Combined transcriptomics and proteomics studies on the effect of electrical stimulation on spinal cord injury in rats.

Electrical stimulation (ES) of the spinal cord is a promising therapy for functional rehabilitation after spinal cord injury (SCI). However, the specific mechanism of action is poorly understood. We designed and applied an implanted ES device in the SCI area in rats and determined the effect of ES on the treatment of motor dysfunction after SCI using behavioral scores. Additionally, we examined the molecular characteristics of the samples using proteomic and transcriptomic sequencing. The differential molecules between groups were identified using statistical analyses. Molecular, network, and pathway-based analyses were used to identify group-specific biological features. ES (0.5 mA, 0.1 ms, 50 Hz) had a positive effect on motor dysfunction and neuronal regeneration in rats after SCI. Six samples (three independent replicates in each group) were used for transcriptome sequencing; we obtained 1026 differential genes, comprising 274 upregulated genes and 752 downregulated genes. A total of 10 samples were obtained: four samples in the ES group and six samples in the SCI group; for the proteome sequencing, 48 differential proteins were identified, including 45 up-regulated and three down-regulated proteins. Combined transcriptomic and proteomic studies have shown that the main enrichment pathway is the hedgehog signaling pathway. Western blot results showed that the expression levels of Sonic hedgehog (SHH) (P < 0.001), Smoothened (SMO) (P = 0.0338), and GLI-1 (P < 0.01) proteins in the ES treatment group were significantly higher than those in the SCI group. The immunofluorescence results showed significantly increased expression of SHH (P = 0.0181), SMO (P = 0.021), and GLI-1 (P = 0.0126) in the ES group compared with that in the SCI group. In conclusion, ES after SCI had a positive effect on motor dysfunction and anti-inflammatory effects in rats. Moreover, transcriptomic and proteomic sequencing also provided unique perspectives on the complex relationships between ES on SCI, where the SHH signaling pathway plays a critical role. Our study provides a significant theoretical foundation for the clinical implementation of ES therapy in patients with SCI.

Erxian decoction inhibits apoptosis by activating Akt1 and repairs spinal cord injury in rats.

Objective: Spinal cord injury (SCI) often leads to severe physiological and pathological changes in patients. Erxian Decoction (EXD) is effective in the postoperative treatment of spinal cord injury, but its specific mechanism of action is poorly defined. Methods: Network pharmacology and molecular docking were used to predict the potential mechanisms of EXD in SCI. In vivo studies were used to validate the above predictions. For in vivo study, the rats were pretreated with or without EXD (5.76 g/kg, by intragastric gavage). Multiple molecular biological test methods to identify molecular mechanisms. One-way analysis of variance (ANOVA) was used with Bonferroni's post-hoc test to identify the differences between groups. Results: In vivo studies have shown that EXD improved motor function at 7dpi in SCI rats (P < 0.0001), significantly reduced spinal cord edema (P = 0.0139), upregulated 5-HT, GFAP, and TMEM119 expression. Through network pharmacology analysis, we found that Akt1 in EXD plays a role in treating SCI. The underlying mechanism may be the inhibition of apoptosis after activation of Akt1 phosphorylation. Molecular docking revealed that the key compounds could spontaneously bind to the Akt1 protein. Pharmacological inhibition of Akt1 activation by MK-2206, attenuated the anti-apoptotic effect of EXD on SCI in rats (P < 0.0001). Conclusions: EXD inhibits apoptosis by activating Akt1, reduce spinal cord edema and restore behavioral function after SCI in rats.

Single-cell RNA sequencing reveals the role of immune-related autophagy in spinal cord injury in rats.

