The Protein Acetylation after Traumatic Spinal Cord Injury: Mechanisms and Therapeutic Opportunities.

Spinal cord injury (SCI) leads to deficits of various normal functions and is difficult to return to a normal state. Histone and non-histone protein acetylation after SCI is well documented and regulates spinal cord plasticity, axonal growth, and sensory axon regeneration. However, our understanding of protein acetylation after SCI is still limited. In this review, we summarize current research on the role of acetylation of histone and non-histone proteins in regulating neuron growth and axonal regeneration in SCI. Furthermore, we discuss inhibitors and activators targeting acetylation-related enzymes, such as α-tubulin acetyltransferase 1 (αTAT1), histone deacetylase 6 (HDAC6), and sirtuin 2 (SIRT2), to provide promising opportunities for recovery from SCI. In conclusion, a comprehensive understanding of protein acetylation and deacetylation in SCI may contribute to the development of SCI treatment.

TGF-ß signaling pathway in spinal cord injury: Mechanisms and therapeutic potential.

Spinal cord injury (SCI) is a highly disabling central nervous system injury with a complex pathological process, resulting in severe sensory and motor dysfunction. The current treatment modalities only alleviate its symptoms and cannot effectively intervene or treat its pathological process. Many studies have reported that the transforming growth factor (TGF)-ß signaling pathway plays an important role in neuronal differentiation, growth, survival, and axonal regeneration after central nervous system injury. Furthermore, the TGF-ß signaling pathway has a vital regulatory role in SCI pathophysiology and neural regeneration. Following SCI, regulation of the TGF-ß signaling pathway can suppress inflammation, reduce apoptosis, prevent glial scar formation, and promote neural regeneration. Due to its role in SCI, the TGF-ß signaling pathway could be a potential therapeutic target. This article reported the pathophysiology of SCI, the characteristics of the TGF-ß signaling pathway, the role of the TGF-ß signaling pathway in SCI, and the latest evidence for targeting the TGF-ß signaling pathway for treating SCI. In addition, the limitations and difficulties in TGF-ß signaling pathway research in SCI are discussed, and solutions are provided to address these potential challenges. We hope this will provide a reference for the TGF-ß signaling pathway and SCI research, offering a theoretical basis for targeted therapy of SCI.

DNMT3a-mediated methylation of PPARγ promote intervertebral disc degeneration by regulating the NF-κB pathway.

Intervertebral disc degeneration (IVDD) is a common chronic musculoskeletal disease that causes chronic low back pain and imposes an immense financial strain on patients. The pathological mechanisms underlying IVDD have not been fully elucidated. The development of IVDD is closely associated with abnormal epigenetic changes, suggesting that IVDD progression may be controlled by epigenetic mechanisms. Consequently, this study aimed to investigate the role of epigenetic regulation, including DNA methyltransferase 3a (DNMT3a)-mediated methylation and peroxisome proliferator-activated receptor γ (PPARγ) inhibition, in IVDD development. The expression of DNMT3a and PPARγ in early and late IVDD of nucleus pulposus (NP) tissues was detected using immunohistochemistry and western blotting analyses. Cellularly, DNMT3a inhibition significantly inhibited IL-1ß-induced apoptosis and extracellular matrix (ECM) degradation in rat NP cells. Pretreatment with T0070907, a specific inhibitor of PPARγ, significantly reversed the anti-apoptotic and ECM degradation effects of DNMT3a inhibition. Mechanistically, DNMT3a modified PPARγ promoter hypermethylation to activate the nuclear factor-κB (NF-κB) pathway. DNMT3a inhibition alleviated IVDD progression. Conclusively, the results of this study show that DNMT3a activates the NF-κB pathway by modifying PPARγ promoter hypermethylation to promote apoptosis and ECM degradation. Therefore, we believe that the ability of DNMT3a to mediate the PPARγ/NF-κB axis may provide new ideas for the potential pathogenesis of IVDD and may become an attractive target for the treatment of IVDD.

