Targeting transcription factor pu.1 for improving neurologic outcomes after spinal cord injury.

Background: After spinal cord injury (SCI), lipid metabolism dysregulation at the lesion site exacerbates secondary damage. The transcription factor pu.1 has been implicated as a negative regulator of multiple lipid metabolism-related genes and pathways. However, its role in post-SCI lipid metabolism remains unclear. Methods: We employed a mouse model of complete T10 crush SCI. Non-targeted metabolomics and bioinformatics analysis were utilized to investigate lipid metabolism at the lesion site after SCI. Polarized light imaging was used to evaluate the presence of cholesterol crystals. DB1976, a specific inhibitor of pu.1, was administered to examine its impact on local lipid metabolism after SCI. Immunofluorescence staining was performed to assess pu.1 expression and distribution, and to evaluate lipid droplet formation, astrocytic/fibrotic scar development, inflammatory cell infiltration, and tight junctions within the vasculature. Results: Non-targeted metabolomics and bioinformatics analyses revealed significant alterations in lipid metabolism components after SCI. Moreover, immunofluorescence staining and polarized light imaging demonstrated substantial BODIPY+ lipid droplet accumulation and persistent cholesterol crystal formation at the lesion site after SCI. Increased pu.1 expression was predominantly observed within macrophages/microglia at the lesion site after SCI. DB1976 treatment significantly mitigated lipid droplet accumulation and cholesterol crystal formation, reduced CD68+ macrophage/microglial infiltration, and attenuated fibrotic scar formation. Moreover, DB1976 treatment promoted the expression of claudin-5 and zonula occludens-1 between vascular endothelial cells and enhanced GFAP+ glial connectivity after SCI. Conclusion: Our study reveals a significant correlation between lipid metabolism disturbance post-SCI and transcription factor pu.1 upregulation, specifically in macrophages/microglia at the lesion site. Thus, targeted pu.1 modulation has the potential to yield promising results by substantially diminishing the deposition of lipid metabolism byproducts at the lesion site and fostering a milieu conducive to SCI repair.

Collagen I is a critical organizer of scarring and CNS regeneration failure.

Although axotomized neurons retain the ability to initiate the formation of growth cones and attempt to regenerate after spinal cord injury, the scar area formed as a result of the lesion in most adult mammals contains a variety of reactive cells that elaborate multiple extracellular matrix and enzyme components that are not suitable for regrowth 1,2 . Newly migrating axons in the vicinity of the scar utilize upregulated LAR family receptor protein tyrosine phosphatases, such as PTPσ, to associate with extracellular chondroitin sulphate proteoglycans (CSPGs), which have been discovered to tightly entrap the regrowing axon tip and transform it into a dystrophic non-growing endball. The scar is comprised of two compartments, one in the lesion penumbra, the glial scar, composed of reactive microglia, astrocytes and OPCs; and the other in the lesion epicenter, the fibrotic scar, which is made up of fibroblasts, pericytes, endothelial cells and inflammatory cells. While the fibrotic scar is known to be strongly inhibitory, even more so than the glial scar, the molecular determinants that curtail axon elongation through the injury core are largely uncharacterized. Here, we show that one sole member of the entire family of collagens, collagen I, creates an especially potent inducer of endball formation and regeneration failure. The inhibitory signaling is mediated by mechanosensitive ion channels and RhoA activation. Staggered systemic administration of two blood-brain barrier permeable-FDA approved drugs, aspirin and pirfenidone, reduced fibroblast incursion into the complete lesion and dramatically decreased collagen I, as well as CSPG deposition which were accompanied by axonal growth and considerable functional recovery. The anatomical substrate for robust axonal regeneration was provided by laminin producing GFAP + and NG2 + bridging cells that spanned the wound. Our results reveal a collagen I-mechanotransduction axis that regulates axonal regrowth in spinal cord injury and raise a promising strategy for rapid clinical application.

hBcl2 overexpression in BMSCs enhances resistance to myelin debris-induced apoptosis and facilitates neuroprotection after spinal cord injury in rats.

