Multi-omics Data Integration Analysis Identified Therapeutic Targets and Potential Reuse Drugs for Osteoporosis.

AIMS: To facilitate drug discovery and development for the treatment of osteoporosis. BACKGROUND: With global aging, osteoporosis has become a common problem threatening the health of the elderly. It is of important clinical value to explore new targets for drug intervention and develop promising drugs for the treatment of osteoporosis. OBJECTIVE: To understand the major molecules that mediate the communication between the cell populations of bone marrow-derived mesenchymal stem cells (BM-MSCs) in osteoporosis and osteoarthritis patients and identify potential reusable drugs for the treatment of osteoporosis. METHODS: Single-cell RNA sequencing (scRNA-seq) data of BM-MSCs in GSE147287 dataset were classified using the Seurat package. CellChat was devoted to analyzing the ligand-receptor pairs (LR pairs) contributing to the communication between BM-MSCs subsets. The LR pairs that were differentially expressed between osteoporosis samples and control samples and significantly correlated with immune score were screened in the GSE35959 dataset, and the differentially expressed gene in both GSE35959 and GSE13850 data sets were identified as targets from a single ligand or receptor. The therapeutic drugs for osteoporosis were screened by network proximity method, and the top-ranked drugs were selected for molecular docking and molecular dynamics simulation with the target targets. RESULTS: Twelve subsets of BM-MSCs were identified, of which CD45-BM-MSCS_4, CD45-BM- MSCS_5, and CD45+ BM-MSCs_5 subsets showed significantly different distributions between osteoporosis samples and osteoarthritis samples. Six LR pairs were identified in the bidirectional communication between these three BM-MSCs subsets and other BM-MSCs subsets. Among them, MIF-CD74 and ITGB2-ICAM2 were significantly correlated with the immune score. CD74 was identified as the target, and a total of 48 drugs targeting CD47 protein were identified. Among them, DB01940 had the lowest free energy binding score with CD74 protein and the binding state was very stable. CONCLUSION: This study provided a new network-based framework for drug reuse and identified initial insights into therapeutic agents targeting CD74 in osteoporosis, which may be meaningful for promoting the development of osteoporosis treatment.

Rehabilitation training enhanced the therapeutic effect of calycosin on neurological function recovery of rats following spinal cord injury.

BACKGROUND: Calycosin (CA), a flavonoids component, has demonstrated potential neuroprotection effects by inhibiting oxidative stress in spinal cord injury (SCI) models. This study aims to investigate the impact of combined rehabilitation training (RT) and calycosin therapy on neurological function following SCI, primarily by assessing changes in motor function recovery, neuronal survival, neuronal oxidative stress levels, and neural proliferation, in order to provide novel insights for the treatment of SCI. MATERIALS AND METHODS: The SCI model was constructed by compressing the spinal cord using vascular clamps. Calycosin was injected intraperitoneally into the SCI model rats, and a group of 5 rats underwent RT. The motor function of rats after SCI was evaluated using the Basso Beattle Bresnaha (BBB) score and the inclined plate test. Histopathological changes were evaluated by NeuN immunohistochemistry, HE and Nissl staining. Apoptosis was detected by TUNEL staining. The antioxidant effect of combined treatment was assessed by measuring changes in oxidative stress markers after SCI. Western blot analysis was conducted to examine changes in Hsp90-Akt/ASK1-p38 pathway-related proteins. Finally, cell proliferation was detected by BrdU and Ki67 assays. RESULTS: RT significantly improved the BBB score and angle of incline promoted by calycosin, resulting in enhanced motor function recovery in rats with SCI. Combining rehabilitation training with calycosin has a positive effect on morphological recovery. Similarly, combined RT enhanced the Nissl and NeuN staining signals of spinal cord neurons increased by calycosin, thereby increasing the number of neurons. TUNEL staining results indicated that calycosin treatment reduced the apoptosis signal in SCI, and the addition of RT further reduced the apoptosis. Moreover, RT combined with calycosin reduced oxidative stress by increasing SOD and GSH levels, while decreasing MDA, NO, ROS, and LDH expressions compared to the calycosin alone. RT slightly enhanced the effect of calycosin in activating Hsp90 and Akt and inhibiting the activation of ASK1 and p38, leading to enhanced inhibition of oxidative stress by calycosin. Additionally, the proliferation indexes (Ki67 and BrdU) assays showed that calycosin treatment alone increased both, whereas the combination treatment further promoted cell proliferation. CONCLUSION: Our research findings demonstrate that rehabilitation training enhances the ability of calycosin to reduce oxidative stress, resulting in a decrease in neuronal apoptosis and an increase in proliferation, ultimately promoting neuronal survival.