[Researchers' Knowledge About the Ethical Norms of National Natural Science Foundation of China Funded Medical Research:Current Status and Suggestions].

Objective To understand the current status of Chinese medical researchers' knowledge regarding the ethical norms of the research involving humans or laboratory animals,and provide reference for further improving the ethics review norms. Methods The questionnaire method was employed to survey the applicants for the 2019 projects supported by the Department of Medical Sciences,National Natural Science Foundation of China (NSFC) about their knowledge of ethical requirements.Furthermore,the ethical supervision of the NSFC and affiliations at the project application and implementation stages was analyzed. Results The survey showed that 29.9% medical researchers were familiar with NSFC's ethical requirements for research involving human or laboratory animals.During the project application stage,59.0% affiliations adopted the simplified review method.Regarding the ethical supervison,95.5% medical researchers believed that the affiliations should fulfill the ethical supervision obligations and take relevant measures during the project implementation period.In addition,55.0% medical researchers fully agreed to discuss with the review experts about the ethical issues involved in the project. Conclusions The NSFC should establish rules and regulations to improve institutional management responsibilities and institutionalize the training about research ethics to comprehensively strengthening the training.Taking the management of research project ethics as a starting point,the NSFC should form a multi-party linkage between project funding and management and establish an accountability mechanism for ethics management.Furthermore,the NSFC should double the endeavors at the review of ethical issues during expert review and process management and attach importance to the research,judgment,and prevention of ethical risks.

Roles of pyroptosis in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD), the key pathological process in low back pain, is characterized by chronic inflammation and progressive cell death. Pyroptosis is a type of pro-inflammatory programmed necrosis mediated by inflammasomes that is dependent on the gasdermin family of proteins. An in-depth study of the pathological mechanisms of IDD has revealed that pyroptosis plays an important role in its occurrence and development. The molecular characteristics and activation signaling mechanisms of pyroptosis are reviewed in this paper. Moreover, the specific roles of pyroptosis in IDD pathology are outlined and various targeted drugs for its treatment are highlighted.

Intrathecal Injection of Botulinum Toxin Type A has an Analgesic Effect in Male Rats CCI Model by Inhibiting the Activation of Spinal P2X4R.

Purinergic receptor P2X4 (P2X4R) plays an essential role in neuropathic pain. However, the specific mechanism needs to be clarified. Botulinum toxin type A is a neurotoxin produced by Clostridium botulinum type A. This study found that intrathecal injection of botulinum toxin type A produced an excellent analgesic effect in a rat model of chronic constriction sciatic nerve injury and inhibited the activation of P2X4R, microglia, and astrocytes. The administration of a P2X4R activator can up-regulate the expression of P2X4R and eliminate the analgesic effect of intrathecal injection of botulinum toxin type A. In addition, we found that microglia and astrocytes in the spinal cord of rats injected with botulinum toxin type A were reactivated after administration of the P2X4R activator. Our results suggest that intrathecal injection of botulinum toxin type A has an analgesic effect in a rat model of chronic constriction sciatic nerve injury by inhibiting the activation of P2X4R in the spinal cord.

Bibliometric and visual analysis of microglia-related neuropathic pain from 2000 to 2021.

Background: Microglia has gradually gained researchers' attention in the past few decades and has shown its promising prospect in treating neuropathic pain. Our study was performed to comprehensively evaluate microglia-related neuropathic pain via a bibliometric approach. Methods: We retrospectively reviewed publications focusing on microglia-related neuropathic pain from 2000 to 2021 in WoSCC. VOS viewer software and CiteSpace software were used for statistical analyses. Results: A total of 2,609 articles were finally included. A steady increase in the number of relevant publications was observed in the past two decades. China is the most productive country, while the United States shares the most-cited and highest H-index country. The University of London, Kyushu University, and the University of California are the top 3 institutions with the highest number of publications. Molecular pain and Pain are the most productive and co-cited journals, respectively. Inoue K (Kyushu University) is the most-contributed researcher and Ji RR (Duke University) ranks 1st in both average citations per article and H-index. Keywords analyses revealed that pro-inflammatory cytokines shared the highest burst strength. Sex differences, neuroinflammation, and oxidative stress are the emerging keywords in recent years. Conclusion: In the field of microglia-related neuropathic pain, China is the largest producer and the United States is the most influential country. The signaling communication between microglia and neurons has continued to be vital in this field. Sexual dimorphism, neuroinflammation, and stem-cell therapies might be emerging trends that should be closely monitored.