After spinal cord injury (SCI), the accumulation of myelin debris at the lesion exacerbates cell death and hinders axonal regeneration. Transplanted bone marrow mesenchymal stem cells (BMSCs) have been proven to be beneficial for SCI repair, but they are susceptible to apoptosis. It remains unclear whether this apoptotic process is influenced by myelin debris. Here, we constructed rat BMSCs overexpressing human B-cell lymphoma 2 (hBcl2) alone (hBcl2 group), BMSCs overexpressing hBcl2 with an endoplasmic reticulum-anchored segment (hBcl2-cb) (cb group), and a negative control group (NC group) for transplantation in this study. Immunocytochemistry staining validated the successful expression of hBcl2 in BMSCs within the hBcl2 group and cb group. All BMSCs from each group exhibited the ability to phagocytize myelin debris. Nevertheless, only BMSCs derived from the hBcl2 group exhibited heightened resistance to apoptosis and maintained prolonged viability for up to 5 days when exposed to myelin debris. Notably, overexpression of hBcl2 protein, rather than its endoplasmic reticulum-anchored counterpart, significantly enhanced the resistance of BMSCs against myelin debris-induced apoptosis. This process appeared to be associated with the efficient degradation of myelin debris through the Lamp1+ lysosomal pathway in the hBcl2 group. In vivo, the hBcl2 group exhibited significantly higher numbers of surviving cells and fewer apoptotic BMSCs compared to the cb and NC groups following transplantation. Furthermore, the hBcl2 group displayed reduced GFAP+ glial scarring and greater preservation of NF200+ axons in the lesions of SCI rats. Our results suggest that myelin debris triggers apoptosis in transplanted BMSCs, potentially elucidating the low survival rate of these cells after SCI. Consequently, the survival rate of transplanted BMSCs is improved by hBcl2 overexpression, leading to enhanced preservation of axons within the injured spinal cord.

Emerging therapeutic potential of adeno-associated virus-mediated gene therapy in liver fibrosis.

Liver fibrosis is a wound-healing response that results from various chronic damages. If the causes of damage are not removed or effective treatments are not given in a timely manner, it will progress to cirrhosis, even liver cancer. Currently, there are no specific medical therapies for liver fibrosis. Adeno-associated virus (AAV)-mediated gene therapy, one of the frontiers of modern medicine, has gained more attention in many fields due to its high safety profile, low immunogenicity, long-term efficacy in mediating gene expression, and increasingly known tropism. Notably, increasing evidence suggests a promising therapeutic potential for AAV-mediated gene therapy in different liver fibrosis models, which helps to correct abnormally changed target genes in the process of fibrosis and improve liver fibrosis at the molecular level. Moreover, the addition of cell-specific promoters to the genome of recombinant AAV helps to limit gene expression in specific cells, thereby producing better therapeutic efficacy in liver fibrosis. However, animal models are considered to be powerless predictive of tissue tropism, immunogenicity, and genotoxic risks in humans. Thus, AAV-mediated gene therapy will face many challenges. This review systemically summarizes the recent advances of AAV-mediated gene therapy in liver fibrosis, especially focusing on cellular and molecular mechanisms of transferred genes, and presents prospective challenges.

D-4F, an apolipoprotein A-I mimetic, promotes the clearance of myelin debris and the reduction of foamy macrophages after spinal cord injury.

After spinal cord injury (SCI), a large number of blood-derived macrophages infiltrate the lesion site and phagocytose myelin debris to become foamy macrophages, which leads to chronic inflammation. The drug D-4F, an apolipoprotein A-I peptidomimetic made of D-amino acids, has been reported to promote the lipid metabolism of foamy macrophages in atherosclerosis. However, the role and mechanism of D-4F in SCI are still unclear. In this study, we found that D-4F can promote the removal of myelin debris, reduce the formation of foamy macrophages in the lesion core and promote neuroprotection and recovery of motor function after SCI. These beneficial functions of D-4F may be related to its ability to upregulate the expression of ATP-binding cassette transporter A1 (ABCA1), the main transporter that mediates lipid efflux in foamy macrophages because inhibiting the activity of ABCA1 can reverse the effect of D-4F in vitro. In conclusion, D-4F may be a promising candidate for treating SCI by promoting the clearance of myelin debris by foamy macrophages via the ABCA1 pathway.

Exosomal LncRNAs and hepatocellular Carcinoma: From basic research to clinical practice.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with poor prognosis. The incidences of HCC and HCC-related deaths have increased over the last several decades. However, the treatment options for advanced HCC are very limited. Long noncoding RNAs (lncRNAs) wrapped in exosomes can change the expression of their target genes in recipient cells, thereby regulating the behavior of recipient cells. Increasing evidence has demonstrated that there is a correlation between the activation of exosomal lncRNAs and the development of HCC. In this review article, we highlighted the functions of exosomal lncRNAs in the development of HCC, showing that exosomal lncRNAs play a vital role in the growth and progression of HCC and are targets for HCC.

Nitric Oxide Induces Immunogenic Cell Death and Potentiates Cancer Immunotherapy.