Spinal cord injury refers to damage to the spinal cord due to trauma, disease, or degeneration; and the number of new cases is increasing yearly. Significant cellular changes are known to occur in the area of spinal cord injury. However, changes in cellular composition, trajectory of cell development, and intercellular communication in the injured area remain unclear. Here, we used single-cell RNA sequencing to evaluate almost all the cell types that constitute the site of spinal cord injury in rats. In addition to mapping the cells of the injured area, we screened the expression of immune autophagy-related factors in cells and identified signaling pathways by the measuring the expression of the receptor-ligand pairs to regulate specific cell interactions during autophagy after spinal cord injury. Our data set is a valuable resource that provides new insights into the pathobiology of spinal cord injury and other traumatic diseases of the central nervous system.

A modified impactor for establishing a graded contusion spinal cord injury model in rats.

Background: Given the indispensable role of animal models in preclinical studies of spinal cord injury (SCI) and the current state of available impactors, we designed a modified impactor for establishing contusion SCI in rats. The major improvement is the replacement of the impactor rod with a weight and an impactor tip. Methods: Preoperatively, radiographs of 8-week-old female Wistar rats were taken to establish a protocol for locating the target spinal segment. A total of 72 rats were randomly divided into 4 groups: the sham, 12.5-, 25- and 50-mm groups. Within 35 days postinjury (dpi), the Basso, Beattie, and Bresnahan locomotor rating scale (BBB) was used to evaluate the hindlimb motor function of the rats. At 7 dpi, the rats were sacrificed, and the spinal cord tissue was fixed. Hematoxylin-eosin (HE) staining was used to assess histological changes. Subsequently, immunofluorescence staining was performed to visualize the expression and distribution of GFAP, CD68, MBP, and NeuN. Additionally, rats were sacrificed, and their tissues were extracted for relevant protein assays. At 3 and 7 dpi, electrophysiological function was evaluated by measuring motor evoked potentials (MEPs) and sensory evoked potentials (SEPs). Results: The behavioral results revealed that higher strike heights were associated with lower BBB scores. Over time, the BBB scores of the SCI rats exhibited an improving trend. Quantitative analysis of the lesions indicated that as the impact height increased, the area of histological destruction, GFAP-negative area, CD68-positive cell count, and MBP-positive destruction area increased, and the number of NeuN-positive cells decreased. Western blot analysis further verified relevant protein changes. Electrophysiology confirmed that the MEP and SEP amplitudes decreased as the strike height increased. Conclusions: Thus, these results confirm that this modified impactor can be used to establish a graded SCI model in rats. The model is clinically relevant, reproducible, stable, accessible, and affordable, providing a practical tool with which to elucidate the pathophysiological mechanisms and potential therapies for contusive SCI.

Blockage of ERCC6 Alleviates Spinal Cord Injury Through Weakening Apoptosis, Inflammation, Senescence, and Oxidative Stress.

Objective: Spinal cord injury (SCI) is a devastating disease resulting in lifelong disability, but the molecular mechanism remains unclear. Our study was designed to observe the role of excision repair cross-complementing group 6 (ERCC6) following SCI and to determine the underlying mechanism. Methods: SCI mouse models and LPS-induced microglia cell models were established. ERCC6 expression was blocked by ERCC6-siRNA-carrying lentivirus. Nissl staining was utilized for detecting neuronal damage, and apoptosis was analyzed with TUNEL and Western blotting (apoptotic markers). Immunofluorescence was used for measuring macrophage markers (CD68 and F4/80) and astrocyte and microglia markers (GFAP and Iba-1). Pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were measured via ELISA. Senescent cells were estimated via SA-ß-Gal staining as well as Western blot (senescent markers p21 and p27). Oxidative stress was investigated by detecting the expression of 4-HNE, Nrf2, and Keap1, and intracellular ROS levels. Results: ERCC6 expression was remarkably upregulated both in the spinal cord of SCI mice and LPS-induced microglia cells. ERCC6 deficiency alleviated neuronal damage and apoptosis. Macrophage infiltration and inflammatory response were suppressed by si-ERCC6 treatment. Moreover, ERCC6 blockage ameliorated astrocyte and microglia activation and cell senescence in the damaged spinal cord. Excessive oxidative stress was significantly decreased by ERCC6 knockdown in SCI. Conclusion: Collectively, ERCC6 exerts crucial functions in mediating physiological processes (apoptosis, inflammation, senescence, and oxidative stress), implying that ERCC6 might act as a prospective therapeutic target against SCI.

