52, the high fall risk proportions in the Jintiange group were significantly lower than those in the control group according to TUG (3.25% vs. 9.55%, p ​= ​0.023) and CRT (20.78% vs. 33.76%, p ​= ​0.01).Jintiange capsules combined with alfacalcidol can effectively improve muscle strength and the balance of the lower extremities and reduce fall risk in patients with primary osteoporosis/osteopenia.Artificial tiger bone powder, a traditional Chinese patent medicine, can improve muscle strength and balance and reduce fall risks effectively among patients with primary osteoporosis. It might be a therapeutic option for osteoporosis individuals combined with sarcopenia to improve their muscle function.

Objective The aim of this study was to search for key genes in ankylosing spondylitis (AS) through comprehensive bioinformatics analysis, thus providing some theoretical support for future diagnosis and treatment of AS and further research. Methods Gene expression profiles were collected from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) by searching for the term "ankylosing spondylitis". Ultimately, two microarray datasets (GSE73754 and GSE11886) were downloaded from the GEO database. A bioinformatic approach was used to screen differentially expressed genes and perform functional enrichment analysis to obtain biological functions and signalling pathways associated with the disease. Weighted correlation network analysis (WGCNA) was used to further obtain key genes. Immune infiltration analysis was performed using the CIBERSORT algorithm to conduct a correlation analysis of key genes with immune cells. The GWAS data of AS were analysed to identify the pathogenic regions of key genes in AS. Finally, potential therapeutic agents for AS were predicted using these key genes. Results A total of 7 potential biomarkers were identified: DYSF, BASP1, PYGL, SPI1, C5AR1, ANPEP and SORL1. ROC curves showed good prediction for each gene. T cell, CD4 naïve cell, and neutrophil levels were significantly higher in the disease group than in the paired normal group, and key gene expression was strongly correlated with immune cells. CMap results showed that the expression profiles of ibuprofen, forskolin, bongkrek-acid, and cimaterol showed the most significant negative correlation with the expression profiles of disease perturbations, suggesting that these drugs may play a role in AS treatment. Conclusion The potential biomarkers of AS screened in this study are closely related to the level of immune cell infiltration and play an important role in the immune microenvironment. This may provide help in the clinical diagnosis and treatment of AS and provide new ideas for further research.

Objective: The aim of this study is to search for key genes in ankylosing spondylitis through comprehensive bioinformatics analysis, thus providing some theoretical support for future diagnosis and treatment of AS and further research. Methods: The expression matrix of ankylosing spondylitis was downloaded and integrated through public libraries. A bioinformatic approach was used to screen differential genes and perform functional enrichment analysis to obtain biological functions and signaling pathways associated with the disease. Weighted correlation network analysis (WGCNA) was used to further obtain key genes. Immune infiltration analysis was performed using the CIBERSORT algorithm to obtain the correlation analysis of key genes with immune cells. The GWAS data of AS were analyzed to identify the pathogenic regions of key genes in AS. Finally, potential therapeutic agents for AS were predicted using these key genes. Results: A total of 7 potential biomarkers were identified: DYSF, BASP1, PYGL, SPI1, C5AR1, ANPEP and SORL1.ROC curves showed good prediction of each gene. T cell, CD4 naive, and neutrophil levels were significantly higher in the disease group compared to the paired normal group, and key gene expression was strongly correlated with immune cells.CMap results showed that the expression profiles of ibuprofen, forskolin, bongkrek-acid, and cimaterol showed the most significant negative correlation with the expression profiles of disease perturbations, suggesting that these drugs may play a role in AS play a good role in the treatment. Conclusion: The potential biomarkers of AS screened in this study are closely related to the level of immune cell infiltration and play an important role in the immune microenvironment. This may provide help for clinical diagnosis and treatment of AS and provide new ideas for further research.

Ferroptosis is an iron-dependent regulation of cell death driven by lipid peroxidation, which is intracellularly dependent on iron and independent of other metals, and morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. Ferroptosis is closely related to physiological and pathological processes, such as development, aging, and immunity, and it plays an important role in a variety of diseases. In many departments, traditional Chinese medicine plays an increasingly important role in their clinical treatment. In recent years, an increasing number of studies have been conducted on the mechanism of ferroptosis in traditional Chinese medicine. However, the role of ferroptosis in the clinical treatment of traditional Chinese medicine requires further exploration. This article mainly introduces the application of ferroptosis in studies of the mechanism of traditional Chinese medicine to help clinicians understand the current status of traditional Chinese medicine therapy for the treatment of ferroptosis-related diseases.

Background Low-intensity pulsed ultrasound (LIPUS) is a safe and noninvasive rehabilitative physical therapy with anti-inflammatory effects. The current study investigated the effect of LIPUS on the inflammation of nucleus pulposus (NP) cells and its underlying mechanism. Methods Human NP cells were acquired from lumbar disc herniation tissue samples and cultured for experiments. Human NP cells were treated with LPS and then exposed to LIPUS (15 mW/cm2, 30 mW/cm2 and 60 mW/cm2) for 20 min daily for 3 days to determine the appropriate intensity to inhibit the expression of the inflammatory factors TNF-α and IL-1β. The gene and protein expression of aggrecan, collagen II, MMP-3 and MMP-9 was measured by real‐time PCR and western blotting, respectively. The activity of the nuclear factor‐kappa B (NF‐κB) pathway was examined by western blotting and immunofluorescence. After pretreatment with the NF-κB inhibitor PDTC, the expression of TNF-α, IL-1β, MMP-3 and MMP-9 was measured by real‐time PCR. Results LIPUS at intensities of 15 mW/cm2, 30 mW/cm2 and 60 mW/cm2 inhibited LPS-induced NP cell expression of the inflammatory factors TNF-α and IL-1β, especially at 30 mW/cm2. LIPUS significantly upregulated the gene and protein expression of aggrecan and collagen II and downregulated the gene and protein expression of MMP-3 and MMP-9 in LPS-induced NP cells. The NF‐κB signaling pathway was inhibited by LIPUS through inhibiting the protein expression of p-P65 and the translocation of P65 into the nucleus in LPS-induced NP cells. In addition, LIPUS had similar effects as the NF-κB inhibitor PDTC by inhibiting the NF-κB signaling pathway, inflammation and catabolism in LPS-induced human degenerative nucleus pulposus cells. Conclusion LIPUS inhibited inflammation and catabolism through the NF‐κB pathway in human degenerative nucleus pulposus cells.

Background: Spondylolysis is a bony defect of the pars interarticularis that most commonly occurs in the lower lumbar spine. However, the condition is rare in the mid-lumbar region. Here, we report on a new technique that involves the use of a U-rod and pedicle screw with iliac bone grafting for the direct intrasegmental repair of mid-lumbar spondylolysis in a teenager. Case Report: A 17-year-old male presented with complaints of mild low back pain after physical exercise, and the pain had aggravated in the last 3 months. Magnetic resonance imaging did not show compression of the intracanal neural tissue, and the lumbar discs did not demonstrate any obvious degeneration. He was diagnosed with L3 spondylolysis and lumbar sacralization. U-rod and screw construct with iliac bone grafting were used to directly repair the spondylolysis in L3. The low back pain totally disappeared 3 months after the surgery. A computerized tomography scan revealed the normal structure of L3 2 years post-surgically, and the internal fixation was removed 3 years later. Conclusions: This novel technique involving the use of a U-rod and pedicle screw associated with iliac bone grafting for the direct repair of mid-lumbar spondylolysis in the teenager yielded a satisfactory outcome, which was confirmed by favorable clinical and radiographic outcomes after 3 years. Key words: Mid-lumber spondylolysis, U-rod, direct repair

The intervertebral disc degeneration (IDD) is considered to be an initiator of a series of spinal diseases, among which changes in the nucleus pulposus (NP) are the most significant. NP cells reside in a microenvironment with a lack of blood vessels, hypoxia, and low glucose within the intervertebral disc. Due to the strong activity of HIF-1α, glycolysis is the main pathway for energy metabolism in NP cells. Our previous study found that higher SIRT1 expression is beneficial to delay the degeneration of NP cells. In order to find the downstream genes by which SIRT1 acts on NP cells, we used iTRAQ sequencing to detect the differences between degenerated NP cells overexpressing SIRT1 and a control group (human NP cells were derived from surgery) and found that the expression of LDHA changed in the same direction with SIRT1. This suggests that SIRT1 may delay the degeneration of NP cells by regulating glycolysis. We then used a Seahorse XFe24 analyzer to measure the bioenergetic parameters of NP cells and obtained three findings: (a) glycolysis is the main energy metabolism pathway in NP cells, (b) there is a large difference in ATP production between senescent cells and young cells, and (c) SIRT1 can regulate the production of ATP from glycolysis by regulating LDHA. We also found that SIRT1 in NP cells has a positive regulatory effect on c-Myc which is an upstream gene of LDHA. Through observing IDD-related indicators such as apoptosis, proliferation, senescence, and extracellular matrix, we found that SIRT1 can delay degeneration, and interference with c-Myc and LDHA, respectively, weakens the protective effect of SIRT1. Interfering with LDHA alone can also inhibit glycolysis and accelerate degeneration. Overall, we found that the inhibition of glycolysis in Np cells significantly affects their normal physiological functions and determined that LDHA is a potential therapeutic target for the treatment of IDD.

