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.

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.