Pan-Cancer Transcriptome and Immune Infiltration Analyses Reveal the Oncogenic Role of Far Upstream Element-Binding Protein 1 (FUBP1).

Despite increasing evidence to support the relationship between FUBP1 and tumorigenesis in some types of cancers, there have been no analyses from a pan-cancer perspective. Here, we are the first to investigate the putative oncogenic role of FUBP1 in 33 cancer types based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Dysregulated FUBP1 expression was observed in most cancer types, and high FUBP1 expression suggests poor prognosis in cancers such as ACC, KICH, LIHC, LUAD, LUSC, SARC, CESC, and SKCM. Missense mutation is the most common type of FUBP1 mutation, and R430 in KH_4 is a predominant mutation site. Enhanced phosphorylation of FUBP1 at the S120 site has been observed in clear cell RCC, lung adenocarcinoma, and pediatric brain cancer specimens from African-American and Asian individuals. The expression of FUBP1 was found to be negatively correlated with the infiltration of CD8+ T lymphocytes in GBM, HNSC-HPV- and UCEC but positively correlated with that of tumor-associated fibroblasts in CESC, ESCA, HNSC, LIHC, LUAD, PAAD, and THYM. Furthermore, RNA splicing and spliceosome signaling were predominantly enriched in both GO and KEGG analyses of the functional mechanism of FUBP1. Briefly, this pan-cancer analysis comprehensively revealed the multifaceted characteristics and oncogenic role of FUBP1 in different human cancers.

miR-590-3p is a novel microRNA which suppresses osteosarcoma progression by targeting SOX9.

Osteosarcoma is the most common primary bone malignancy and arises primarily in the metaphyseal ends of long bones in children and adolescents. m iR-590 has been found to have anti-tumor effects in many other cancers. However, the role of miR-590-3p in osteosarcoma is poorly understood. In this study, we show that miR-590-3p was significantly decreased both in osteosarcoma tissues and cell lines, suggesting a potential role of miR-590-3p in osteosarcoma. Over-expression of miR-590-3p inhibited U2OS cell viability as shown by the CCK-8 assay and clonogenic assay. Ki-67 immunofluorescence staining and cell cycle analysis revealed that up-regulation of miR-590-3p inhibited U2OS cell proliferation. Transfection with miR-590-3p mimics suppressed PCNA, Cyclin D1 and CDK4 expression and increased p53 and p21 expression. In addition, U2OS cells transfected with miR-590-3p mimics exhibited reduced cell invasion and migration, characterized by the wound healing assay and transwell assay. Furthermore, bioinformatics analysis demonstrated that SOX9 was a potential target of miR-590-3p. SOX9 was up-regulated in osteosarcoma tissues. Transfection with miR-590-3p mimics markedly suppressed SOX9 expression both at the mRNA level and protein level. Dual luciferase assay validated the direct binding site of miR-590-3p on SOX9. Exogenous SOX9 expression in U2OS cells at least partially reversed the effects of miR-590-3p in U2OS cells. Enforced SOX9 expression restored cell viability in osteosarcoma cells transfected with miR-590-3p mimics. In addition, over-expression of SOX9 restored decreased cell metastasis properties caused by transfection with miR-590-3p mimics in osteosarcoma cells. In summary, these results indicated that miR-590-3p is an anti-cancer miRNA that can inhibit proliferation and metastasis in osteosarcoma cells. Our findings provide a novel insight into the biological function of miR-590-3p in osteosarcoma and SOX9 may be a potential therapeutic target for osteosarcoma.