Symptomatic degenerative disc disease (DDD) is considered the leading cause of chronic lower back pain (LBP). As one of the main features of intervertebral disc degeneration (IDD), vascular ingrowth plays a crucial role in the progression of LBP. Stromal cell‑derived factor 1 (SDF1) and its receptor C‑X‑C receptor 4 (CXCR4) were reported to be overexpressed in the degenerated intervertebral discs, suggesting that they may be involved in the pathogenesis of IDD. Moreover, SDF1 has been identified to induce neovascularization in rheumatoid arthritis disease. However, the roles of the SDF1/CXCR4 axis in the neovascularization of IDD remain unclear. Therefore, the objective of the present study was to elucidate whether the SDF1/CXCR4 axis takes part in neovascularization in degenerated intervertebral discs and its underlying mechanisms. Adenovirus infection was used to upregulate SDF1 expression in primary nucleus pulposus cells (NPCs). The effects of SDF1 on the proliferation and angiogenesis of vascular endothelial cells (VECs) were assessed by Cell Counting Kit‑8 and tube formation assays after VECs were treated with the supernatants derived from SDF1 overexpressed or not treated NPCs. Transwell chambers using the supernatants from NPCs as chemokines were applied to assess VEC migration and invasion. AMD3100, MK‑2206 and SF1670 were used to antagonize CXCR4, AKT serine/threonine kinase 1 (AKT) and phosphatase and tensin homolog (PTEN) in VECs. The results revealed that SDF1 overexpression significantly increased the ratio of phosphorylated AKT to AKT and decreased PTEN expression in NPCs, as well as enhanced the proliferation, migration, invasion and angiogenesis abilities of VECs. However, these effects induced by SDF1 overexpression in NPCs were all reversed when VECs were pretreated with AMD3100 or MK‑2206, whereas enhanced by SF1670 treatment. Collectively, the present study indicated that enhancement of the SDF1/CXCR4 axis in NPCs can significantly accelerate angiogenesis by regulating the PTEN/phosphatidylinositol‑3‑kinase/AKT pathway.

Digital PCR (polymerase chain reaction) is a powerful and attractive tool for the quantification of nucleic acids. However, the multiplex detection capabilities of this system are limited or require expensive instrumentation and reagents, all of which can hinder multiplex detection goals. Here, we propose strategies toward solving these issues regarding digital PCR. We designed and tested a self-priming digital PCR chip containing 6-plex detection capabilities using monochrome fluorescence, which has six detection areas and four-layer structures. This strategy achieved multiplex digital detection by the use of self-priming to preintroduce the specific reaction mix to a certain detection area. This avoids competition when multiple primer pairs coexist, allowing for multiplexing in a shorter time while using less reagents and low-cost instruments. This also prevents the digital PCR chip from experiencing long sample introduction time and evaporation. For further validation, this multiplex digital PCR chip was used to detect five types of EGFR (epidermal growth factor receptor) gene mutations in 15 blood samples from lung cancer patients. We conclude that this technique can precisely quantify EGFR mutations in high-performance diagnostics. This multiplex digital detection chip is a simple and inexpensive test intended for liquid biopsies. It can be applied and used in prenatal diagnostics, the monitoring of residual disease, rapid pathogen detection, and many other procedures.

Articular cartilage defects are common in the clinic but difficult to treat. Exploring the chondrogenic molecular mechanisms of mesenchymal stem cells (MSCs) is of great theoretical interest and industrial significance. Bone morphogenetic protein 2 (BMP2) is a key factor that induces cartilage differentiation and can induce stem cell chondrogenic differentiation. However, the oxidative stress in the microenvironment during cartilage injury and degeneration inhibits cartilage regeneration and homeostasis. Silent mating type information regulator 2 homolog-1 (SIRT1) is an important histone deacetylase that regulates proliferation, differentiation, aging, and inflammation processes; moreover, it is an essential factor for chondrogenesis. The specific mechanism of SIRT1 in cartilage differentiation and homeostasis is still unclear. First, we investigated whether SIRT1 could coordinate BMP2-induced chondrogenic differentiation. Second, we investigated the protective effect of SIRT1 on BMP2-induced MSCs under oxidative stress. The results showed that SIRT1 could promote BMP2-induced chondrogenic differentiation of MSCs, and reduce the apoptosis and decomposition of extracellular matrix under oxidative stress. In summary, these results suggested that SIRT1 plays an important coordination role in BMP2-induced chondrogenic differentiation of stem cells and cartilage maintenance under oxidative stress, establishing the experimental basis for exploring the use of SIRT1 in cartilage defect repair.

Intervertebral disc degeneration (IDD) is characterized by the decrease of nucleus pulposus cells (NPCs). With the increase of the degree of degeneration, the reactive oxygen species (ROS) in nucleus pulposus tissue increases. Pyroptosis is a newly discovered form of cell death and its relationship with oxidative stress in NPCs remains unclear. This study was performed to investigate the mechanisms of pyroptosis of NPCs under oxidative stress. NPCs were isolated from IDD patients by surgical treatment. Pyroptosis related proteins like NLR family pyrin domain containing 3(NLRP3) and PYD and CARD domain containing (PYCARD) were detected by western blot, and membrane pore formation was observed by hochest33342/PI double staining or scanning electron microscope. The results showed that ROS induced the pyroptosis of NPCs and it depended on the expression of NLRP3 and PYCARD. The increased ROS level also increased transcription factor nuclear factor, erythroid 2 like 2 (NFE2L2, Nrf2) and the autophagy of NPCs, both of which attenuated the pyroptosis. In summary, ROS induces the pyroptosis of NPCs through the NLRP3/ PYCARD pathway, and establishes negative regulation by increasing autophagy and NFE2L2. These findings may provide a better understanding of the mechanism of IDD and potential therapeutic approaches for IDD treatment.

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.

Inflammatory stress of nucleus pulposus cells (NPCs) plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). Pyroptosis and NLRP3 inflammasome activation have been reported aggravating IVDD. SIRT1 is essential for mammalian cell survival and longevity by participating in various cellular processes. However, few studies analyzed the potential mechanism of SIRT1 in NLRP3- activated pyroptosis in NPCs. In this study, we confirmed that IL-1β could induce pyroptosis and NLRP3 inflammation activation, meanwhile, resulted in mitochondrial oxidative stress injury and dysfunction in NPCs. When the mitochondrial ROS was inhibited by Mito-Tempo, the pyroptosis and NLRP3 inflammation activation was also inhibited. SIRT1 overexpression could ameliorate IL-1β induced mitochondrial dysfunction and ROS accumulation, inhibit NLRP3 inflammasome activation by promoting PINK1/Parkin mediated mitophagy, however, these protective phenomena reversed by autophagy inhibitor 3-MA pretreatment. In vivo, SIRT1 agonist (SRT1720) treatment decreased the expression of NLRP3, p20, and IL-1β, increased the expression of PINK1 and LC3, delayed IVDD process in the rat model. Taken together, our results indicate that SIRT1 alleviates IL-1β induced NLRP3 inflammasome activation via mitophagy in NPCs, SIRT1 may be a potential therapeutic target to alleviate NLRP3- activated pyroptosis in the inflammatory stress related IVDD.

