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.

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 facemask oxygenation with and without positive pressure ventilation on gastric volume during anesthesia induction in patients undergoing laparoscopic cholecystectomy or partial hepatectomy: a randomized controlled trial.

BACKGROUND: Studies focusing on the relationship between gastric volume and facemask oxygenation without ventilation during apnea in anesthesia induction are scarce. This study compared the change in gastric volume during apnea in anesthesia induction using facemask ventilation and facemask oxygenation without ventilation in adults undergoing laparoscopic surgery. METHODS: In this prospective, randomized, double-blinded trial, 70 adults undergoing laparoscopic surgery under general anesthesia were divided into two groups to receive facemask oxygenation with and without ventilation for 60 seconds after loss of consciousness. Before anesthesia induction and after endotracheal intubation, the gastric antral cross-sectional area was measured with ultrasound imaging. Arterial blood gases were tested at baseline (T1), after preoxygenation (T2), after loss of consciousness (T3), and before and after endotracheal intubation (T4 and T5, respectively). RESULTS: Sixty patients were included (ventilation n = 30; non ventilation n = 30, 10 patients were excluded). The median [IQR] change of gastric antral cross-sectional area in ventilation group was significantly higher than in non ventilation group (0.83 [0.20 to 1.54] vs. 0.10 [- 0.11 to 0.56] cm2, P = 0.001). At T4 and T5, the PaO2 in ventilation group was significantly higher than in non ventilation group (T4: 391.83 ± 61.53 vs. 336.23 ± 74.99 mmHg, P < 0.01; T5: 364.00 ± 58.65 vs. 297.13 ± 86.95 mmHg, P < 0.01), while the PaCO2 in non ventilation group was significantly higher (T4: 46.57 ± 5.78 vs. 37.27 ± 6.10 mmHg, P < 0.01; T5: 48.77 ± 6.59 vs. 42.63 ± 6.03 mmHg, P < 0.01) and the pH value in non ventilation group was significantly lower (T4: 7.35 ± 0.029 vs 7.42 ± 0.047, P < 0.01; T5: 7.34 ± 0.033 vs 7.39 ± 0.044, P < 0.01). At T4, the HCO3- in non ventilation group was significantly higher (25.79 ± 2.36 vs. 23.98 ± 2.18 mmol l- 1, P < 0.01). CONCLUSIONS: During apnoea, the increase in gastric volume was milder in patients undergoing facemask oxygenation without ventilation than with positive pressure ventilation. TRIAL REGISTRATION: ChiCTR2100054193, 10/12/2021, Title: "Effect of positive pressure and non-positive pressure ventilation on gastric volume during induction of general anesthesia in laparoscopic surgery: a randomized controlled trial". Website: https://www.chictr.ogr.cn .

Suvorexant with or without ramelteon to prevent delirium: a systematic review and meta-analysis.

Delirium is a common and serious neurobehavioral syndrome, associated with prolonged hospital stays, and increased morbidity and mortality. As it remains unclear whether suvorexant with or without ramelteon prevents delirium in elderly hospitalized patients, we conducted a systematic review and meta-analysis to evaluate, searching the PubMed, Cochrane Library, Web of Science, EMBASE, and EBSCOhost databases for all randomized controlled trials (RCTs), case-control studies, and cohort studies that investigated the effects of suvorexant with or without ramelteon on delirium in adult hospitalized patients. The primary outcome was the incidence of delirium. Two randomized controlled trials, 7 cohort studies and 2 case-control studies involving 2594 patients were included in this meta-analysis. The results showed that both suvorexant alone (odds ratio (OR) = 0.30, 95% confidence interval (CI): 0.14-0.65, P = 0.002) and suvorexant with ramelteon (OR = 0.39, 95% CI 0.23-0.65, P = 0.0003) reduced the incidence of delirium in adult hospitalized patients. Six studies involved the use of benzodiazepines; subgroup analysis performed separately in the suvorexant alone and suvorexant with ramelteon groups indicated that when benzodiazepine was administered, suvorexant with ramelteon was effective at reducing the incidence of delirium (OR = 0.53, 95% CI 0.37-0.74, P = 0.0002), but no significant difference was observed for suvorexant alone (OR = 0.40, 95% CI 0.11-1.53, P = 0.18). The current literature thus supports the effectiveness of suvorexant with or without ramelteon for delirium prevention, although suvorexant alone failed to significantly reduce the incidence of delirium when benzodiazepine was administered. The present study was limited by the significant heterogeneity among the included studies, and caution should be exercised when interpreting the results. This study was registered in the PROSPERO database (CRD4202017964).

Inhibition of endotoxin-induced acute lung injury in rats by bone marrow-derived mesenchymal stem cells: Role of Nrf2/HO-1 signal axis in inhibition of NLRP3 activation.

Both the Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway and Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) pathway are considered essential for the development of acute lung injury (ALI)/ARDS induced by sepsis. Our aim was to study the role of Nrf2/HO-1 pathway on activation of the NLRP3 in the protective effect of marrow mesenchymal stem cells (BMSCs) on LPS-induced ALI. We found that BMSCs ameliorated ALI as evidenced by 1) decreased histopathological injury, wet/dry ratio, and protein permeability index in lung; 2) decreased reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl content and restored the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in lung tissue; 3) reduced LPS-induced increase in inflammatory cell count and promotion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels in bronchoalveolar lavage fluid (BALF); 4) improvement in the four-day survival rate of animals; and 5) enhanced expression of Nrf2 and HO-1 and decreased expression of NOD-like receptor protein 3(NLRP3) and caspase-1 (p20) in lung tissue. Of note, Nrf2 transcription factor inhibitor brusatol and HO-1 inhibitor tin protoporphyrin IX (SnppIX) reversed BMSCs induced down-expression of NLRP3 and caspase-1 (p20), and inhibited the protective effects of BMSCs. These findings demonstrated that the Nrf2-mediated HO-1 signaling pathway plays a critical role in the protective effects of BMSCs on LPS-induced ALI. BMSCs may play an anti-inflammatory effect partly through the Nrf2/HO-1-dependent NLRP3 pathway.