Acid-sensing Ion Channels: Implications for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is the leading cause of lower back pain and seriously affects the quality of life of patients. The intervertebral disc (IVD) is an environment of hypoxia, ischemia, acidity, and hypertonicity. Matrix acidity potentially negatively affects gene expression, activity, proliferation, and apoptosis of IVD cells. Acid-sensing ion channels (ASICs) are a group of proton-gated ion channels that play important roles in physiological and pathological conditions. The distribution of ASICs in the nucleus pulposus (NP), annulus fibrosus, cartilage endplate, and nucleus pulposus mesenchymal stem cells (NP-MSCs), as well as the special functions of ASIC1a and ASIC3, show that ASICs play an important role in IDD. In this review, we comprehensively discuss the roles of ASICs in the development and basic pathology of IDD and their potential relevance as therapeutic targets. A deeper understanding of the roles of ASICs in these processes may provide novel therapeutic targets for IDD prevention and treatment.

Epitope-based minigene vaccine targeting fibroblast activation protein α induces specific immune responses and anti-tumor effects in 4 T1 murine breast cancer model.

Fibroblast activation protein (FAPα) is a tumor stromal antigen expressed by cancer-associated fibroblasts (CAFs) in more than 90 % of malignant epithelial carcinomas. FAPα-based immunotherapy has been reported and showed that FAPα-specific immune response can remold immune microenvironment and contribute to tumor regression. Many FAPα-based vaccines have been investigated in preclinical trials, which can elicit strong and durable cytolytic T lymphocytes (CTL) with good safety. However, epitope-based FAPα vaccines are rarely reported. To break tolerance against self-antigens, analogue epitopes with modified peptides at the anchor residues are typically used to improve epitope immunogenicity. To investigate the feasibility of a FAPα epitope-based vaccine for cancer immunotherapy in vivo, we conducted a preclinical study to identify a homologous CTL epitope of human and mouse FAPα and obtained its analogue epitope in BALB/c mice, and explored the anti-tumor activity of their minigene vaccines in 4 T1 tumor-bearing mice. By using in silico epitope prediction tools and immunogenicity assays, immunodominant epitope FAP.291 (YYFSWLTWV) and its analogue epitope FAP.291I9 (YYFSWLTWI) were identified. The FAP.291-based epitope minigene vaccine successfully stimulated CTLs targeting CAFs and exhibited anti-tumor activity in a 4 T1 murine breast cancer model. Furthermore, although the analogue epitope FAP.291I9 enhanced FAP.291-specific immune responses, improvement of anti-tumor immunity effects was not observed. Check of immunosuppressive factors revealed that the high levels of IL-10, IL-13, myeloid-derived suppressor cells and iNOS induced by FAP.291I9 increased, which considered the main cause of the failure of the analogue epitope-based vaccine. Thus, we demonstrated for the first time that the FAP.291 minigene vaccine could induce mouse CTLs and also function as a tumor regression antigen, providing the basis for future studies of FAPα epitope-based vaccines. This study may also be valuable for further improvement of the immunogenicity of analogue epitope vaccines.

[Effect and mechanism of thymosin beta 4 on spinal cord-derived neural stem /progenitor cell injury induced by oxidative stress].

