Zinc remodels mitochondrial network through SIRT3/Mfn2-dependent mitochondrial transfer in ameliorating spinal cord injury.

Spinal cord injury (SCI) is a traumatic neuropathic condition that results in motor, sensory and autonomic dysfunction. Mitochondrial dysfunction caused by primary trauma is one of the critical pathogenic mechanisms. Moderate levels of zinc have antioxidant effects, promote neurogenesis and immune responses. Zinc normalises mitochondrial morphology in neurons after SCI. However, how zinc protects mitochondria within neurons is unknown. In the study, we used transwell culture, Western blot, Quantitative Real-time Polymerase Chain Reaction (QRT-PCR), ATP content detection, reactive oxygen species (ROS) activity assay, flow cytometry and immunostaining to investigate the relationship between zinc-treated microglia and injured neurons through animal and cell experiments. We found that zinc promotes mitochondrial transfer from microglia to neurons after SCI through Sirtuin 3 (SIRT3) regulation of Mitofusin 2 protein (Mfn2). It can rescue mitochondria in damaged neurons and inhibit oxidative stress, increase ATP levels and promote neuronal survival. Therefore, it can improve the recovery of motor function in SCI mice. In conclusion, our work reveals a potential mechanism to describe the communication between microglia and neurons after SCI, which may provide a new idea for future therapeutic approaches to SCI.

Naringin may promote functional recovery following spinal cord injury by modulating microglial polarization through the PPAR-γ/NF-κB signaling pathway.

OBJECTIVE: The flavonoid Naringin (Nar) has been extensively investigated and found to have multiple pharmacological properties, including neuroprotection. Although recent reports have shown that Nar can effectively treat spinal cord injury (SCI), its potential mechanism remains unknown. This study aimed to investigate the effects of Nar on motor recovery and inflammatory responses after SCI and to elucidate its mechanism. METHODS: SCI rat models were established using Allen's weight-drop method. The rats were intragastrically given Nar (40 mg/kg) for 21 d, and their motor function before surgery and on the 1st, 3rd, 7th, 14th, 21st days after surgery was assessed by the Basso-Beattie-Bresnahan (BBB) scale and examined by the grid walking test (GWT). The enzyme linked immunosorbent assay (ELISA) was used to detect the interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 levels in rat spinal cord tissues, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure the mRNA expression levels of microglial activation markers CD68 and ionized calcium binding adaptor molecule 1 (Iba-1), M1 markers inducible nitric oxide synthase (iNOS) and IL-6, and M2 markers CD206 and Arginase 1 (Arg1). The expression levels of peroxisome proliferator-activated receptor gamma/nuclear factor kappa B (PPAR-γ/NF-κB) pathway-related proteins in rat spinal cord tissues were determined using western blotting. RESULTS: Nar significantly increased the BBB score and decreased the mean error rate of GWT in SCI rats. Additionally, Nar effectively inhibited microglial activation and expression of M1 markers in spinal cord tissues. It also elevated M2 polarization-related gene expression and significantly lowered the levels of inflammatory factors. Further investigation showed that Nar enhanced the expression of PPAR-γ protein and inhibited NF-κB pathway activity. CONCLUSION: Nar promotes functional recovery by regulating microglial polarization and inhibiting the inflammatory response in SCI, and its mechanism may be related to the PPAR-γ/NF-κB signaling pathway activity.

Ginsenoside Rb1 improves energy metabolism after spinal cord injury.

Mitochondrial damage caused by oxidative stress and energy deficiency induced by focal ischemia and hypoxia are important factors that aggravate diseases. Studies have shown that ginsenoside Rb1 has neurotrophic and neuroprotective effects. However, whether it influences energy metabolism after spinal cord injury remains unclear. In this study, we treated mouse and cell models of spinal cord injury with ginsenoside Rb1. We found that ginsenoside Rb1 remarkably inhibited neuronal oxidative stress, protected mitochondria, promoted neuronal metabolic reprogramming, increased glycolytic activity and ATP production, and promoted the survival of motor neurons in the anterior horn and the recovery of motor function in the hind limb. Because sirtuin 3 regulates glycolysis and oxidative stress, mouse and cell models of spinal cord injury were treated with the sirtuin 3 inhibitor 3-TYP. When Sirt3 expression was suppressed, we found that the therapeutic effects of ginsenoside Rb1 on spinal cord injury were remarkably inhibited. Therefore, ginsenoside Rb1 is considered a potential drug for the treatment of spinal cord injury, and its therapeutic effects are closely related to sirtuin 3.

Curcumin Conjugated Gold Nanoclusters as Perspective Therapeutics for Diabetic Cardiomyopathy.