Tumor cells undergoing immunogenic cell death (ICD) release immunogenic damage-associated molecular patterns (DAMPs) to trigger a long-term protective antitumor response. ICD can be induced by certain pathogens, chemotherapeutics, and physical modalities. In this work, we demonstrate that a gaseous molecule, specifically nitric oxide (NO), can induce a potent ICD effect. NO exerts cytotoxic effects that are accompanied by the emission of DAMPs based on the endoplasmic reticulum stress and mitochondrial dysfunction pathways. Released DAMPs elicit immunological protection against a subsequent rechallenge of syngeneic tumor cells in immunocompetent mice. We prepare polynitrosated polyesters with high NO storage capacity through a facile polycondensation reaction followed by a postsynthetic modification. The polynitrosated polyesters-based NO nanogenerator (NanoNO) that enables efficient NO delivery and controlled NO release in tumors induces a sufficient ICD effect. In different immune-intact models of tumors, the NanoNO exhibits significant tumor growth suppression and increases the local dose of immunogenic signals and T cell infiltrations, ultimately prolonging survival. In addition, the NanoNO synergizes with the PD-1 blockade to prevent metastasis. We conclude not only that NO is a potent ICD inducer for cancer immunotherapy but also that it expands the range of ICD inducers into the field of gaseous molecules.

STING-mediated inflammation contributes to Gao binge ethanol feeding model.

Alcohol metabolism causes hepatocytes to release damage-associated molecular patterns (DAMPs). This includes mitochondrial DNA (mtDNA), which is generated and released from damaged hepatocytes and contributes to liver injury by producing proinflammatory cytokines. STING is a pattern recognition receptor of DAMPs known to control the induction of innate immunity in various pathological processes. However, the expression profile and functions of STING in the Gao binge ethanol model remain poorly understood. We demonstrated that STING is upregulated in the Gao binge ethanol model. STING functions as an mtDNA sensor in the Kupffer cells of the liver and induces STING-signaling pathway-dependent inflammation and further aggravates hepatocyte apoptosis in the Gao binge ethanol model. This study provides novel insights into predicting disease progression and developing targeted therapies for alcoholic liver injury.

CD3D, GZMK, and KLRB1 Are Potential Markers for Early Diagnosis of Rheumatoid Arthritis, Especially in Anti-Citrullinated Protein Antibody-Negative Patients.

Early diagnosis and monitoring of rheumatoid arthritis (RA) progress are critical for effective treatment. In clinic, the detection of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) are usually combined to diagnose early RA. However, the poor specificity of RF and high heterogeneity of ACPA make the early diagnosis of RA still challenging. Bioinformatics analysis based on high-throughput omics is an emerging method to identify novel and effective biomarkers, which has been widely used in many diseases. Herein, utilizing an integrated strategy based on expression correlation analysis and weighted gene coexpression network analysis (WGCNA), we identified 76 RA-trait different expression genes (DEGs). Combined with protein-protein interaction (PPI) network construction and clustering, new hub genes associated in RA synovia, CD3D, GZMK, and KLRB1, were identified. We verified the specificity of these genes in the synovium of RA patients through three external datasets. We also observed high sensitivity and specificity of them for ACPA-negative patients. CD3D, GZMK, and KLRB1 are potentially key mediators of RA pathogenesis and markers for RA diagnosis.

The stabilization of yes-associated protein by TGFß-activated kinase 1 regulates the self-renewal and oncogenesis of gastric cancer stem cells.

Gastric cancer (GC) is the most frequent digestive system malignant tumour and the second most common cause of cancer death globally. Cancer stem cell (CSC) is a small percentage of cancer cells in solid tumours that have differentiation, self-renewal and tumorigenic capabilities. They have an active participation in the initiation, development, metastasis, recurrence and resistance of tumours to chemotherapy and radiotherapy. Gastric cancer stem cells (GCSCs) have been shown to be correlated with GC initiation and metastasis. In this study, we found that TAK1 expression level in GC tissues was significantly increased compared to the adjacent non-cancerous tissues by RT-qPCR, Western blot and immunohistochemistry. TAK1 has been identified as a critical molecule that promoted a variety of malignant GC phenotypes both in vivo and in vitro and promoted the self-renewal of GCSCs. Mechanistically, TAK1 was up-regulated by IL-6 and prevented the degradation of yes-associated protein (YAP) in the cytoplasm by binding to YAP. Thus, TAK1 promoted the SOX2 and SOX9 transcription and the self-renewal and oncogenesis of GCSCs. Our findings provide insights into the mechanism of self-renewal and tumorigenesis of TAK1 in GCSCs and have broad implications for clinical therapies.

Design, synthesis and cell imaging of a simple peptide-based probe for the selective detection of RNA.

Here we present a novel peptide-based fluorescent "turn-on" molecule P1 for detecting RNA, in a double or single strand, AU-rich or CG-rich. Both computational and experimental studies indicate that the detection efficiency depends on the binding affinity of P1 and conformational changes. P1 could be applied for cell imaging without any additional transfection vectors. Selective detection of RNA in cells was determined by RNase digestion. Successful application of P1 for RNA imaging in cell mitosis reveals that it may have broad applications in research, biotechnology and medical science.

The Prognosis Of Peroxiredoxin Family In Breast Cancer.