OBJECTIVE: To investigate the role and mechanism of thymosin beta 4 (Tß4) in oxidative stress injury of spinal cord-derived neural stem/progenitor cells (NSPCs) induced by hydrogen peroxide (H2O2). METHODS: NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), H2O2 group (NSPCs cells damaged by 500 µM H2O2), Tß4 -3 groups (NSPCs were treated with 1, 2.5, 5 µg/ml Tß4 on the basis of H2O2 treatment) and TAK-242 group [NSPCs were treated with 5 µg/ml Tß4 and Toll-like receptor 4(TLR4) inhibitor TAK-242 on the basis of H2O2 treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with H2O2 and Tß4. The expression of Tß4, TLR4, MyD88 were detected by qRT-PCR and Western blot. Cell viability was detected by MTT assay and lactate dehydrogenase(LDH) assay kit. Ca2+ concentration was detected by Fluo-3/AM probe method. The apoptosis of NSPCs was detected by flow cytometry and Caspase-3 and Caspase-9 kits;reactive oxygen species (ROS), superoxi dedismu-tase dismutase(SOD) activity and glutathione (GSH) content were detected by corresponding kits. Interleukin(IL)-6 and IL-1ß were detected by enzyme-linked immunosorbent assay. RESULTS: The expression of Tß4 was decreased in H2O2 injured NSPCs(P<0.05). Compared with H2O2 group, the cell viability and Ca2+ concentration was significantly increased, release of LDH and apoptosis were significantly decreased, production of ROS and pro-inflammatory cytokines were significantly decreased, and the expression levels of TLR4 and MyD88 protein were significantly decreased in Tß4-3 groups and TAK-242 group (P<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (P<0.05), while after treatment with Tß4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (P<0.05). CONCLUSION: Tß4 attenuates H2O2-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways.

Efficacy of Single-Position Oblique Lateral Interbody Fusion Combined With Percutaneous Pedicle Screw Fixation in Treating Degenerative Lumbar Spondylolisthesis: A Cohort Study.

Objective: To investigate the surgical outcomes of single-position oblique lateral interbody fusion (OLIF) combined with percutaneous pedicle screw fixation (PPSF) in treating degenerative lumbar spondylolisthesis (DLS). Methods: We retrospectively analyzed 85 patients with DLS who met the inclusion criteria from April 2018 to December 2020. According to the need to change their position during the operation, the patients were divided into a single-position OLIF group (27 patients) and a conventional OLIF group (58 patients). The operation time, intraoperative blood loss, hospitalization days, instrumentation accuracy and complication rates were compared between the two groups. The visual analog scale (VAS) and Oswestry Disability Index (ODI) were used to evaluate the clinical efficacy. The surgical segment's intervertebral space height (IDH) and lumbar lordosis (LL) angle were used to evaluate the imaging effect. Results: The hospital stay, pedicle screws placement accuracy, and complication incidence were similar between the two groups (P > 0.05). The operation time and intraoperative blood loss in the single-position OLIF group were less than those in the conventional OLIF group (P < 0.05). The postoperative VAS, ODI, IDH and LL values were significantly improved (P < 0.05), but there was no significant difference between the two groups (P > 0.05). Conclusions: Compared with conventional OLIF, single-position OLIF combined with PPSF is also safe and effective, and it has the advantages of a shorter operation time and less intraoperative blood loss.

Long non­coding RNA PART1: dual role in cancer.

Increasing evidence has shown that long non-coding RNAs (lncRNAs), which are non-coding endogenous single-stranded RNAs, play an essential role in various physiological and pathological processes through transcriptional interference, post-transcriptional regulation, and epigenetic modification. Moreover, lncRNAs, as oncogenes or tumor suppressor genes, play an important role in the occurrence and development of human cancers. Prostate androgen-regulated transcript 1 (PART1) was initially identified as a carcinogenic lncRNA in prostate adenomas. The upregulated expression of PART1 plays a tumor-promoting role in liver, prostate, lung cancers, and other tumors. In contrast, the expression of PART1 is downregulated in esophageal squamous cell carcinoma, glioma, and other tumors, which may inhibit the tumor. PART1 plays a dual role in cancer and regulates cell proliferation, apoptosis, invasion, and metastasis through a variety of potential mechanisms. These findings suggest that PART1 is a promising tumor biomarker and therapeutic target. This article reviews the biological functions, related mechanisms, and potential clinical significance of PART1 in a variety of human cancers.