Summary In this population-based, cross-sectional study, we investigated vertebral fracture (VF) prevalence among Chinese postmenopausal women. We found 14.7% of population had VFs, which increased with age. Age ≥ 65 years, hip fracture, and densitometric osteoporosis were significantly associated with VFs. The prevalence of osteoporosis was remarkably high. Purpose To investigate VF prevalence among Chinese postmenopausal women in this population-based, randomized-sampling, cross-sectional study. Methods The investigator obtained lists of women from communities. Randomization was performed using SAS programming based on age group in each region. Postmenopausal women aged ≥ 50 years in the urban community were included. The investigator interviewed subjects to collect self-reported data and measured BMD. Spine radiographs were adjudicated by Genant’s semi-quantitative method. VFs were defined as fractures of at least one vertebra classified by Genant’s score 1–3 and were analyzed using descriptive statistics. Results A total of 31,205 women listed for randomized sampling from 10 Tier-3 hospitals at 5 regions. Of 2634 women in the full analysis set, 14.7% (388/2634, 95% CI: 13.4, 17.1) had prevalent VFs. VF prevalence increased with age (Cochran–Armitage test p Conclusion VFs are prevalent among Chinese postmenopausal women who were ≥ 50 years and community-dwelled. Osteoporosis prevalence is remarkable when fragile fractures were part of clinical diagnosis.

In this study, we investigated the role of heme oxygenase-1 (HO-1) in intervertebral disc degeneration (IDD) by assessing the effects of HO-1 overexpression on IL-1β-induced apoptosis in nucleus pulposus cells (NPCs). Immunohistochemical staining showed HO-1 expression to be lower in NPCs from IDD patients than from patients with lumbar vertebral fractures (LVF). Western blot analysis showed HO-1 and LC3-II/I levels to be lower in NP tissues from IDD patients than from LVF patients, suggesting suppression of autophagy in degenerative intervertebral disc. Consistent with that idea, autophagy was increased in HO-1-overexpressing NPCs while IL-1β-induced apoptosis was reduced. These effects were reversed by treatment with the early autophagy inhibitor 3-methyl adenine, which suggests HO-1-induced autophagy suppresses IL-1β-induced apoptosis in NPCs. HO-1 overexpression promoted autophagy by increasing levels of Beclin-1/PI3KC3 complex. Phospho-P65 levels were lower in HO-1-overexpressing NPCs, suggesting inhibition of NF-κB-mediated apoptosis. Our study thus demonstrates that HO-1 promotes autophagy by enhancing formation of Beclin-1/PI3KC3 complex and suppresses IL-1β-induced apoptosis by inhibiting NF-κB. We suggest that HO-1 is a potential therapeutic target to alleviate IDD.

Degenerative disc disease (DDD) is the main cause of low back pain, and the ingrowth of new blood vessels is one of its pathological features. The stromal cell-derived factor 1 (SDF1)/CXCR7 signaling axis plays a role in these physiological and pathological activities. The aims of this study were to explore whether this signaling axis participates in the angiogenesis of degenerated intervertebral discs (IVDs) and to define its underlying mechanism. In this study, we cocultured human nucleus pulposus cells (NPCs) and vascular endothelial cells (VECs) and regulated the expression of SDF1/CXCR7 to investigate the effect of VEC angiogenesis by NPCs. The results revealed that angiogenesis was enhanced with increased SDF1 and that angiogenesis was weakened with the inhibition of CXCR7. We found that PI3K/AKT was involved in the downstream pathway in the coculture. VEC angiogenesis induction by NPCs was enhanced with an increase in pAKT or a decrease in PTEN. We conclude that the SDF1/CXCR7 signaling axis plays a role in the angiogenesis of degenerated IVD through the PI3K/AKT pathway.

Intervertebral disk degeneration (IDD) is a common disease that is caused by degeneration of the nucleus pulposus (NP). One goal in the treatment of IDD is delaying or reversing the degeneration of NP via the transformation of exogenous genes. This study first investigated the role of BMP9 in the extracellular matrix (ECM) of nucleus pulposus cells (NPCs) and its mechanism. We found that BMP9 promotes the expression of ECM in NPCs, and the key molecules of Notch signaling, namely, NICD-1, hes and hey, and it was significantly altered in BMP9-transfected NPCs, which suggests that BMP9 may regulate the ECM via the Notch signaling pathway. We verified the expression of Notch ligands and receptors in NPCs infected with Ad-BMP9 and demonstrated a significant decrease in DLL1 and Notch1; then, NPCs were transfected with Ad-dnNotch1, Ad-Jagged1, and Ad-DLL1, and different multiple groups were established to further identify the ligands or receptors that affected ECM expression. The results demonstrated that Ad-dnNotch1, Jagged1 and DLL1 inhibited ECM expression, and dnNotch1 promoted expression. Therefore, we demonstrated that BMP9 promoted the expression of ECM in NPCs via inhibition of Notch1 and DLL1. This study provides a possible method for IDD treatment.

Growing evidence has shown that microRNAs (miRNAs) play crucial roles in the physiopathology of spinal cord injury (SCI). Recent studies have confirmed that miR-338-5p regulates myelination, suggesting a potential role in the treatment of SCI. However, the molecular mechanism of miR-338-5p on SCI is still unknown. Recently, exosomes have emerged as an ideal vector to deliver therapeutic molecules such as miRNAs. Here, we explored the effects of miR-338-5p-overexpressing exosomes derived from bone marrow-derived mesenchymal stromal cells (BMSCs) on SCI. In vivo, a model of contusion SCI in rats was established, and we observed that overexpression of miR-338-5p in exosomes profoundly increased the expression levels of neurofilament protein-M and growth-associated protein-43 and decreased those of myelin-associated glycoprotein and glial fibrillary acidic protein, which provided neuroprotective effects after acute SCI. In an in vitro study, we found that overexpression of miR-338-5p in exosomes repressed cell apoptosis following H2O2-induced oxidative stress injury in PC12 cells. Additionally, we confirmed that cannabinoid receptor 1 (Cnr1) was the target gene of miR-338-5p by dual-luciferase reporter assays and that Rap1 was the downstream gene by the KEGG pathway analysis. We found that miR-338-5p increased cAMP accumulation as a consequence of downregulated expression of the target gene Cnr1, and then, Rap1 was activated by cAMP. Eventually, the activation of the PI3K/Akt pathway attenuated cell apoptosis and promoted neuronal survival by cAMP-mediated Rap1 activation. In brief, these findings showed that exosomes overexpressing miR-338-5p were a promising treatment strategy for SCI.

Ferroptosis is a new form of regulated cell death, which is mediated by intracellular iron. Although it is reported that bavachin has antitumour effects on several tumour cells and prompts the reactive oxygen species (ROS) generation, it is unclear whether ferroptosis can be induced by bavachin in osteosarcoma (OS) cells. In this study, we found that bavachin inhibits the viability of MG63 and HOS OS cell lines along with an increase in the ferrous iron level, ROS accumulation, malondialdehyde overexpression, and glutathione depletion. Moreover, iron chelators (deferoxamine), antioxidants (Vit E), and ferroptosis inhibitors (ferrostatin-1 and liproxstatin-1) reverse bavachin-induced cell death. Bavachin also altered the mitochondrial morphology of OS cells, leading to smaller mitochondria, higher density of the mitochondrial membrane, and reduced mitochondrial cristae. Further investigation showed that bavachin upregulated the expression of transferrin receptor, divalent metal transporter-1, and P53, along with downregulating the expression of ferritin light chain, ferritin heavy chain, p-STAT3 (705), SLC7A11, and glutathione peroxidase-4 in OS cells. More importantly, STAT3 overexpression, SLC7A11 overexpression, and pretreatment with pifithrin-α (P53 inhibitor) rescued OS cell ferroptosis induced by bavachin. The results show that bavachin induces ferroptosis via the STAT3/P53/SLC7A11 axis in OS cells.

