A novel microglia-targeting strategy based on nanoparticle-mediated delivery of miR-26a-5p for long-lasting analgesia in chronic pain.

Accumulating evidence supports the notion that microglia play versatile roles in different chronic pain conditions. However, therapeutic strategies of chronic pain by targeting microglia remain largely overlooked. This study seeks to develop a miRNA-loaded nano-delivery system by targeting microglia, which could provide a decent and long-lasting analgesia for chronic pain. Surface aminated mesoporous silica nanoparticles were adopted to load miR-26a-5p, a potent analgesic miRNA, by electrostatic adsorption, which can avoid miR-26a-5p is rapidly released and degraded. Then, targeting peptide MG1 was modified on the surface of aminated mesoporous silica particles for microglia targeting. In peripheral nerve injury induced neuropathic pain model, a satisfactory anti-allodynia effect with about 6 weeks pain-relief duration were achieved through targeting microglia strategy, which decreased microglia activation and inflammation by Wnt5a, a non-canonical Wnt pathway. In inflammatory pain and chemotherapy induced peripheral neuropathic pain, microglia targeting strategy also exhibited more efficient analgesia and longer pain-relief duration than others. Overall, we developed a microglia-targeting nano-delivery system, which facilitates precisely miR-26a-5p delivery to enhance analgesic effect and duration for several chronic pain conditions.

Epigenetic combined with transcriptomic analysis of the m6A methylome after spared nerve injury-induced neuropathic pain in mice.

Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.

Celastrol-encapsulated microspheres prepared by microfluidic electrospray for alleviating inflammatory pain.

Inflammatory pain is induced by trauma, infection, chemical stimulation, etc. It causes severe physical and psychological agony to patients. Celastrol has powerful anti-inflammatory property and has achieved good results in various inflammation-related diseases. However, the low water solubility and multi-system toxicity limit its clinical application. Herein, we proposed alginate microspheres with core-shell structure which encapsulated celastrol by microfluidic electrospray to effectively overcome the shortcomings and improve the therapeutic effect. The microspheres had uniform size and good biocompatibility, and could release the loaded drugs in the gut. The behavioral tests showed that the celastrol-loaded microspheres effectively alleviated inflammatory pain, and the hematoxylin and eosin staining (HE staining), immunofluorescence and detection of inflammatory cytokines showed the anti-inflammatory effect. These results indicated that the microspheres could reduce dose and toxicity without affecting efficacy, and facilitate the application of celastrol in different clinical situations.

miR-26a-5p alleviates CFA-induced chronic inflammatory hyperalgesia through Wnt5a/CaMKII/NFAT signaling in mice.

BACKGROUND: Inflammation often leads to the occurrence of chronic pain, and many miRNAs have been shown to play a key role in the development of inflammatory pain. However, whether miR-26a-5p relieves pain induced by inflammation and its possible mechanism are still unclear. METHODS: The complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model was employed. Intrathecal or subcutaneous injection of miR-26a-5p agomir was performed after modeling to study its antinociceptive effect and the comparison of different administration methods. Bioinformatics analysis of miRNAs was performed to study the downstream mechanisms of miR-26a-5p. HE staining, RT-qPCR, Western blotting, and immunofluorescence were used for further validation. RESULTS: A single intrathecal and subcutaneous injection of miR-26a-5p both reversed mechanical hypersensitivity and thermal latency in the left hind paw of mice with CFA-induced inflammatory pain. HE staining and immunofluorescence studies found that both administrations of miR-26a-5p alleviated inflammation in the periphery and spinal cord. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. Wnt5a was mainly expressed in neurons and microglia in the spinal cord of mice with inflammatory pain. Intrathecal injection of miR-26a-5p could significantly reduce the expression level of Wnt5a and inhibit the downstream molecules of noncanonical Wnt signaling Camk2/NFAT, inhibiting the release of spinal cord inflammatory factors and alleviating the activation of microglia. In addition, miR-26a-5p could also inhibit lipopolysaccharide (LPS)-stimulated BV2 cell inflammation in vitro through a noncanonical Wnt signaling pathway. CONCLUSIONS: miR-26a-5p is a promising therapy for CFA-induced inflammatory pain. Both intrathecal and subcutaneous injections provide relief for inflammatory pain. miR-26a-5p regulated noncanonical Wnt signaling to be involved in analgesia partly through antineuroinflammation, suggesting a pain-alleviating effect via noncanonical Wnt signaling pathway in the CFA-induced inflammatory pain model in vivo.

Human PMSCs-derived small extracellular vesicles alleviate neuropathic pain through miR-26a-5p/Wnt5a in SNI mice model.