Formononetin Inhibits Microglial Inflammatory Response and Contributes to Spinal Cord Injury Repair by Targeting the EGFR/MAPK Pathway.

Zhenbao Pill contains many Chinese herbal medicinal ingredients and has been proven to have therapeutic effects on the repair of spinal cord injury (SCI). This study attempts to investigate the role of formononetin (FMN), an ingredient of Zhenbao Pill, in regulating neuroinflammation after SCI and the underlying mechanism. Primary microglia isolated from the spinal cord of newborn rats and human microglial clone 3 (HMC3) cells were stimulated with IL-1ß followed by FMN incubation. The cell viability and inflammatory cytokine levels were detected. The target of FMN was predicted and screened using databases. By silencing or overexpression of epidermal growth factor receptor (EGFR), the anti-neuroinflammatory effect of FMN was assessed in vitro. In vivo, FMN was intraperitoneally injected into rats after SCI followed by the neurological function and histopathology examination. The isolated microglia were in high purity, and the different concentrations of FMN incubation had no toxic effects on primary microglia and HMC3 cells. FMN reduced the inflammatory cytokine levels (TNF-α and IL-6) in a concentration-dependent manner. EGFR silencing or FMN incubation decreased p-EGFR and p-p38 levels and down-regulated inflammatory cytokine levels in IL-1ß-stimulated cells or supernatants. Nevertheless, the effects of FMN on microglial inflammation were reversed by EGFR overexpression. In vivo, FMN treatment improved the neuromotor function, repaired tissue injury, and inhibited EGFR/p38MAPK phosphorylation. Formononetin inhibits microglial inflammatory response and contributes to SCI repair via the EGFR/p38MAPK signaling pathway.

Calycosin ameliorates spinal cord injury by targeting Hsp90 to inhibit oxidative stress and apoptosis of nerve cells.

BACKGROUND: Zhenbao pill is effective in protecting against spinal cord injury (SCI). We attempt to explore the characteristics of calycosin (a main monomer of Zhenbao pill) in SCI and its relative mechanism. METHODS: The target of calycosin was screened using pharmacological network analysis. The SCI cell model was constructed using hydrogen peroxide (H2O2), and the animal model was developed by compressing spinal cord with a vascular clamp. Flow cytometry was conducted to test reactive oxygen species (ROS) levels and cell apoptosis. Detection of malondialdehyde (MDA) activity and Superoxide dismutase (SOD) activity were performed using relative kits. Heat shock protein 90 (HSP90) was examined using western blot and quantitative real-time PCR. Motor function tests were carried out. The hematoxylin-eosin and Nissl staining were conducted. RESULTS: In SCI models, ROS, MDA, and cell apoptosis were elevated, SOD and HSP90 levels were restrained, while calycosin addition reversed the above results. Besides, calycosin application or HSP90 overexpression enhanced phosphorylation of protein kinase B (Akt) but weakened that of apoptosis signal-regulating kinase 1 (ASK1) and p38, while HSP90 inhibitor 17-AAG treatment restrained the above results. Meanwhile, the injection of calycosin improved the motor function in SCI model rats. Furthermore, the pathologic results also clarified the positive effect of calycosin on SCI. CONCLUSION: HSP90 was lowly expressed in SCI models. Calycosin alleviated SCI by promoting HSP90 up-regulation and inhibiting oxidative stress and apoptosis of nerve cells.

Recent Advances in the Role of Nuclear Factor Erythroid-2-Related Factor 2 in Spinal Cord Injury: Regulatory Mechanisms and Therapeutic Options.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a pleiotropic transcription factor, and it has been documented that it can induce defense mechanisms both oxidative stress and inflammatory injury. At present, more and more evidences show that the Nrf2 signaling pathway is a key pharmacological target for the treatment of spinal cord injury (SCI), and activating the Nrf2 signaling pathway can effectively treat the inflammatory injury and oxidative stress after SCI. This article firstly introduces the biological studies of the Nrf2 pathway. Meanwhile, it is more powerful to explain that activating the Nrf2 signaling pathway can effectively treat SCI by deeply exploring the relationship between Nrf2 and oxidative stress, inflammatory injury, and SCI. In addition, several potential drugs for the treatment of SCI by promoting Nrf2 activation and Nrf2-dependent gene expression are reviewed. And some other treatment strategies of SCI by modulating the Nrf2 pathway are also summarized. It will provide new ideas and directions for the treatment of SCI.