MicroRNA expression levels highly correlate with the occurrence, diagnosis and prognosis of disease. However, challenges remain in establishing a multiplex and fast detection method. Here, we developed a multiplex and fast detection platform for microRNAs based on a self-priming microfluidic chip and duplex-specific nuclease. It can detect three types of miRNAs, including miR-100, miR-155, and Let-7a, simultaneously at 50 °C within 1 h. The probes are pre-introduced into the chip using the self-priming method and cross-contamination can be avoided by using a screw valve. The reagent consumption and cost have been largely reduced in this work compared to the traditional detection method. This chip also exhibits good quantitative performance and specificity. As a proof of concept, we detect three types of microRNAs from different cancer cell lines, which demonstrates its potential in real sample analysis. In summary, this microfluidic chip shows great advantages in multiplex, fast and simple detection of microRNAs, and possesses great potential in the early diagnosis of miRNA-related diseases, especially for point-of-care application.

During the work of sports competitions and social groups, it is necessary to evaluate and optimize their teamwork capabilities, so that they can effectively improve related capabilities. First, this paper establishes a Bayesian algorithm model, analyzes and simulates the formation of teams in different dimensions and analyzes multi-dimensional analysis of time and space to determine the effectiveness of team cooperation. Different players have their own emphasis on two-person combinations, three-person combinations and other combinations, so they should be arranged according to the actual performance of the players. But on the whole, the three-person combination is more suitable for most players. Under different time scales, their performance is also relatively a big difference. For most players, a match time of 100–1000 is reasonable. Finally, the paper makes error analysis and evaluation of the model to make it more perfect.

Background/Objective: This study is a case–control study to explore risk and protective factors, including clinical data and bone mineral density (BMD), affecting vertebral body fragility fracture in elderly men and postmenopausal women. In addition, we investigate the effectiveness of lumbar spine BMD by quantitative computed tomography (QCT) in discriminating vertebral fragility fracture. Methods: In this case–control study, 52 males and 198 females with vertebral fragility fracture were compared with sex- and age-matched healthy controls to analyse the risk factors that may affect vertebral fragility fracture. The L1–L3 vertebral BMDs were measured by QCT. The difference in risk factors between fracture cases and controls were analysed using student t test and Mann–Whitney U test. The correlation between BMD, age, height and weight were analysed using univariate analysis. Multiple logistic regression analysis was used to study statistically significant indexes. The receiver operating characteristic curve was used to calculate the cut-off values for positive and negative predictive values of BMD for vertebral fracture discrimination. Results: In males, body weight and BMD were significantly different between the fracture group and the control group, whereas BMD was only weakly correlated with age (r = −0.234). In females, only BMD was significantly different between the fracture and control groups. BMD was weakly correlated with height (r = 0.133) and weight (r = 0.120) and was moderately correlated with age (r = −0.387). There was no correlation between BMD and the remaining variables in this study. In both men and women, the BMD (p = 0.000) was the independent protective factor against vertebral fracture. The cut-off values of vertebral BMD for fractures were 64.16 mg/cm3 for males and 55.58 mg/cm3 for females. QCT-measured BMD has a high positive predictive value and negative predictive value for discriminating vertebral fragility fracture across a range of BMD values. Conclusion: This study suggests that BMD is closely related to vertebral fragility fracture and that QCT is an effective technique to accurately discriminate vertebral fragility fracture. The translational potential of this article: The spine BMD measured by QCT is closely related to fracture, which may allow clinicians to more accurately discriminate which individuals are likely to experience vertebral fragility fracture. Keywords: Bone mineral density, Quantitative computed tomography, Vertebral fragility fracture

A digital polymerase chain reaction (dPCR) using fluorescence images for collecting quantitative information needs efficient software tools to automate the image analysis process. However, due to the broad range of fluorescence image characteristics, such as the uneven fluorescence intensity distribution, irregular structures of microarrays, and diverse and unpredictable morphologies of microchambers, existing tools fail to extract signals from these images properly, thus posing challenges for the improvement of detection accuracy. In this paper, a deep learning method based on the Mask R-CNN model was used for image processing to achieve more accurate quantification of nucleic acids in both microarray and droplet dPCR. This Mask R-CNN based method uses massive dPCR fluorescence image data to train a model that has the ability to recognize target signals in dPCR images precisely and automatically, regardless of the non-uniform luminosity or spot impurities appearing in dPCR images. When the Mask R-CNN model is used to process images with non-uniform luminosity, the true positive rate of this model can reach 97.56%. By contrast, the true positive rate of threshold segmentation is only 68.29%. As for dealing with images with spot impurities, which caused a 7.25% fault of the positive points in the threshold segmentation method, the error rate decreased to zero using the Mask R-CNN model. In addition, the labeling of annotated pictures is time-consuming. Therefore, an iteration method was used in the annotation stage to reduce time spent on labeling. With proper modifications, this method has great potential to serve as an alternative to achieve a highly efficient fluorescence image process for dPCR or other digital assays.

Recent evidence suggests that Propionibacterium acnes (P. acnes) is a novel pathogenic factor promoting intervertebral disc degeneration (IVDD), however, whose mechanism remains unclear. A key component of inflammatory responses to P. acnes appears to be interleukin (IL)-1β, which has been proved to be high expression in degenerative nucleus pulposus cells (NPCs). This study aimed to explore the inflammatory mechanism driving the host response to P. acnes infection in IVDD. Our data demonstrated that the number of nod-like receptor protein 3 (NLRP3)-positive cells was significantly increased in the P. acnes-infected nucleus pulposus (NP) tissue. Meanwhile, the up-regulated expressions of NLRP3, caspase-1, caspase-5, IL-1β, IL-18, Gasdermin D (GSDMD) were observed in NPCs after co-culturing with P. acnes, which suggested NPCs pyroptosis activation induced by P. acnes. To further investigate the underlying mechanisms, NLRP3 inflammasome inhibitor MCC950 and thioredoxin binding protein (TXNIP)-siRNA were used. With the addition of MCC950 to NPCs co-cultured with P. acnes in vitro, the secretions of mature IL-1β and IL-18 were reduced. Moreover, these MCC950-mediated effects were repeated by siRNA-transfected TXNIP knockdown. These results implied P. acnes activated inflammatory response by the TXNIP–NLRP3 pathway. To further reveal the anti-degeneration role of MCC950 in vivo, MCC950 was injected into the rabbit IVDD models infected by P. acnes. The MRI and histological detection provided more solid evidence that MCC950 treatment effectively retarded the degenerative process of the intervertebral discs in vivo. In summary, these results suggest that P. acnes-induced NPCs pyroptosis activation via the NLRP3-dependent pathway is likely responsible for the inflammatory pathology of IVDD. MCC950 can alleviate inflammatory injury and NPCs pyroptosis under P. acnes infection and may delay the progression of disc degeneration, which provides a new direction for the treatment of IVDD.

Backgrounds Adipose derived stem cells (ADSCs) could undergo osteogenesis via focal adhesion kinase (FAK) and bone morphogenetic protein (BMP) 9 signals, both of which could affect Wnt-β-catenin signal, a signal pathway closely related to ADSCs osteogenesis. It’s still enigma whether FAK and BMP-9 contribute to osteogenesis. Here, we examined the effect of FAK on BMP9-inducedosteogenic differentiation, unveiled the possible molecular mechanism underling this process. Methods In the present study, ADSCs were isolated and purified, and cells of passage 3 underwent virus mediated transfection to prepare ADSCs with stable FAK shRNA expression. Cell viability and migration were detected by MTT and transwell assay, respectively. Expression of osteogenic gene, phosphorylation of FAK and GSK were detected by western blot. Osteogenic potential was evaluated by activity of alkaline phosphatase (ALP) and calcium deposition by ALP staining and Alizarin Red S staining. Results BMP-9 administration promoted ADSCs osteogenesis. Knocking down FAK attenuated this process, inhibited osteogenic proteins expression through Wnt-β-catenin signal. BMP-9 also triggered ADSCs proliferation and migration, and shFAK antagonized such effects too. Although Wnt signal is affected by FAK shRNA, Smad signal remains intact in ADSCs with shFAK. Conclusion FAK and BMP-9 could cross talk on Wnt signal pathway and promote ADSCs osteogenesis. FAK could participate in BMP-9 induced ADSCs osteogenesis via Wnt signal pathway other than Smads signals (see in graph).