BACKGROUND: Mesenchymal stem cell (MSCs)-derived small Extracellular Vesicles (sEVs) are considered as a new cell-free therapy for pain caused by nerve injury, but whether human placental mesenchymal stem cell-derived sEVs relieve pain in sciatic nerve injury and its possible mechanism are still unclear. In this study, we investigated the roles of hPMSCs-derived sEVs and related mechanisms in neuropathic pain. METHODS: The spared nerve injury (SNI) mouse model was employed. Intrathecal injection of sEVs or miR-26a-5p agomir was performed on the seventh day of modeling, to study its anti-nociceptive effect. sEVs' miRNA sequencing (miRNA-Seq) and bioinformatics analysis were performed to study the downstream mechanisms of miRNAs. RT-qPCR, protein assay and immunofluorescence were used for further validation. RESULTS: A single intrathecal injection of sEVs durably reversed mechanical hypersensitivity in the left hind paw of mice with partial sciatic nerve ligation. Immunofluorescence studies found that PKH26-labeled sEVs were visible in neurons and microglia in the dorsal horn of the ipsilateral L4/5 spinal cord and more enriched in the ipsilateral. According to miRNA-seq results, we found that intrathecal injection of miR-26a-5p agomir, the second high counts microRNA in hPMSCs derived sEVs, significantly suppressed neuropathic pain and neuroinflammation in SNI mice. Bioinformatics analysis and dual-luciferase reporter gene analysis identified Wnt5a as a direct downstream target gene of miR-26a-5p. The results showed that overexpression of miR-26a-5p in vivo could significantly reduce the expression level of Wnt5a. In addition, Foxy5, a mimetic peptide of Wnt5a, can significantly reverse the inhibitory effect of miR-26a-5p on neuroinflammation and neuropathic pain, and at the same time, miR-26a-5p can rescue the effect of Foxy5 by overexpression. CONCLUSIONS: We reported that hPMSCs derived sEVs as a promising therapy for nerve injury induced neuropathic pain. In addition, we showed that the miR-26a-5p in the sEVs regulated Wnt5a/Ryk/CaMKII/NFAT partly take part in the analgesia through anti-neuroinflammation, which suggests an alleviating pain effect through non-canonical Wnt signaling pathway in neuropathic pain model in vivo.

Effect of Celastrol on LncRNAs and mRNAs Profiles of Cerebral Ischemia-Reperfusion Injury in Transient Middle Cerebral Artery Occlusion Mice Model.

Celastrol plays a significant role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that celastrol post-treatment has a protective effect on ischemic stroke, the therapeutic effect of celastrol on ischemic stroke and the underlying molecular mechanism remain unclear. In the present study, focal transient cerebral ischemia was induced by transient middle cerebral artery occlusion (tMCAO) in mice and celastrol was administered immediately after reperfusion. We performed lncRNA and mRNA analysis in the ischemic hemisphere of adult mice with celastrol post-treatment through RNA-Sequencing (RNA-Seq). A total of 50 differentially expressed lncRNAs (DE lncRNAs) and 696 differentially expressed mRNAs (DE mRNAs) were identified between the sham and tMCAO group, and a total of 544 DE lncRNAs and 324 DE mRNAs were identified between the tMCAO and tMCAO + celastrol group. Bioinformatic analysis was done on the identified deregulated genes through gene ontology (GO) analysis, KEGG pathway analysis and network analysis. Pathway analysis indicated that inflammation-related signaling pathways played vital roles in the treatment of ischemic stroke by celastrol. Four DE lncRNAs and 5 DE mRNAs were selected for further validation by qRT-PCR in brain tissue, primary neurons, primary astrocytes, and BV2 cells. The results of qRT-PCR suggested that most of selected differentially expressed genes showed the same fold change patterns as those in RNA-Seq results. Our study suggests celastrol treatment can effectively reduce cerebral ischemia-reperfusion injury. The bioinformatics analysis of lnRNAs and mRNAs profiles in the ischemic hemisphere of adult mice provides a new perspective in the neuroprotective effects of celastrol, particularly with regards to ischemic stroke.

Experimental study on remediation of low permeability Cu-Zn contaminated clay by vacuum enhanced leaching combined with EDTA and hydrochloric acid.

To improve the washing efficiency for low permeable clay, the method of vacuum enhanced washing technology was proposed. In this study, ethylene diamine tetraacetic acid (EDTA) and hydrochloric acid (HCl) as eluents, the experiments of remediation of Cu-Zn contaminated cohesive soil by traditional soil column leaching and vacuum enhanced leaching was carried out. Results show that compared with traditional leaching method, the vacuum enhanced leaching method can significantly shorten the leaching time and prominently improve the removal rate of heavy metals Cu-Zn. When concentration of EDTA is 0.1 mol/L, the removal rate of Cu-Zn can reach 95.7% and 82.9%, respectively by vacuum-enhanced leaching. When concentration of HCl is 0.2 mol/L, the removal rate of Cu-Zn by vacuum enhanced leaching method can reach 96.2% and 90.2%, respectively. Whereas, the higher the concentration of EDTA and HCl, the slower the leaching rate. At the same concentration, the leaching rate of HCl was faster. After HCl leaching, the soil structure was seriously corroded, and the relative content of mineral composition changed considerably. After EDTA leaching, the corrosion of soil structure is light, and the relative content of mineral composition changes little. The conclusions obtained are of crucial theoretical value and technical support for environmental engineering.