Identification of Hub Genes to Regulate Breast Cancer Spinal Metastases by Bioinformatics Analyses.

Breast cancer (BC) had been one of the deadliest types of cancers in women worldwide. More than 65% of advanced-stage BC patients were identified to have bone metastasis. However, the molecular mechanisms involved in the BC spinal metastases remained largely unclear. This study screened dysregulated genes in the progression of BC spinal metastases by analyzing GSE22358. Moreover, we constructed PPI networks to identify key regulators in this progression. Bioinformatics analysis showed that these key regulators were involved in regulating the metabolic process, cell proliferation, Toll-like receptor and RIG-I-like receptor signaling, and mRNA surveillance. Furthermore, our analysis revealed that key regulators, including C1QB, CEP55, HIST1H2BO, IFI6, KIAA0101, PBK, SPAG5, SPP1, DCN, FZD7, KRT5, and TGFBR3, were correlated to the OS time in BC patients. In addition, we analyzed TCGA database to further confirm the expression levels of these hub genes in breast cancer. Our results showed that these regulators were significantly differentially expressed in breast cancer, which were consistent with GSE22358 dataset analysis. Furthermore, our analysis demonstrated that CEP55 was remarkably upregulated in the advanced stage of breast cancer compared to the stage I breast cancer sample and was significantly upregulated in triple-negative breast cancers (TNBC) compared to other types of breast cancers, including luminal and HER2-positive cancers, demonstrating CEP55 may have a regulatory role in TNBC. Finally, our results showed that CEP55 was the most highly expressed in Basal-like 1 TNBC and Basal-like 2 TNBC samples but the most lowly expressed in mesenchymal stem-like TNBC samples. Although more studies are still needed to understand the functions of key regulators in BC, this study provides useful information to understand the mechanisms underlying BC spinal metastases.

Zhenbao Pill reduces Treg cell proportion in acute spinal cord injury rats by regulating TUG1/miR-214/HSP27 axis.

Background: Acute spinal cord injury (SCI) is one of the weakest pathologies that seriously affect the quality of life of patients. Objective: To study the mechanism of how Zhenbao Pill reduces Treg cell proportion and improves acute SCI. Methods: A rat SCI model was established. Flow cytometry analysis was performed to determine the Treg cell proportion. RNA immunoprecipitation (RIP) and RNA pull-down were applied in confirming taurine up-regulated gene 1 (TUG1) and miR-214 binding. Intrathecal injection of TUG1 siRNA was also conducted to determine the effect of TUG1 in vivoResults: Zhenbao Pill promoted the expression of TUG1 and heat shock protein 27 (HSP27) protein, and reduced the expression of miR-214 and forkhead box protein p3 (Foxp3) as well as Treg cell proportion in a concentration-dependent manner in SCI rats or in vitro cultured CD4+ T cells. Knockdown of TUG1 reversed the high protein expression of HSP27 and the inhibition of Treg cell proportion as well as Foxp3 protein induced by Zhenbao Pill, and miR-214 inhibitor canceled the TUG1 knockdown effect. Further, miR-214 mimic reversed the inhibition of Treg cell proportion and Foxp3 protein expression by Zhenbao Pill, which was abolished by the overexpression of HSP27. The mechanism was validated in animal experiments. Conclusion: Zhenbao Pill regulated TUG1/miR-214/HSP27 signaling pathway to reduce Treg cell proportion and thus relieve acute SCI.

Zhenbao pill protects against acute spinal cord injury via miR-146a-5p regulating the expression of GPR17.

The aim of the present study was to observe the effect of zhenbao pill on the motor function of acute spinal cord injury (ASCI) rats and the molecular mechanisms involving miR-146a-5p and G-protein-coupled receptor 17 (GPR17). ASCI rat model was established by modified Allen method, and then the rats were divided into three groups. SH-SY5Y cells were cultured overnight in hypoxia condition and transfected with miR-146a-5p mimic or miR-146a-5p inhibitor. The hind limb motor function of the rats was evaluated by Basso, Beattie, Bresnahan (BBB) scoring system. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of miR-146a-5p, GPR17, inducible nitric oxide synthase (iNOS), interleukin 1ß (IL-1ß), and tumor necrosis factor α (TNF-α). Neuronal apoptosis was measured using flow cytometry assay. Luciferase reporter assay was performed to determine the regulation of miR-146a-5p on GPR17. Zhenbao pill could enhance hind limb motor function and attenuate the inflammatory response caused by ASCI. Moreover, zhenbao pill increased the level of miR-146a-5p and decreased GPR17 expression in vivo and in vitro Bioinformatics software predicted that GPR17 3'-UTR had a binding site with miR-146a-5p Luciferase reporter assay showed that miR-146a-5p had a negative regulatory effect on GPR17 expression. Knockdown of miR-146a-5p could reverse the effect of zhenbao pill on the up-regulation of GPR17 induced by hypoxia, reversed the inhibitory effect of zhenbao pill on the cell apoptosis induced by hypoxia and the recovery of zhenbao pill on hind limb motor function in ASCI rats. Zhenbao pill could inhibit neuronal apoptosis by regulating miR-146a-5p/GPR17 expression, and then promoting the recovery of spinal cord function.