The goal of this study was to investigate the impact of repeated use of a metacognitive self-regulation inventory (MSRI) in translator self-training. Designed by the researchers, the MSRI includes the cognitive management strategies of planning, monitoring and evaluation. A pre-post comparison study was conducted with two groups of students. The data obtained from students’ responses to the inventory, think-aloud protocols (TAPs), post-task interviews, and translation products assessments were analysed for triangulation purposes. The results show that an MSRI can be used as an effective tool for translator self-training. Specifically, repeated practice with the inventory in students’ self-training processes can effectively increase their awareness of metacognitive self-regulation by diagnosing their strengths and weaknesses, assist them to transform declarative knowledge into procedural knowledge, promote top-down processing mode, and eventually influence the balance of their patterns of metacognitive self-regulation. Furthermore, the strengthening of metacognitive self-regulation encourages students to pay more attention to the communicative function of the translation, and ultimately enhances their translation quality, particularly in the aspects of clarity, vocabulary, morphosyntax, genre conventions, and translation’s purpose and target audience.

Background: MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation and chondrification in vivo. The purpose of the present study was to explore the potential mechanism of miR-128 in osteoporosis (OP). Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-128 in femoral neck trabecular bones of OP patients (n=40) and non-OP patients (n=40). C2C12 cells were transfected with miR-128 mimic or inhibitor to determine the effect of miR-128 on osteoblastic differentiation of C2C12 cells. Bioinformatics and luciferase reporter genes were used to determine the molecular mechanism of miR-128 in osteoblastic differentiation of C2C12 cells. Results: The qRT-PCR results showed that the expression level of miR-128 in bone samples of OP patients was significantly higher than that of non-OP patients, while miR-128 was significantly down-regulated during the osteogenic differentiation of C2C12 cells. In addition, the results showed that overexpression of miR-128 significantly inhibited the mRNA and protein expression levels of osteocalcin (OC), alkaline phosphatase (ALP) and collagen I type-α1 (COL1A1) in C2C12 cells, while miR-128 inhibitor could reverse this effect. Bioinformatics analysis and dual-luciferase reporter assay found that silencing information regulatory protein 6 (SIRT6) was a direct target of miR-128. The qRT-PCR and Western Blot results found that miR-128 significantly down-regulated the mRNA and protein expressions of SIRT6. Furthermore, silencing SIRT6 significantly inhibited the promoting effect of the miR-128 inhibitor on the expression of osteoblast markers. Conclusion: The above results confirmed that miR-128 inhibited osteoblast differentiation in OP by down-regulating SIRT6 expression, thus accelerating the development of OP.

Chip-based digital assays such as the digital polymerase chain reaction (digital PCR), digital loop-mediated amplification (digital LAMP), digital enzyme-linked immunosorbent assay (digital ELISA) and digital proximity ligation assay (digital PLA) need high-throughput quantification of the captured fluorescence image data. However, traditional methods that are mainly based on image segmentation using either a fixed threshold or an automated hard threshold failed to extract valid signals over a broad range of image characteristics. In this study, we introduce a new method for automated image analysis to extract signals applied to chip-based digital assays. This approach precisely locates each micro-compartment based on the structure design of the chip, thereby eliminating the interference of non-signal noise in the image. Utilizing the principle that the human eyes can distinguish between the positive micro-compartments and the negative micro-compartments, we take the parameters of each micro-compartment together with its surrounding micro-compartments as the training dataset of the Random Forest classifier to classify the micro-compartments and extract valid signals, thus solving the problem caused by the differences among images. Furthermore, we adopted the iteration methodology that adds the output of a model's prediction to the input of the next model's training dataset, until the output of a model's prediction reaches the accuracy we expected, which improves the work efficiency during data training greatly. We demonstrate the method on the dPCR dataset and it performs well without any manual adjustment of settings. The results show that our proposed method can recognize the positive signals from the fluorescence images with an accuracy of 97.78%. With minor modification, bio-instrument companies or researchers can integrate this method into their digital assay devices' software conveniently.

An optical microring resonator model that allows for the measurement of low concentrations of hexavalent chromium was numerically investigated with the finite element method. The optical microring resonator based on whispering gallery modes was optimized in terms of microring radius, microring width, and gap distance. Thus, the optimization of these geometric parameters has been illustrated. A free spectral range of 3.2 nm, full width at half maximum of 0.10 nm, quality factor of 5,485, and finesse of 31.66 make the high-performance device suitable for the measurement of hexavalent chromium. The reduction in optical intensity at the resonance wavelength of 542 nm strongly depends on the imaginary part of the refractive index. The excellent performance of the sensor clearly showed a linear trend of optical intensity with increasing concentration of hexavalent chromium.

Schisandrin B is an active monomer of the Chinese magnolia vine (Schisandra chinensis) that can reduce transaminase activity in liver cells, inhibit lipid peroxidation, enhance antioxidant status, has protective effects in the liver and has antitumor effects. The present study investigated the potential protective effects of schisandrin B on the p53 signaling pathway in attenuating the inflammatory response, oxidative stress and apoptosis induced by traumatic spinal cord injury (TSCI) in adult rats. Behavioral examination, inclined plate test and spinal cord water content were used to evaluate the protective effect of schisandrin B in TSCI rats. The expression levels of superoxide dismutase (SOD), malondialdehyde (MDA), nuclear factor (NF)‑κB subunit p65 and tumor necrosis factor (TNF)‑α were examined using ELISA kits. Western blot analysis was performed to analyze the protein expression of caspase‑3 and phosphorylated (p)‑p53 in TSCI rats. In the present study, schisandrin B improved behavioral examination results and the maximum angle of inclined plate test, and inhibited spinal cord water content in rats with TSCI. Notably, schisandrin B reduced the activation of traumatic injury‑associated pathways, including SOD, MDA, NF‑κB p65 and TNF‑α, in TSCI rats. In addition, schisandrin B suppressed the TSCI‑induced expression of caspase‑3 and p‑p53 in TSCI rats. These results indicated that schisandrin B may attenuate the inflammatory response, oxidative stress and apoptosis in TSCI rats by inhibiting the p53 signaling pathway in adult rats.

Background: Low back pain is a common worldwide health problem and has a significant socioeconomic impact on public health. Internal disc disruption has been considered as the most common cause of low back pain. Various therapies, including interbody fusion, disc replacement, injection therapies, and thermal annular procedures have been utilized for the treatment of discogenic low back pain. Recently, a new method of intradiscal methylene blue injection has been introduced to treat discogenic low back pain, but the clinical outcomes are controversial. Objectives: To investigate the clinical outcomes and magnetic resonance imaging changes of intradiscal methylene blue injection for the treatment of discogenic low back pain. Study Design: Observational study. Setting: An interventional low back pain management practice in a university hospital. Methods: A total of 33 patients were selected to be treated with intradiscal methylene blue injection. The clinical outcomes were evaluated by numeric rating scale and Oswestry Disability Index at pretreatment, one month, 3, 6, and 12 months after treatment. The magnetic resonance imaging changes of involved intervertebral discs were assessed by apparent diffusion coefficient and T2 values at pretreatment, 3, 6, and 12 months after treatment. Results: All of the patients got a follow-up period up to 12 months. The mean numeric rating scale scores at pretreatment, one month, 3, 6, and 12 months after treatment were 6.54, 2.98, 3.23, 3.66, and 4.72, respectively. There was a minimum of 2 points reduction at one month, 3, and 6 months after treatment, but less than 2 points reduction at 12 months. There was at least 50% improvement on the Oswestry Disability Index at one month, 3, and 6 months after treatment, but not at 12 months. The mean apparent diffusion coefficient and T2 value were significantly higher at 6 and12 months after treatment compared to pretreatment, but there was no significant difference between pretreatment and 3 months after treatment. Limitations: This is an observational study with a relatively small sample size and short-term follow-up. Conclusions: The intradiscal methylene blue injection might be an effective therapy for discogenic low back pain for the short-term and could improve disc degeneration condition to some extent. Key words: Low back pain, discogenic pain, internal disc disruption, provocation discography, methylene blue, intradiscal injection, disc degeneration, magnetic resonance, imaging