Mechanisms and functions of long noncoding RNAs in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a key pathological process underlying low back pain. Although, to date, specific molecular mechanisms have not been elucidated, at the cellular level, it is mainly due to pathological changes in the life process of nucleus pulposus (NP) cells in the intervertebral disc (IVD). These changes are closely related to cell proliferation, apoptosis, senescence, autophagy, inflammation, and extracellular matrix (ECM) remodeling. Long noncoding RNAs (lncRNAs) have gradually become a focus of scientific research because of their functional complexity and local tissue specific expression. Moreover, they mediate a series of cellular signaling pathways in NP cells by competing for microRNA (miRNA) or directly targeting gene expression by mRNA adsorption, thereby regulating cell life activities that play a vital role in the mechanism underlying IDD. In-depth studies on lncRNAs can help identify new therapeutic targets or aid in developing IDD treatment strategies at the gene level and those based on regenerative medicine, thus providing new ideas for researchers. This article reviews the classification, biological functions, mechanisms of action, and therapeutic potential of lncRNAs in IDD.

Ski Regulates the Inflammatory Response of Reactive Astrocytes Induced by Oxygen Glucose Deprivation/Reoxygenation (OGD/R) Through the NF-κB Pathway.

Spinal cord injury (SCI) is a common disease of the nervous system, including primary and secondary injuries. Neuronal inflammation after SCI is the most important pathological process of SCI and a chemical barrier to nerve function recovery after injury. Ski, an evolutionarily conserved functional transcriptional regulator protein, is upregulated in reactive astrocytes after SCI and regulates the biological characteristics of astrocytes. However, its role in the glial inflammatory response triggered by reactive astrocytes after spinal cord ischemia and its exact mechanism remains unclear. This study investigated the role and mechanism of Ski in the inflammatory response triggered by reactive astrocytes induced by oxygen and sugar deprivation/reoxygenation (OGD/R) model in vitro. In the ODG/R model, Ski expression was upregulated. In contrast, Ski upregulation was accompanied by increased levels of iNOS, IL-1ß, IL-6, TNF-α, and other inflammation-related factors. These results indicated that the inflammatory response triggered by astrocytes was significantly enhanced in OGD/R-stimulated astrocytes. Astrocytes were transfected with Ski specific siRNA to knock out Ski and subsequently attenuate OGD-induced astrocyte-triggered inflammation. Our results also suggest that Ski downregulation downregulates the expression of iNOS, IL-1ß, IL-6, and TNF-α in OGD/R-induced reactive astrocytes by inhibiting the activity of the NF-κB signaling pathway. In conclusion, downregulation of Ski can effectively inhibit glial inflammation in SCI by inhibiting the activity of the NF-κB pathway. These findings suggest that Ski is a promising therapeutic target for inflammatory responses after SCI.In conclusion, Ski downregulation can effectively inhibit glial inflammation in SCI by inhibiting the activity of the NF-κB pathway. These findings suggest that Ski might serve as a promising target for the treatment of inflammatory responses after SCI.

SKI knockdown suppresses apoptosis and extracellular matrix degradation of nucleus pulposus cells via inhibition of the Wnt/ß-catenin pathway and ameliorates disc degeneration.