Zhenbao Pill reduces the percentage of Treg cells by inducing HSP27 expression.

CONTEXT: Zhenbao pill containing Nacre, Safflower, Musk and Cornu Bubahas has been proved to have a good therapeutic effect on the repair of spinal cord injury (SCI). However, its complex mechanism of repairing SCI is not yet known. OBJECTIVE: This study attempts to investigate the role of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the mechanism of the action of Zhenbao pill, and further explore the relationship between Tregs and HSP27 expression in repair mechanism. MATERIALS AND METHODS: Treatment of human peripheral blood mononuclear cells (PBMCs) with different concentrations (0, 0.5, 2.5, 5, 10mg/mL) of Zhenbao pill, flow cytometry was used to detect the expression of the specific factors CD4+CD25+Foxp3+ in Tregs, and detection of Tregs related regulatory factor TGF-ß content was performed with ELISA assay. The relationship between miR-214 and HSP27 was assessed by Luciferase assay, and the level of miR-214 was detected by qPCR. The expression of HSP27 was examined with qPCR and western blotting. RNA interference technology and gene recombination were used to inhibit and up-regulate the expression of HSP27. RESULTS: Zhenbao pill with 11.61mg/mL of IC50 for Tregs can significantly inhibit the differentiation into Tregs in human PBMCs and up-regulate by more than 1-fold of HSP27 expression. Essentially, it enhanced the expression of HSP27 by inhibiting miR-214 expression (50%). Inhibition of HSP27 expression, followed by the differentiation into Tregs, was promoted in human PBMCs. When the HSP27 expression was up-regulated, the differentiation into Tregs was decreased by 30%. It indicated that the expression of HSP27 regulated the differentiation into Tregs. Inhibition of HSP27 expression and Zhenbao pill treatment, the differentiation into Tregs was decreased but remained at a higher level than that of the group was only treated with pill. Under the action of Zhenbao pill, the expression of HSP27 was not completely interfered, and its expression level was still increased. CONCLUSIONS: In the process of repairing the SCI, Zhenbao pill inhibits reduces numbers of Treg lymphocytes as well as TGF-ß levels by inducing HSP27 expression.

Minimally invasive one-level lumbar decompression and fusion surgery with posterior instrumentation using a combination of pedicle screw fixation and transpedicular facet screw construct.

BACKGROUND: Minimally invasive lumbar spine fusion surgery has gained popularity in recent years. Routinely, this technique requires bilateral parasagittal incisions for decompression, interbody fusion, and posterior instrumentation. The following study is a description of a new minimally invasive technique for one-level transforaminal lumbar interbody fusion (TLIF) using a unilateral parasagittal incision (Wiltse approach), with placement of pedicle screws and then a percutaneous transpedicular facet screw insertion on the contralateral side. The biomechanical stability of this posterior construct will be discussed while the efficacy and complications of this technique have been examined. METHODS: Forty patients underwent this new technique of one-level TLIF with posterior instrumentation using unilateral pedicle screw fixation supplemented with contralateral percutaneous transpedicular facet screw construct. Data regarding surgical time, estimated blood loss (EBL), hospital length of stay (LOS), and complications related to the posterior instrumentation are recorded. RESULTS: The average surgical time of this new procedure was 124 minutes; average EBL was 140 cc; average hospital LOS was 3 days. Two patients developed new leg pain on the side where the facet screw had been placed. Both patients had the facet screw removed. CONCLUSION: This novel technique of unilateral pedicle screw fixation combined with contralateral percutaneous transpedicular facet screw construct has further reduced the amount of normal tissue injury while maintaining the same biomechanical advantages of bilateral pedicle screw fixation. However, caution is needed during the placement of the percutaneous facet screw in order to avoid nerve root injury.