Polymethyl methacrylate (PMMA) bone cement has been commonly used for percutaneous injection into collapsed vertebral bodies due to malignant tumor. The purpose of this study was to investigate the possible mechanisms of PMMA’s cytotoxcity on primary cultured spinal metastastic cells (SMCs) in vitro. PMMA specimens were prepared in standard discs made of dough and polymerization stages, and the eluates were prepared following the ISO standard. Primary SMCs were obtained and isolated from 7 patients with spinal metastatic tumors undergoing vertebroplasty. Primary cultured SMCs were treated with PMMA specimens of different stages for 24 h, or co-cultured with extracted medium for successive 3 days. The temperatures in two locations from cement discs were recorded by K-type thermocouples. Furthermore, cell proliferation, apoptosis and cycles were determined by MTT and flow cytometry, respectively. The modulation of apoptotic proteins (bcl-2, bax, caspase-3, caspase-8, Fas and PARP) and cell cycle proteins (cyclin D1, P21 and P27) were analyzed by western blot and real time PCR. The results of this study demonstrated that PMMA treatment was able to suppress SMCs proliferation, induce cell apoptosis and inhibit cell cycle arrest when compared to the control group in vitro (P

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

RSK2 (90 kDa ribosomal S6 kinase) is a downstream effector of the Ras/ERK (extracellular signal-regulated kinase) signaling pathway that has major functions in cell biological activities, including regulating nuclear signaling, cell cycle progression, cell proliferation, cell growth, protein synthesis, cell migration and cell survival, and is expressed in most types of human malignant tumors, including lung cancer, prostate and breast tumors, skin cancer and osteosarcomas (OS). RSK2 was found to be essential for osteosarcoma formation. To investigate whether RSK2 is expressed at high levels in human osteosarcome tissues and whether its expression is correlated with the aggressive biological behavior of osteosarcoma cell line (OCLs), we assessed the association between RSK2 expression and OS cell progression, as well as the effects of RSK2 inhibition on the biological activities of osteosarcoma cells. We performed immunohistochemistry to analyze the expression of RSK2 in specimens from 30 humans with osteosarcoma, and 15 normal tissues. RSK2 gene expression levels in 30 specimens with osteosarcoma were significantly higher than those of normal tissues. We performed RNA interference on three OCLs to evaluate cell apoptosis, cell growth, cell proliferation, cell motility, chemosensitivity and oncogenicity. After transfection with RSK2 shRNA, increased cell apoptosis, cell growth inhibition, cell cycle progression, weaker cell proliferation, cell migration and weaker tumor formation were observed in all OCLs. These results suggested that RSK2 expression may mediate the biological activities of OS cells and RSK2 may be an effective therapeutic target for the treatment of osteosarcomas. The AKT/mTOR, MAPK/ERK/c-Fos and Bcl2/Bax pathways were analysed to clarify the mechanisms involved.

STUDY DESIGN In vitro experimental study. OBJECTIVE To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the extracellular matrix (ECM) synthesis of degenerative human nucleus pulposus cells and explore the molecular mechanism. SUMMARY OF BACKGROUND DATA LIPUS has been used successfully for bone fracture healing and been proved to be effective in stimulating ECM metabolism in animal intervertebral disc cells. However, whether LIPUS also exerts an anabolic effect on degenerative human nucleus pulposus cells and the possible molecular mechanism is still unclear. METHODS The degenerative human nucleus pulposus cells were cultured in calcium alginate beads. In the LIPUS group, cells were exposed to an average temporal intensity of 30 mW/cm2 and a frequency of 1.5 MHz of LIPUS 20 minutes daily for 1 week. The control group was cultured in the same way but without LIPUS stimulation. The LY294002 group was stimulated by LIPUS and treated with LY294002 simultaneously. The expression of aggrecan, collagen-II, Sox9, tissue inhibitor of metalloproteinase-,1 and matrix metalloproteinase-3 were evaluated by Enzyme-Linked Immunosorbent Assay, Western blot or RT-PCR. Expression of signaling proteins involved in FAK/PI3K/Akt pathway was studied by Western blot analysis. RESULTS LIPUS significantly upregulated expression of aggrecan, collagen-II, Sox9, and tissue inhibitor of metalloproteinase-1 compared with control group, but inhibited secretion of matrix metalloproteinase-3. The study further demonstrated that the upregulation of aggrecan, collagen-II, and Sox9 was related to the activation of focal adhesion kinase (FAK)//PI3K/Akt pathway caused by LIPUS. Moreover, inhibition of PI3K/Akt significantly suppressed the special biological effect activated by LIPUS. CONCLUSION LIPUS promotes the ECM synthesis of degenerative human nucleus pulposus cells through activation of FAK/PI3K/Akt pathway. LEVEL OF EVIDENCE N/A.

Intervertebral disc degeneration (IDD) is a main cause of diseases such as discogenic low back pain, cervical and lumbar disc herniation, degenerative spinal stenosis, and lumbar spondylolisthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcription factor, regulates antioxidant genes and induces cellular defense mechanisms against oxidative stress. In this study, the protective effect of plant antioxidant lycopene on nucleus pulposus cells (NPCs) under oxidative stress was investigated. The results indicated that Nrf2 expression decreased in degenerated NPCs. We further found that lycopene was protective in NP tissue under oxidative stress and alleviated oxidative stress-induced apoptosis of degenerative human NPCs via Nrf2. The results also showed that lycopene reduced H2O2-induced decomposition of cartilage extracellular matrix in NPCs. In conclusion, our findings suggested that lycopene may alleviate disc degeneration under oxidative stress through the Nrf2 signaling pathway.

Misclassification of positive partitions in microfluidic digital polymerase chain reaction (dPCR) can cause the false positives and false negatives, which significantly alter the resulting estimate of target DNA molecules. To address this issue, establishing real-time fluorescence interrogation of each partition in microfluidic arrays is an effective way in which false positive and false negative partitions can be eliminated. However, currently available devices for real-time fluorescence interrogation are either not competent for microfluidic digital array, or they are bulky, expensive and entail peripheral equipment due to low integration. Therefore, in this study, a Raspberry Pi based, low-cost and highly integrated device is presented to achieve real-time fluorescence detection for microfluidic digital array, termed real-time dPCR device. In the device, uniform thermocycler, streamlined real-time fluorescence imaging setup, and compact data processing system are all integrated to undergo on-chip dPCR amplification, real-time fluorescence detection, and data analysis. Using this real-time dPCR device, the accuracy of DNA absolute quantification by dPCR is improved, since the misclassification of positive partitions is efficiently reduced based on the characteristic real-time fluorescence curves of positive partitions in a self-priming microfluidic chip. Compared with end-point dPCR on our device and commercialized QuantStudio™ 3D dPCR system, the real-time dPCR on our device exhibits a higher accuracy for DNA quantification. In addition, this real-time dPCR device is much smaller and cheaper than the commercialized Digital PCR system, but not sacrificing the capability of error correction for absolute quantitation analysis. Conclusively, this highly integrated real-time dPCR device is very beneficial for DNA quantitative analysis where the determination accuracy is pivotal.

To evaluate the results of percutaneous endoscopic transforaminal diskectomy (PETD) in comparison with percutaneous endoscopic interlaminar diskectomy (PEID) for axillar herniation at L5-S1.From January 2017 to March 2018, 80 patients admitted with axillar herniation at L5-S1 were randomly recruited into 2 groups: 40 cases in the PETD group and 40 in the PEID group. Each group separately underwent PETD or PEID. Patient sex, age, body mass index, axillar herniation size, number of C-arm fluoroscopies, operation time, postoperative bed time, complications, and clinical effect were compared. Both groups were followed-up using the Oswestry Disability Index (ODI), visual analog scale (VAS), and Macnab criteria.Except for 1 case in the PETD group that switched to the PEID group, the patients completed the study as expected. All patients were followed-up. Preoperative demographics were not significantly different (P0.05) between the 2 groups. The mean number of C-arm fluoroscopies (12.44 ± 3.21) and the operation time (66.49 ± 16.29 minutes) of the PETD group were significantly improved compared with the PEID group (number of fluoroscopies: 3.41 ± 0.81, P0.001; operation time: 53.56 ± 10.82 minutes, P0.001), but the postoperative bed rest time and complication rate were not (P0.05). The postoperative ODI and VAS scores were obviously improved in both groups when compared with preoperation (P0.001). There were no significant differences between the 2 groups in the Macnab criteria or VAS and ODI scores at the same time point (P0.05).For axillar herniation at L5-S1, PEID can ignore the anatomic obstruction with advantages including a shorter operation time and less intraoperative radiation exposure. PETD has a clinical effect similar to that of PEID, but the process of it is more dangerous and harder than PEID.