This study aimed to determine the effects of SKI on interleukin (IL)-1ß-induced apoptosis of nucleus pulposus (NP) cells, intervertebral disc degeneration (IDD), and the Wnt signaling pathway. NP tissue specimens of different Pfirrmann grades (II-V) were collected from patients with different grades of IDD. Real-time polymerase chain reaction and western blotting were used to compare SKI mRNA and protein expression in NP tissues from patients. Using the IL-1ß-induced IDD model, NP cells were infected with lentivirus-coated si-SKI to downregulate the expression of SKI and treated with LiCl to evaluate the involvement of the Wnt/ß-catenin signaling pathway. Western blotting, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect NP cell apoptosis, extracellular matrix (ECM) metabolism, and related protein expression changes in the Wnt/ß-catenin signaling pathway. To investigate the role of SKI in vivo, a rat IDD model was established by needle puncture of the intervertebral disc. Rats were injected with lentivirus-coated si-SKI and evaluated by magnetic resonance imaging (MRI), and hematoxylin and eosin (HE) and safranin O staining. SKI expression positively correlated with the severity of human IDD. In the IL-1ß-induced NP cell degeneration model, SKI expression increased significantly and reached a peak at 24 h. SKI knockdown protected against IL-1ß-induced NP cell apoptosis and ECM degradation. LiCl treatment reversed the protective effects of si-SKI on NP cells. Furthermore, lentivirus-coated si-SKI injection partially reversed the NP tissue damage in the IDD model in vivo. SKI knockdown reduced NP cell apoptosis and ECM degradation by inhibiting the Wnt/ß-catenin signaling pathway, ultimately protecting against IDD. Therefore, SKI may be an effective target for IDD treatment.

Ski regulates proliferation and migration of reactive astrocytes induced by lipopolysaccharide (LPS) through PI3K/Akt pathway.

Spinal cord injury (SCI) is a leading cause of disability and death worldwide. Reactive astrogliosis, a typical feature of SCI, undergoes various molecular and morphological changes and contributes to glial scar formation, which impedes axonal regeneration. Ski is a novel molecule that regulates the biological characteristics of astrocytes after spinal cord injury, but its function and the exact mechanism of its overexpression in reactive astrocyte proliferation and migration after SCI remain unclear. The purpose of this study was to elucidate the effect and mechanism of Ski on the proliferation and migration of reactive astrocytes, and to regulate the spatiotemporal formation of glial scars after SCI. In an in vitro lipopolysaccharide (LPS)-induced astrocyte injury model, the expression of Ski was upregulated in a time-dependent manner in LPS-induced astrocytes, and the upregulation of Ski was accompanied by that of PCNA, CDK4, CyclinD1, and other proliferation-related proteins. Our findings suggest that Ski promotes the proliferation and migration of reactive astrocytes. Next, astrocytes were transfected with a specific lentivirus to cause the overexpression of Ski, which significantly enhanced the proliferation and migration of reactive astrocytes and LPS-induced activation of the PI3K/Akt pathway. The PI3K/Akt pathway inhibitor LY294002 significantly inhibited the proliferation and migration of LPS-induced reactive astrocytes after Ski overexpression. In conclusion, Ski regulates LPS-induced astrocyte proliferation and migration through the PI3K/Akt pathway, making Ski a promising target for strategies to combat glial scarring after SCI.

The role of Wnt/mTOR signaling in spinal cord injury.

Spinal cord injury (SCI) is the most common disabling spinal injury, a complex pathologic process that can eventually lead to severe neurological dysfunction. The Wnt/mTOR signaling pathway is a pervasive signaling cascade that regulates a wide range of physiological processes during embryonic development, from stem cell pluripotency to cell fate. Numerous studies have reported that Wnt/mTOR signaling pathway plays an important role in neural development, synaptogenesis, neuron growth, differentiation and survival after the central nervous system (CNS) is damaged. Wnt/mTOR also plays an important role in regulating various pathophysiological processes after spinal cord injury (SCI). After SCI, Wnt/mTOR signal regulates the physiological and pathological processes of neural stem cell proliferation and differentiation, neuronal axon regeneration, neuroinflammation and pain through multiple pathways. Due to the characteristics of the Wnt signal in SCI make it a potential therapeutic target of SCI. In this paper, the characteristics of Wnt/mTOR signal, the role of Wnt/mTOR pathway on SCI and related mechanisms are reviewed, and some unsolved problems are discussed. It is hoped to provide reference value for the research field of the role of Wnt/mTOR pathway in SCI, and provide a theoretical basis for biological therapy of SCI.