Accumulation of lipids in the myocardium contributes to the development of cardiac dysfunctions and various chronic diseases, such as diabetic cardiomyopathy (DCM). Curcumin (Cur) can relieve lipid accumulation problems, but its efficiency is limited by poor water solubility and biocompatibility. Herein, gold nanoclusters (AuNCs) were used to improve the efficiency of Cur, and the conjugates Curcumin-AuNCs (AuCur) were developed. In the treatment of high-fat-induced myocardial cell damage, we found that AuCur could effectively reduce intracellular lipid accumulation, the increase of reactive oxygen species (ROS), the increase of mitochondrial division, and the increase of apoptosis compared with Cur. AuCur decreased the expression of the peroxisome proliferator-activated receptors-α subtype (PPARα), and the therapeutic effect of AuCur was canceled when the expression of PPARα was enhanced. For the above reasons, AuCur treated the toxic effect of high lipid on cardiomyocytes by regulating PPARα, providing a new idea and method for the treatment of DCM.

Zinc attenuates ferroptosis and promotes functional recovery in contusion spinal cord injury by activating Nrf2/GPX4 defense pathway.

AIM: Spinal cord injury (SCI) involves multiple pathological processes. Ferroptosis has been shown to play a critical role in the injury process. We wanted to explore whether zinc can inhibit ferroptosis, reduce inflammation, and then exert a neuroprotective effect. METHODS: The Alice method was used to establish a spinal cord injury model. The Basso Mouse Scale (BMS), Nissl staining, hematoxylin-eosin staining, and immunofluorescence analysis were used to investigate the protective effect of zinc on neurons on spinal cord neurons and the recovery of motor function. The regulation of the nuclear factor E2/heme oxygenase-1 (NRF2/HO-1) pathway was assessed, the levels of essential ferroptosis proteins were measured, and the changes in mitochondria were confirmed by transmission electron microscopy and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. In vitro experiments using VSC4.1 (spinal cord anterior horn motor neuroma cell line), 4-hydroxynonenal (4HNE), reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), lipid peroxides, and finally the levels of inflammatory factors were detected to assess the effect of zinc. RESULTS: Zinc reversed behavioral and structural changes after SCI. Zinc increased the expression of NRF2/HO-1, thereby increasing the content of glutathione peroxidase 4 (GPX4), SOD, and GHS and reducing the levels of lipid peroxides, MDA, and ROS. Zinc also rescued injured mitochondria and effectively reduced spinal cord injury and the levels of inflammatory factors, and the NRF2 inhibitor Brusatol reversed the effects of zinc. CONCLUSION: Zinc promoted the degradation of oxidative stress products and lipid peroxides through the NRF2/HO-1 and GPX4 signaling pathways to inhibit ferroptosis in neurons.

Berberine-loaded M2 macrophage-derived exosomes for spinal cord injury therapy.

Spinal cord injury (SCI) causes immune activation of resident macrophages/microglia. Activated macrophages/microglia have two different phenotypes, the pro-inflammatory classically activated (M1) phenotype and the anti-inflammatory alternatively activated (M2) phenotype. M1 phenotype macrophages/microglia are the key factor in inflammation. The treatment of SCI remains a huge challenge due to the nontargeting and inefficiency of anti-inflammatory drugs through the blood-brain barrier (BBB). The purpose of this experiment was to design M2-type primary peritoneal macrophages exosomes (Exos) as a drug carrier for berberine (Ber), which can be efficiently targeted to deliver drugs to the injured spinal cord due to the natural advantage of Exos across the BBB. The Exos with particle size of 125±12 nm were loaded with by an ultrasonic method and the drug loading reached 17.13 ±1.64%. The Ber release experiment showed that the loaded sample (Exos-Ber) exhibited sustained release effect, and the cumulative release amount reached 71.44±2.86% within 48 h. In vitro and in vivo experiments confirmed that the Exos-Ber could decrease the M1 protein marker iNOS, elevate the M2 protein marker CD206 and reduce inflammatory and apoptotic cytokines (TNF-α, IL-1ß, IL-6, Caspase 9, Caspase 8), which showed that Exos-Ber had a good anti-inflammatory and anti-apoptotic effect by inducing macrophages/microglia from the M1 phenotype to M2 phenotype polarization. Moreover, the motor function of SCI mice was significantly improved after Exos-Ber treatment, indicating that Exos-Ber is a potential agent for SCI therapy. STATEMENT OF SIGNIFICANCE: Efficient targeting strategy for drug delivery. In addition to good biocompatibility and stealth ability, M2 macrophage-derived Exosomes present natural inflammatory targeting ability. The inflammatory microenvironment after spinal cord injury provides motivation for the targeting of exosomes. Natural drug carrier with higher safety. With the rapid development of nanomaterials, drug carriers have become more selective. However, due to the special microenvironment after central nervous system damage, some non-degradable inorganic materials will increase the pressure of self-healing and even secondary damage to neurons, which has been solved by the emergence of exosomes. Some previous studies used tumor cell line exosomes as drug carriers, but the carcinogenic factors carried by themselves have extremely high hidden dangers, and endogenous macrophage exosomes have absolute advantages over their safety.