Objective Extensive incision associated with large-scale callus exfoliation and internal fixation is the common therapeutic approach employed by the majority of orthopaedists in the treatment of old femoral fractures. Inspired by the surgical techniques of intramedullary fixation and reduction by traction, the present study attempted to treat old femoral fractures with minimally invasive methods utilising the principles of biological osteosynthesis (BO). Methods A retrospective analysis involving 16 patients with old femoral fractures treated with combined traction, small incision, limited callus treatment, reduction by leverage and intramedullary fixation was conducted. The operative effect was evaluated by the operation time, intraoperative blood loss, bone grafting, healing time of fractures during follow-up, VAS score, and Harris hip score. Results Intraoperative observation revealed an average operation time of 1.53 ± 0.34 h and average blood loss of 268.13 ± 97.29 ml without bone grafting in all patients. All enrolled patients had outcomes resulting in effective fixation restoration of limb alignment. Of the 16 enrolled patients, 13 patients completed follow-up with an average follow-up time of 7.42 ± 3.29 months. The average healing time for proximal femoral fractures was 3 months. The average healing time of femoral shaft fractures was 4 ± 1.09 months; two of these cases took 4 months to heal, whereas 1 case demonstrated a delayed healing time of 6 months. The VAS score was 1.15 ± 1.70, 1 patient experienced sciatica, and the Harris hip score was 92.92 ± 5.42. There were no complications of malunion, nonunion or infection among any of the patients who completed follow-up. Conclusions Minimally invasive treatment is feasible for most patients with old femoral fractures of the trochanter and femoral shaft. This finding is consistent with BO principles, thereby providing a possible new method for the treatment of old femoral fractures.

Extracellular matrix (ECM) anabolism and catabolism imbalance is key feature of chondrocyte and intervertebral disc nucleus pulposus (NP) cell degeneration. The role of LIF as a multifunctional cytokine in the ECM metabolism of chondrocytes is controversial, but no relevant research in the ECM metabolism of NP cells. This study aimed to explore the biofunction and related mechanisms of LIF in the degenerative NP cells. We obtained an increase in the expression of LIF in the human degenerated NP specimens. The addition of recombinant human leukemia inhibitory factor (rhLIF) to the degenerated NP cells cultured in vitro was found to stimulate the synthesis of ECM, and rhLIF could activate the ERK1/2 signaling pathway. However, coculture with PD98059, a signal inhibitor of ERK1/2, blocked the effect of rhLIF on the synthesis of ECM. To furtherly clarify the role of LIF, we carried out animal experiments and found that rhLIF treatment could successfully delay the degree of degeneration of the intervertebral disc in a rabbit model; but with the addition of PD98059, the function of rhLIF for degeneration protection disappeared. In summary, this study demonstrates that LIF plays a role in promoting ECM synthesis in the degenerated NP cells as a protective role in intervertebral disc degeneration (IDD), which is related to the activation of ERK1/2 signaling pathway.

Intervertebral disc degeneration (IDD) is closely related with aging, whereas mitochondrial dysfunction is a common feature of aging in which results cell senescence. Phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1 (PINK1) is a mitochondrial-targeted serine/threonine kinase, which plays a protective role against mitochondrial dysfunction with mitochondrial quality control by activating PINK1/Parkin mediated mitophagy. This study aimed to investigate the protective role of PINK1 against mitochondrial dysfunction and human nucleus pulposus cell (NPC) senescence. We found that mitochondrial dysfunction and NPC senescence could be induced under sublethal oxidative stress by 150 μM H2O2. Moreover, down-regulation of PINK1 tended to aggravate NPC senescence under oxidative stress. Therefore, mitophagy was evaluated in NPCs to further reveal the underlying mechanism. Results showed that sublethal oxidative stress induced mitochondria dysfunction and mitophagy in NPCs. Furthermore, depletion of PINK1 utilizing short hairpin RNA targeting PINK1 (PINK1-shRNA) impaired mitophagy, and exasperated NPC senescence under oxidative stress. In summary, these results suggested that PINK1 played as a protective role in clearance of damaged mitochondrial and alleviating cell senescence under oxidative stress, whose mechanism is associated with regulating mitophagy. These findings may provide a better understanding in pathomechanism of IDD and potential therapeutic approaches for IDD treatment.

OBJECTIVE: To investigate the regulatory effect of resveratrol (RES) on the extracellular matrix (ECM) expression of nucleus pulposus cells (NPC), and its relative molecular mechanism. METHODS: Ten patients receiving discectomy were collected, of which 5 patients were young with spinal burst fracture, classified as control group; the rest 5 patients were senile with lumbar disc herniation, classified as degenerative group. The nucleus pulposus tissue of 2 groups were collected, the in situexpression of β-catenin was detected by immunohistochemistry, and the protein expressions of collagen type Ⅱ and Aggrecan were detected by Western blot. The NPC were isolated and cultured from degenerative nucleus pulposus tissues. RES treated the third-passage NPC with (group B) or without IL-1β (group C), to further determine the protein expressions of collagen type Ⅱ and Aggrecan by Western blot, the unstimulated cells were set up as blank control group (group A). Moreover, NPC treated with small interfering RNA (siRNA) targeted silent SIRT1 or β-catenin were used to determine the protein and gene expressions of β-catenin and SIRT1 by Western blot and real-time fluorescence quantitative PCR. In addition, the third-passage NPC treated with complete medium (group 1), IL-1β (group 2), RES+IL-1β (group 3), and SIRT1-siRNA+RES+IL-1β (group 4) for 24 hours were used to detect the nuclear translocation of β-catenin by cell immunofluorescence staining. Finally, the third-passage NPC treated with complete medium (group Ⅰ), IL-1β (group Ⅱ), IL-1β+β-catenin-siRNA (group Ⅲ), IL-1β+RES (group Ⅳ), and IL-1β+RES+SIRT1-siRNA (group Ⅴ) for 24 hours were used to detect the protein expressions of collagen type Ⅱ and Aggrecan by Western blot. RESULTS: Immunohistochemical staining and Western blot detection showed that when compared with control group, the cell proportion of expression of β-catenin were significantly increased in degenerative group (t=4.616, P=0.010); the protein expression of β-catenin was also significantly increased and the protein expressions of collagen type Ⅱ and Aggrecan were significantly decreased (P