Seaweed farms provide refugia from ocean acidification.

Seaweed farming has been proposed as a strategy for adaptation to ocean acidification, but evidence is largely lacking. Changes of pH and carbon system parameters in surface waters of three seaweed farms along a latitudinal range in China were compared, on the weeks preceding harvesting, with those of the surrounding seawaters. Results confirmed that seaweed farming is efficient in buffering acidification, with Saccharina japonica showing the highest capacity of 0.10 pH increase within the aquaculture area, followed by Gracilariopsis lemaneiformis (ΔpH = 0.04) and Porphyra haitanensis (ΔpH = 0.03). The ranges of pH variability within seaweed farms spanned 0.14-0.30 unit during the monitoring, showing intense fluctuations which may also help marine organisms adapt to enhanced pH temporal variations in the future ocean. Deficit in pCO2 in waters in seaweed farms relative to control waters averaged 58.7 ± 15.9 µatm, ranging from 27.3 to 113.9 µatm across farms. However, ΔpH did not significantly differ between day and night. Dissolved oxygen and Ωarag were also elevated in surface waters at all seaweed farms, which are benefit for the survival of calcifying organisms. Seaweed farming, which unlike natural seaweed forests, is scalable and is not dependent on suitable substrate or light availability, could serve as a low-cost adaptation strategy to ocean acidification and deoxygenation and provide important refugia from ocean acidification.

Experimental study on treating landfill sludge by preconditioning combined with vacuum preloading: Effects of freeze-thaw and FeCl3 preconditioning.

The deep dewatering of landfill sludge (LS) mainly uses the methods of chemical preconditioning and mechanical dewatering, which is easy to cause environmental pollution and is not conducive to the subsequent recycling treatment of sludge. To find a more environment-friendly and efficient method for LS's deep dewatering and volume reduction, an in-situ sludge treatment method combining freeze-thaw (F/T) preconditioning and vacuum preloading was proposed. Firstly, the F/T test of LS was carried out to explore the optimum freezing temperature. FeCl3, the most widely used agent, was selected as the chemical preconditioning. Then carry out vacuum preloading model box test. The data were compared after the test. The mechanisms of the two different sludge preconditioning methods on the LS's consolidation were analyzed. The results show that: after freezing, the specific resistance of LS decreases obviously, the overall particle size increases, the content of small particles decreases. Too fast freezing rate is not conducive to the LS's dewatering. After preconditioning (F/T and FeCl3) combined with vacuum preloading, the volume reduction ratio was 57.1% and 41.1% respectively, the water content was reduced from 73.4% to 53.7% and 58.1%, and the unconfined compressive strength(UCS) was improved from 15.5 kPa to 50.9 kPa and 77.3 kPa. The total water discharge, drainage rate, volume reduction, and water content of freeze-thaw preconditioned LS are better than FeCl3 preconditioned, while FeCl3 preconditioned LS has higher UCS. F/T can aggregate small sludge particles but the acidification and hydrolysis of FeCl3 always produce small particle, which is not conducive to the consolidation of LS during vacuum preloading.

Insight into conditioning landfill sludge with ferric chloride and a Fenton reagent: Effects on the consolidation properties and advanced dewatering.

The landfill sludge in storage reservoirs needs to be dewatered and disposed of for environmental and engineering purposes. The key factors are the high organic matter content and low permeability. Chemical conditioning is considered an efficient method for adjusting the properties of sludge. In this paper, two typical chemical agents, FeCl3 and a Fenton reagent with different additive amounts, are studied and compared for dewatering and consolidation purposes. Compression experiments and consolidation experiments are compared, and the coefficient of compressibility and compression index are obtained and compared. Then, the sludge permeability, grain size distribution variations, specific resistance to filtration (SRF) and morphology observations are considered to analyse the treatment mechanism. The results indicate that the properties of landfill sludge will change as the curing time increases. FeCl3 and Fenton are both effective in improving the consolidation and permeability properties of sludge. For the conditioning process, the optimum FeCl3 content is 20%, and the process is dominated by coagulation if FeCl3 is less than 20%; otherwise, it is dominated by hydrolysis. For the Fenton reagent, the optimum Fe2+ content and H2O2 content are 4% and 12%, respectively. The depolymerization effect of the Fenton reagent leads to the oxidation and recombination of the polar group on extracellular polymeric substances (EPSs). The results can be used to explain the conditioning mechanism of the effective agents of FeCl3 and Fenton and compare the corresponding consolidation properties. The consolidation characteristics provide a reference for further application of vacuum preloading in the sludge disposal process.