Quantitative analysis of types I and II collagen in the disc annulus in adolescent idiopathic scoliosis.

OBJECTIVE: To find the possible roles of collagen in the pathogenesis of AIS through studies of the distribution of the collagen I and II in the disc annulus fibrous in adolescent idiopathic scoliosis (AIS). METHODS: The disc annulus fibrous of apex curve were harvested during anterior surgery and divided into concave and convex samples. 25 AIS cases were covered in this study including 6 males and 19 females, with an average age of 14.6 years (range 12-18 years). 11 specimens were intervertebral disc from the thoracic region (from T8 to T11) and 14 sample were from lumbar discs (from L1 to L2). RT-PCR was employed to investigate the distribution and content of collagen I (220 bp) and collagen II (359 bp) in the intervertebral disc specimens. 1% agarose gel electrophoresis and the Gelwork image analysis system was used in the semi-quantitative analysis of the product. RESULTS: For the AIS group, type I collagen and type II collagen significantly increased on convex side compared with that of the concave side (p<0.01). The same trend was observed in both thoracic and lumber disc annulus fibrosis, but there was no statistical difference expect for the expression of type II collagen in convex side of disc annulus fibrous (p<0.05). CONCLUSION: The asymmetric expression of collagen I and II collagen showing that there was degeneration in the intervertebral disc of AIS. The abnormal collagen metabolism may be one of reasons in the development of AIS and probably an important factor in the progression of AIS.

Association of estrogen receptor gene polymorphisms with susceptibility to adolescent idiopathic scoliosis.

STUDY DESIGN: A case-control study is presented. OBJECTIVE: To investigate the association of estrogen receptor gene polymorphisms with adolescent idiopathic scoliosis (AIS) risk. SUMMARY OF BACKGROUND DATA: Previous studies have shown that genetic factors are important in the pathogenesis of idiopathic scoliosis. Only 1 publication suggested that XbaI site polymorphism was associated with curve severity of idiopathic scoliosis. However, to our knowledge, the relationship of estrogen receptor gene polymorphisms and the individual susceptibility to idiopathic scoliosis has not been studied. METHODS: This study included 202 patients with AIS and 174 healthy controls. Height, menarche status, curve pattern, Cobb angle, and Risser sign in female patients were recorded. There were 2 polymorphic loci, PvuII and XbaI locus, of estrogen receptor analyzed by restriction fragment length polymorphisms. RESULTS: The frequency of XX genotype was significantly higher in patients than that in controls (P = 0.005). The X allele appeared to be overrepresented in patients compared with controls (P = 0.001). Furthermore, the frequencies of XX genotype in female patients whose height was > or = 160 cm and Cobb angle > or = 40 degrees were higher than those whose height was <160 cm and Cobb angle <40 degrees (P = 0.001 and P < 0.001, respectively). CONCLUSIONS: The XbaI site polymorphism of estrogen receptor gene may be associated with a risk of AIS.

[The effect of titanium mesh cage on maintenance of thoracolumbar and lumbar lordosis in anterior instrumentation for thoracolumbar and lumbar scoliosis].

OBJECTIVE: To evaluate the clinical importance of titanium mesh cage (TMC) in anterior instrumentation for scoliosis. METHODS: Thirty-six consecutive patients with thoracolumbar/lumbar adolescent idiopathic scoliosis were treated with anterior derotation and instrumentation using autologous bone grafting and TMC for interbody fusion. The average age of patients was 17 years (ranged from 14 to 22 years), in which 5 cases were male and 31 cases were female. RESULTS: The coronal Cobb angle before surgery, 2 weeks after surgery, and 13 months at the follow-up was measured as 56 degrees , 15 degrees , and 18 degrees respectively, while the thoracic kyphosis from T(5) to T(12) was 30 degrees , 33 degrees , and 37 degrees respectively, and the lumbar lordosis from L(1) to S(1) was 46 degrees , 56 degrees , 51 degrees . There were no death, infection, implant failures or collapse of intervertebral space. Intraoperative injury of lacteal occurred in one patient, but no postoperative complications happened. One of two patients who had pleural effusion was managed with close drainage. Exudative pleurisy occurred in one patient, the sympathectomy effect occurred in three patients who presented asymmetry of skin temperature and resolved in short term. CONCLUSIONS: Coronal and sagittal alignment can be well corrected and maintained with TMC in anterior scoliosis surgery, collapse of intervertebral space and lumbar kyphosis can be prevented.