To investigate the role and relative mechanism of stromal cell derived factorl (SDF-1) secreted by nucleus pulposus cells (NPCs) on the proliferation of vascular endothelial cells (VECs).The NPCs were isolated from the degenerated disc specimens after discectomy. NPCs at passage 1 were transfected with lentivirus-mediated SDF-1 over-expression; transfected and untransfected NPCs at passage 2 were cultured in the three-dimensional alvetex® scaffold, then they were co-cultured with HMEC-1 cells. The morphology of NPCs was observed by scanning electron microscope (SEM), and the apoptosis of HMEC-1 cells was detected by Annexin V/propidiumiodide staining after 72 hours co-culutre. The proliferation of HMEC-1 cells was detected by cell counting kit 8 at 12, 24, 48, and 72 hours in transfected group and untransfected group, respectively. ELISA was used to measure the vascular endothelial growth factor (VEGF) expression level. The virus transfection efficiency and relative Akt pathway were determined by Western blot.The NPCs maintained cell phenotype and secreted much extracellular matrix in three-dimensional-culture by SEM observation. In the co-culutre system, after NPCs were transfected with SDF-1 over-expression lentivirus, the proliferation of HMEC-1 cells was significantly increased, while the apoptosis was decreased obviously. The ELISA results demonstrated that the amount of VEGF was remarkably increased in the culture medium. Furthermore, SDF-1 promoted the up-regulation of phosphorylate Akt expression; after inhibition of Akt expression by GSK690693, the proliferation rate of VECs decreased significantly.Over-expression of SDF-1 by NPCs is beneficial for VECs proliferation, which is involved in SDF-1-Akt signalling pathway.探讨人退变髓核细胞（nucelus pulposus cells，NPCs）分泌的基质细胞衍生因子 1（stromal cell derived factorl，SDF-1）对血管内皮细胞（vascular endothelial cells，VECs）的增殖影响及其相关分子机制。.将术中摘取的椎间盘组织分离、培养人退变 NPCs，取第 1 代转染 SDF-1 过表达慢病毒。取第 2 代细胞，转染或未转染过表达 SDF-1 慢病毒后，种植于 alvetex® 三维培养支架上行三维培养，并与人 HMEC-1 细胞（即 VECs）共培养。共培养 72 h 扫描电镜观察 NPCs 在支架上的生长情况；细胞计数试剂盒 8（cell counting kit 8，CCK-8）法分别检测 12、24、48、72 h 转染组与非转染组 HMEC-1 增殖情况，以及加入 Akt 特异性抑制剂 GSK690693 后 24 h HMEC-1 增殖情况；膜联蛋白Ⅴ/碘化丙啶法分别检测共培养 72 h 后转染组和非转染组中 VECs 凋亡率；ELISA 法分别检测转染组和非转染组培养基中 VEGF 含量；Western blot 法检测转染组、未转染组和抑制剂组磷酸化 Akt（p-Akt）和总 Akt 蛋白表达。.扫描电镜观察示 NPCs 在三维支架中维持了细胞表型，分泌大量细胞外基质；慢病毒成功转染后，Western blot 检测示 SDF-1 在 NPCs 中表达明显升高。在共培养体系中，NPCs 过表达 SDF-1 后可使 HMEC-1 增殖上升，凋亡率下降，并在细胞外培养基检测中发现 VEGF 含量显著上升；而在进一步分子机制检测中发现，SDF-1 的过表达使 HMEC-1 中 p-Akt 水平升高，采用 GSK690693 特异性抑制 Akt 表达后，SDF-1 对 HMEC-1 促细胞增殖作用明显降低。.人退变 NPCs 高表达的 SDF-1 有利于 VECs 增殖，这一调控机制或许与 SDF-1-Akt 信号通路有关。.

Aim: To improve nucleus pulposus cell-targeted therapy for intervertebral disc degeneration (IDD) by fabricating a novel kind of ultrasound (US)-mediated poly(lactic-co-glycolic acid) nanobubbles (NBs) as a means of targeted drug delivery. Materials & methods: The resveratrol (RES)-embedded NBs were synthesized using a double-emulsion method. The active NP cell-targeting biomarker CDH2 antibody (AbCDH2) was further conjugated to the NBs using a carbodiimide method. Then, this RES/AbCDH2 NBs were examined by physical properties, specifc cell-targeting ability, anticatabolism effect in vitro and in vivo. Results: RES/AbCDH2 NBs exhibited high RES-loading efficiency, and US triggered accelerated RES release. Furthermore, RES/AbCDH2 NB treatment exhibited excellent anticatabolic ability in vitro; and in an IDD rabbit model, US-mediated RES/AbCDH2 NB injection effectively retarded the degenerative process of the intervertebral disc in vivo. Conclusion: The combination of US irradiation and drug delivery through RES/AbCDH2 NBs can be considered as a novel treatment option for IDD.

The present study was performed to compare the clinical efficacy of lateral plate and lateral and medial double-plating fixation of distal femoral fractures and explore the indication of lateral and medial double-plating fixation of the distal femoral fractures. From March 2006 to April 2014, 48 and 12 cases of distal femoral fractures were treated with lateral plate (single plate) and lateral and medial plates (double plates), respectively. During the surgery, after setting the lateral plate for the distal femoral fractures, if the varus stress test of the knee was positive and the lateral collateral ligament rupture was excluded, lateral and medial double-plating fixation was used for the stability of the fragments. All the patients were followed up at an average period of 15.9 months. The average operation time, the intraoperative hemorrhage and the fracture union time of the two groups were compared. One year after operation, knee function was evaluated by the Kolmert’s standard. There was no significant difference in the average operation time, intraoperative hemorrhage, fracture healing time and excellent and good rates of postoperative knee function between two groups. Positive Varus stress test during operation can be an indication for lateral and medial double-plating fixation of distal femoral fractures.

Background: Low back pain (LBP), one of the most prominent problems worldwide, lacks effective disease-modifying medical therapy. Intervertebral disc degeneration (IVDD) is a major cause of LBP, and proinflammatory cytokines are the key factors involved in the development of IVDD-induced back pain. Sirtuin 1 (SIRT1) is implicated in the molecular control of aging and immune responses in various diseases; however, its effect on IVDD is not clearly understood. Objective: To investigate the effects of SIRT1 on proinflammatory stress and signal transduction pathways induced by interleukin-1β (IL-1β) in human degenerative nucleus pulposus (NP) cells. Study Design: Research study. Setting: Chongqing Key Laboratory of Ophthalmology. Methods: Anti-apoptotic and anti-catabolic effects of SIRT1 on IL-1β were investigated using a three-dimensional cell culture model of prestimulated human NP cells transfected with a lentiviral vector overexpressing SIRT1 or a small-interfering RNA (siRNA) against the gene encoding SIRT1. In addition, molecular mechanisms underlying the association of SIRT1 with Toll-like receptor-2 (TLR2) and nuclear factor kappa B (NF-κB) were investigated. Results: Our results indicated that decreased SIRT1 expression was associated with IVDD. Direct regulation of SIRT1 expression did not affect the synthesis of extracellular matrix (ECM). However, SIRT1 overexpression mediated by the lentiviral vector suppressed IL-1β-induced ECM degradation and cell apoptosis. In contrast, knockdown of the gene encoding SIRT1 by the siRNA increased MMP expression and cell apoptosis induced by IL-1β. Furthermore, SIRT1 deacetylated RelA/p65 to inhibit the nuclear translocation of NF-κB, thus inhibiting inflammation. On the other hand, IL-1β downregulated SIRT1 expression by activating TLR2. However, inhibition of TLR2 expression by an siRNA did not inhibit IL-1β-induced nuclear translocation of NF-κB. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of SIRT1 on IVDD. Conclusions: Our results indicated that SIRT1 exerted anti-inflammatory effects against IL-1βmediated degeneration of NP cells through the TLR2/SIRT1/NF-κB pathway, suggesting that it could be used as a potential candidate for treating IVDD-mediated back pain. Key words: SIRT1, TLR2, NF-κB, intervertebral disc degeneration

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering. [BMB Reports 2016; 49(3): 179-184].

Various gene delivery systems have been widely studied for the acute spinal cord injury (SCI) treatment. In the present study, a novel type of brain-derived neurotrophic factor (BDNF)-loaded cationic nanobubbles (CNBs) conjugated with MAP-2 antibody (mAbMAP-2/BDNF/CNBs) was prepared to provide low-intensity focused ultrasound (LIFU)-targeted gene therapy. In vitro experiments, the ultrasound-targeted tranfection to BDNF overexpressioin in neurons and efficiently inhibition neuronal apoptosis have been demonstrated, and the elaborately designed mAbMAP-2/BDNF/CNBs can specifically target to the neurons. Furthermore, in a acute SCI rat model, LIFU-mediated mAbMAP-2/BDNF/CNBs transfection significantly increased BDNF expression, attenuated histological injury, decreased neurons loss, inhibited neuronal apoptosis in injured spinal cords, and increased BBB scores in SCI rats. LIFU-mediated mAbMAP-2/BDNF/CNBs destruction significantly increase transfection efficiency of BDNF gene both in vitro and in vivo, and has a significant neuroprotective effect on the injured spinal cord. Therefore, the combination of LIFU irradiation and gene therapy through mAbMAP-2/BDNF/CNBs can be considered as a novel non-invasive and targeted treatment for gene therapy of SCI.