PURPOSE: PRDX (Peroxiredoxin) family has involved in breast cancer tumorigenesis from the evidence obtained from cell lines, human tissues and mouse models. Nonetheless, the diversified expression patterns, coupled with the prognostic values of PRDX family, still require explanation. This study aimed at investigating the clinical importance and biological of PRDXs in breast cancer. PATIENTS AND METHODS: Specimens of paraffin sections used for immunohistochemistry were collected from the hospital and the remaining patient information was retrieved from online databases. The expression and survival data of PRDXs in patients with breast cancer were from ONCOMINE, GEPIA, Kaplan-Meier Plotter. cBioPortal, Metascape, String, Cytoscape and DAVID were used to predict functions and pathways of the changes in PRDXs and their frequently altered neighbor genes. Immunohistochemistry was used to detect the expression of PRDXs in breast cancer. RESULTS: We discovered the expression levels of PRDX1-5 were higher in breast cancer tissues than in normal tissues, whereas the expression level of PRDX6 was observed as lower in the former one in comparison with that of the latter one. There existed a correlation between the expression levels of PRDX4, 5 and the advanced tumor stage. Survival analysis revealed that the expression of PRDXs were all associated with relapse-free survival (RFS) in all of the patients with breast cancer. Eventually, we discovered significant regulation of the cellular oxidant detoxification and detoxification of ROS by the PRDX changes, together with obtaining the core modules of genes (TXN, TXN2, TXNRD1, TXNRD2, GPX1 and GPX2) linked to the PRDX family of genes in breast cancer. CONCLUSION: The PRDX family is widely involved in the development of breast cancer and affects the prognosis of patients. The functions and pathways of the changes in PRDXs and their frequently altered neighbor genes can be further verified by wet experiments.

Methylation of RCAN1.4 mediated by DNMT1 and DNMT3b enhances hepatic stellate cell activation and liver fibrogenesis through Calcineurin/NFAT3 signaling.

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis. Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-ß1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-ß1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner. Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

Effect of Shikonin on Spinal Cord Injury in Rats Via Regulation of HMGB1/TLR4/NF-kB Signaling Pathway.

BACKGROUND/AIMS: Shikonin, a compound extracted from Zicao, has been demonstrated to hold anti-bacterial, anti-inflammatory, and anti-tumor activities in various diseases and it has been shown to protect human organs from injuries. However, the effect of shikonin on the recovery of spinal cord injury (SCI) remains unknown. This study was designed to estimate the potential therapeutic effect and underlying mechanism of shikonin on SCI in vivo. METHODS: In the study, we used HE staining, ELISA assay, transfection assay, TUNEL assay, real time PCR and Western blot to detect the effects of shikonin on spinal cord injury in rats. RESULTS: we showed that shikonin could promote the recovery of motor function and tissue repair after SCI treatment in rats SCI model. Moreover, we demonstrated that shikonin inhibited the spinal cord edema in SCI model of rats. According to further investigation, shikonin induced the reduction of inflammatory response through decreasing the expression levels of HMGB1, TLR4 and NF-κB after SCI injury. In addition, we also found that shikonin could suppress the apoptosis and expression of caspase-3 protein in SCI model of rats. CONCLUSION: Our results demonstrated that shikonin induced the recovery of tissue repair and motor function via inactivation of HMGB1/TLR4/NF-κB signaling pathway in SCI model of rats. Meanwhile, shikonin regulated the inflammation response in SCI by suppressing the HMGB1/TLR4/NF-κB signaling pathway. The described mechanism sheds novel light on molecular signaling pathway in spinal cord injury and secondary injury including inflammatory response.

IL-17 induces reactive astrocytes and up-regulation of vascular endothelial growth factor (VEGF) through JAK/STAT signaling.

Spinal cord injury is a grave neurological disability resulting in neuron degeneration and permanent paralysis. The inflammation triggered by the injury would promote the spinal cord lesion in turn. Activated astrocytes during inflammatory response could promote glial scar formation and contribute to the progression of the spinal cord injury. Interleukin 17 (IL-17) was upregulated in inflammatory responses to contusion or compression of the spinal cord. in this study, IL-17 could induce reactive astrocytes which was indicated by a well-known hallmark glial fibrillary acidic protein (GFAP) in vitro and in vivo. Moreover, we demonstrated that the upregulation of VEGF was induced by IL-17 human astrocytoma cells. In our further investigation, IL-17 induced the expression of VEGF in spinal cord injury by activating JAK/STAT signaling pathway both in vitro and in vivo. In addition, we also found that IL-17 significantly changed tissue preservation and residual urine volumes and blood-spinal cord-barrier integrity in vivo. This newly found IL-17-JAK/STAT-VEGF axis improves our understanding of the molecular mechanism of spinal cord injury during inflammatory response and provides another potential target of spinal cord injury.