Effects of chrysophanol on hippocampal damage and mitochondrial autophagy in mice with cerebral ischemia reperfusion.

OBJECTIVE: The cerebral ischemia-reperfusion (I/R) model is crucial for the study of cerebral stroke. Chrysophanol (Chry) can protect nerve damage of mice in cerebral ischemia-reperfusion injury. This study aimed at investigating the neuroprotective effects of chrysophanol through mitochondrial autophagy in mice with ischemia-reperfusion injury. MATERIALS AND METHODS: Adult mice were stochastically divided into five groups: sham, I/R (solvent), I/R+Chry (dose, 10.0ml/kg), I/R+Chry (dose, 1.0ml/kg), and I/R+Chry (dose, 0.1ml/kg). The cerebral ischemia-reperfusion model was made in I/R and I/R+Chry groups. The changes in hippocampal formation were observed by hematoxylin and eosin (H&E) staining. The expressions of LC3B-II and LC3B-I protein in hippocampus were demonstrated by western blot (WB). The fluorescence intensities of NIX, LC3B, and mitochondria were detected by immunohistochemistry fluorescent (IF). RESULTS: Comparing with the I/R group, the I/R+Chry groups showed improvements in reducing the damage on the hippocampus, indicated by the reduced ratio of LC3B-II and LC3B-I protein, decreased fluorescence intensity of NIX and LC3B, and increased intensity of mitochondrial fluorescence. CONCLUSION: Our study showed that chrysophanol may regulate mitochondrial autophagy through NIX protein and alleviate the damage of hippocampus through decreasing the level of mitochondrial autophagy.

The Role of Sphingomyelin Metabolism in the Protection of Rat Brain Microvascular Endothelial Cells by Mild Hypothermia.

BACKGROUND: Sphingomyelin, composed of ceramide (CER), sphingosine (Sph), and sphingosine-1-phosphate (S1P), is an essential structural component of cellular membranes and plays an important role in the signal transduction regulating cell proliferation, differentiation, and apoptosis. CER is mainly metabolized to Sph, and under the action of sphingosine kinases (SphKs), Sph produces S1P, which can be converted back to Sph by S1P phosphatase. It is suggested that the fate of cells is controlled partly by the interconversion of CER and intracellular S1P. SphK2 is considered the main kinase of S1P synthesis in the central nervous system. The objective of this study was to explore the hypothesis that SphK2 and sphingomyelin metabolism participated in the process of cell apoptosis and the protection of mild hypothermia. METHODS: Rat brain microvascular endothelial cells were divided into groups for intervention of SphK2 inhibitor, SphK2 small interfering RNA (SiRNA) transfection, ischemia-reperfusion, and mild hypothermia. After interventions, cell apoptosis was detected by 4,6-diamino-2-phenyl indole (DAPI) and flow cytometry, the expression of apoptosis-related protein was detected by Western Blot, and SphK2 enzyme activity and the content of sphingomyelin were determined. RESULTS: ABC294640 and transfection of SphK2 SiRNA could increase apoptosis, accompanied by the increase of the expression of proapoptotic genes Caspase3 and Bax and the decrease of the expression of BCL-2. This effect could be partially reversed with mild hypothermia. Ischemia-reperfusion injury, transfection of SphK2 SiRNA, and the addition of ABC294640 could significantly inhibit the activity of SphK2, accompanied by the increase of CERs and the decrease of S1P. Mild hypothermia could reverse the changes of sphingolipids but have no significant effect on the activity of sphk2. CONCLUSIONS: Mild hypothermia can inhibit the occurrence of apoptosis and reverse the changes of apoptosis-related genes and sphingomyelin content induced by ischemia-reperfusion injury, but the effect on sphk2 enzyme activity was not significant.