Bioengineered Zinc Oxide Nanoparticle-Loaded Hydrogel for Combinative Treatment of Spinal Cord Transection.

Spinal cord injury (SCI) is one of the most destructive diseases. The neuroinflammation microenvironment needs comprehensive mitigation of damages. Thus, regulation of local, microenvironment drugs could be a potential effective treatment. However, clinical studies on SCI with common treatment have reported it to cause systemic toxicity and side effects. Zinc oxide nanoparticles (ZnONPs) have been widely reported to have satisfying anti-inflammation function. Furthermore, green synthesis procedures can improve the capability and possible utilization of ZnONPs. However, the efficient administration and underlying mechanism of ZnONPs in SCI treatment remain unclear. Herein, an innovative approach was built by utilizing ZnONPs loaded in a skeletal muscle-derived adhesive hydrogel (ZnONPs-Gel). Different from the systemic application of ZnONPs, the local administration of ZnONPs-Gel offered the ZnONPs-loaded extracellular matrix with beneficial biocompatibility to the injured spinal cord, thereby promoting effective function recovery. Mechanistically, the ZnONPs-Gel treatment not only markedly reduced ROS production but also decreased apoptosis in the injured spinal cord. Therefore, the strategy based on local administration of the ZnONPs-Gel in the early stage of SCI may be an effective therapeutic treatment.

Zinc provides neuroprotection by regulating NLRP3 inflammasome through autophagy and ubiquitination in a spinal contusion injury model.

AIM: Spinal cord injury (SCI) is a serious disabling injury worldwide, and the excessive inflammatory response it causes plays an important role in secondary injury. Regulating the inflammatory response can be a potential therapeutic strategy for improving the prognosis of SCI. Zinc has been demonstrated to have a neuroprotective effect in experimental spinal cord injury models. In this study, we aimed to explore the neuroprotective effect of zinc through the suppression of the NLRP3 inflammasome. METHOD: Allen's method was used to establish an SCI model in C57BL/6J mice. The Basso Mouse Scale (BMS), Nissl staining were employed to confirm the protective effect of zinc on neuronal survival and functional recovery in vivo. Western blotting (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression levels of NLRP3 inflammasome and autophagy-related proteins. Transmission electron microscopy (TEM) was used to confirm the occurrence of zinc-induced autophagy. In vitro, lipopolysaccharide (LPS) and ATP polarized BV2 cells to a proinflammatory phenotype. 3-Methyladenine (3-MA) and bafilomycin A1 (BafA1) were chosen to explore the relationship between the NLRP3 inflammasome and autophagy. A coimmunoprecipitation assay was used to detect the ubiquitination of the NLRP3 protein. RESULTS: Our data showed that zinc significantly promoted motor function recovery after SCI. In vivo, zinc treatment inhibited the protein expression level of NLRP3 while increasing the level of autophagy. These effects were fully validated by the polarization of BV2 cells to a proinflammatory phenotype. The results showed that when 3-MA and BafA1 were applied, the promotion of autophagy by zinc was blocked and that the inhibitory effect of zinc on NLRP3 was reversed. Furthermore, co-IP confirmed that the promotion of autophagy by zinc also activated the protein expression of ubiquitin and suppressed high levels of NLRP3. CONCLUSION: Zinc provides neuroprotection by regulating NLRP3 inflammasome through autophagy and ubiquitination after SCI.

Buyang Huanwu decoction up-regulates Notch1 gene expression in injured spinal cord.

Expression of genes in the Notch signaling pathway is altered in the injured spinal cord, which indicates that Notch participates in repair after spinal cord injury. Buyang Huanwu decoction, a traditional Chinese herbal preparation, can promote the growth of nerve cells and nerve fibers; however, it is unclear whether Buyang Huanwu decoction affects the Notch signaling pathway in injured spinal cord. In this study, a rat model was established by injuring the T10 spinal cord. At 2 days after injury, rats were intragastrically administered 2 mL of 0.8 g/mL Buyang Huanwu decoction daily until sacrifice. Real-time reverse transcription polymerase chain reaction analysis demonstrated that at 7, 14 and 28 days after injury, the expression of Notch1 was increased in the Buyang Huanwu decoction group compared with controls. These findings confirm that Buyang Huanwu decoction can promote the expression of Notch1 in rats with incomplete spinal cord injury, and may indicate a mechanism to promote the repair of spinal cord injury.

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/ß-catenin signaling pathway.

The Wnt/ß-catenin signaling pathway plays a crucial role in neural development, axonal guidance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/ß-catenin signaling pathway after spinal cord injury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of ß-catenin protein, caspase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/ß-catenin signaling pathway. Rapamycin increased the levels of ß-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental findings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/ß-catenin signaling pathway after spinal cord injury.

Combining Bone Marrow Stromal Cells with Green Tea Polyphenols Attenuates the Blood-Spinal Cord Barrier Permeability in Rats with Compression Spinal Cord Injury.

This study was performed to investigate the effect of bone marrow stromal cells (BMSCs) combined with green tea polyphenols (GTPs) on the blood-spinal cord barrier (BSCB) permeability after spinal cord injury (SCI) in the rat model. In the model of SCI rats, we found that the water content and the BSCB permeability were decreased by BMSCs and GTPs treatment, and their combination had a synergistic effect. Further, the motor function of rats was also greatly improved by BMSCs and GTPs administration. After treated by the combination of BMSCs and GTPs, SCI rats showed the up-regulated expression of tight junction (TJ) associated proteins claudin-5, occludin and ZO-1 by Western blot, which was more remarkable than that in the single treatment. The increased expression levels of claudin-5, occludin, and ZO-1 were the most obvious in the spinal cord microvessels using immunohistochemistry assay. This led to the conclusion that the combination of BMSCs and GTPs could decrease the BSCB permeability by up-regulating protein expression levels of claudin-5, occludin, and ZO-1. In addition, after BMSCs and GTPs administration, the results of Western blot and enzyme-linked immunosorbent assay (ELISA) revealed a significant decrease in protein expression level and the activation of nuclear factor-кB (NF-кB) p65. Our results indicated that combination of BMSCs and GTPs could improve motor function after SCI, which might be correlated with improvements in BSCB integrity, and that NF-кB might be involved in the modulating process.

Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats.

Previous studies have shown that curcumin (Cur) can produce potent neuroprotective effects against damage due to spinal cord injury (SCI). However, whether Cur can preserve the function of the blood-spinal cord barrier (BSCB) is unclear. The present study was performed to investigate the mechanism underlying BSCB permeability changes, which were induced by treatment with Cur (75, 150, and 300 mg/kg, i.p.) after compressive SCI in rats. BSCB permeability was evaluated by Evans blue leakage. Motor recovery of rats with SCI was assessed using the Basso, Beattie, and Bresnahan scoring system every day until the 21st days post-injury. The protein levels of heme oxygenase-1 (HO-1), tight junction protein, and inflammatory factors were analyzed by western blots. The expression of the inflammatory factors tumor necrosis factor-α (TNF-α) and nuclear factor-kappaB (NF-κB) mRNA was determined with reverse transcription-polymerase chain reactions. Treatment with Cur (150 and 300 mg/kg) significantly reduced Evans blue leakage into the spinal cord tissue at 24h after SCI. Cur (150 mg/kg) significantly increased HO-1 protein expression. The levels of TNF-α and NF-κB mRNA and protein greatly increased at 24h after SCI, and this increase was significantly attenuated by Cur treatment. ZO-1 and occludin expression was upregulated by Cur (150 mg/kg) treatment after SCI, and this effect was blocked by the HO-1 inhibitor zinc protoporphyrin. Long-term effects of Cur on motor recovery after SCI were observed. Our results indicated that Cur can improve motor function after SCI, which could correlate with improvements in BSCB integrity.

The protective effect of salvianolic acid B on blood-spinal cord barrier after compression spinal cord injury in rats.

Salvianolic acid B (Sal B), a bioactive compound isolated from the Chinese medicinal herb danshen, is commonly used for the prevention and treatment of cardiovascular disease. The present study was performed to investigate the effect of Sal B on the blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) in a rat model. Sal B (1, 10, and 50 mg/kg i.v.) was administered to rats immediately following SCI. The permeability of the BSCB and spinal cord tissue water content were evaluated. Additionally, the expression levels of tight junction proteins and heme oxygenase-1 (HO-1) were monitored by Western blot analysis. Enzyme-linked immunosorbent assay analysis of spinal cord tissue homogenates was performed 24 h post-SCI to evaluate the expression of inflammation-related cytokines. In addition, the motor recovery of SCI rats was assessed using the Basso, Beattie, and Bresnahan scoring system. Compared to the SCI group, rats treated with Sal B (10, 50 mg/kg) exhibited significantly reduced spinal cord tissue water content and BSCB permeability. Further, the motor function of rats was also greatly improved by Sal B administration. The expression of pro-inflammatory factors TNF-α and NF-κB was found to be greatly increased 24 h post-SCI, and this upregulation was significantly attenuated by Sal B treatment. The expression of ZO-1 and occludin was upregulated by Sal B (10 mg/kg) treatment after SCI, and this effect was blocked by the HO-1 inhibitor ZnPP. Taken together, our results clearly indicate that Sal B attenuates SCI by promoting the repair of the damaged BSCB, demonstrating that this molecule is a novel and promising therapeutic agent for human SCI.