Melatonin and its analogues for the prevention of postoperative delirium: A systematic review and meta-analysis.

It remains unclear whether melatonin and its analogues prevent postoperative delirium (POD). Therefore, we conducted a systematic review and meta-analysis to evaluate the effect of melatonin and its analogues on POD prevention. PubMed, Cochrane Library, Web of Science, Embase and CINAHL databases were searched. Primary outcome was the incidence of POD. Six randomized controlled trials, 2 cohort studies and 1 case-control study were included in this meta-analysis. Results showed that melatonin and its analogue ramelteon decreased the incidence of POD in the entire adult surgical population (odds ratio [OR] = 0.45, 95% confidence interval [CI] 0.24-0.84, P = .01). When administered at a higher dose (5 mg), melatonin was effective in reducing the POD incidence (OR = 0.32, 95% CI 0.20-0.52, P < .00001). Melatonin administered less than 5 elimination half-lives before the surgery significantly reduced the POD incidence (OR = 0.31, 95% CI 0.19-0.49, P < .00001). Current literature supports the effectiveness of melatonin and its analogue ramelteon in POD prevention. However, the present study was limited by the significant heterogeneity of the included studies. More studies are needed to ascertain the preventive effect of melatonin and its analogues on the incidence of delirium after cardiac and noncardiac surgeries.

Effect of propofol on microRNA expression in rat primary embryonic neural stem cells.

BACKGROUND: Propofol is a widely used intravenous anesthetic that is well-known for its protective effect in various human and animal disease models. However, the effects of propofol on neurogenesis, especially on the development of neural stem cells (NSCs), remains unknown. Related microRNAs may act as important regulators in this process. METHODS: Published Gene Expression Omnibus (GEO) DataSets related to propofol were selected and re-analyzed to screen neural development-related genes and predict microRNA (miRNA) expression using bioinformatic methods. Screening of the genes and miRNAs was then validated by qRT-PCR analysis of propofol-treated primary embryonic NSCs. RESULTS: Four differentially expressed mRNAs were identified in the screen and 19 miRNAs were predicted based on a published GEO DataSet. Two of four mRNAs and four of 19 predicted miRNAs were validated by qRT-PCR analysis of propofol-treated NSCs. Rno-miR-19a (Rno, Rattus Norvegicus) and rno-miR-137, and their target gene EGR2, as well as rno-miR-19b-2 and rno-miR-214 and their target gene ARC were found to be closely related to neural developmental processes, including proliferation, differentiation, and maturation of NSCs. CONCLUSION: Propofol influences miRNA expression; however, further studies are required to elucidate the mechanism underlying the effects of propofol on the four miRNAs and their target genes identified in this study. In particular, the influence of propofol on the entire development process of NSCs remains to be clarified.

Proteomic profiling of the phosphoproteins in the rat thalamus, hippocampus and frontal lobe after propofol anesthesia.

BACKGROUND: Propofol is a safe and effective intravenous anesthetic that is widely used for the induction and maintenance of anesthesia during surgery. However, the mechanism by which propofol exerts its anesthetic effect remains unknown. The rapid onset of phosphorylation modifications coincides with that of propofol anesthesia. METHODS: Propofol-anesthetized rat models were built and phosphorylated proteins in the thalamus, hippocampus and frontal lobe were enriched the to analyze the changes in these phosphoproteins after propofol anesthesia. RESULTS: Sixteen of these phosphoprotein spots were successfully identified using MALDI-TOF MS and a subsequent comparative sequence search in the Mascot database. Of these proteins, keratin 18 and the tubulin 2c chain are cytoskeletal proteins; keratin 18 and gelsolin are relevant to alcohol drowsiness. Based on Western blot analysis, we also confirmed that the phosphorylation of these proteins is directly induced by propofol, indicating that propofol anesthesia may be relevant to cytoskeletal proteins and alcohol drowsiness. CONCLUSIONS: These identified propofol-induced phosphorylations of proteins provide meaningful contributions for further studying the anesthetic mechanism of propofol.

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.

Single-cell analyses reveal the dynamic functions of Itgb2+ microglia subclusters at different stages of cerebral ischemia-reperfusion injury in transient middle cerebral occlusion mice model.

Introduction: The underlying pathophysiological mechanisms of cerebral ischemia reperfusion injury (CIRI) is intricate, and current studies suggest that neuron, astrocyte, microglia, endothelial cell, and pericyte all have different phenotypic changes of specific cell types after ischemic stroke. And microglia account for the largest proportion after CIRI. Previous transcriptomic studies of ischemic stroke have typically focused on the 24 hours after CIRI, obscuring the dynamics of cellular subclusters throughout the disease process. Therefore, traditional methods for identifying cell types and their subclusters may not be sufficient to fully unveil the complexity of single-cell transcriptional profile dynamics caused by an ischemic stroke. Methods: In this study, to explore the dynamic transcriptional profile of single cells after CIRI, we used single-cell State Transition Across-samples of RNA-seq data (scSTAR), a new bioinformatics method, to analyze the single-cell transcriptional profile of day 1, 3, and 7 of transient middle cerebral artery occlusion (tMCAO) mice. Combining our bulk RNA sequences and proteomics data, we found the importance of the integrin beta 2 (Itgb2) gene in post-modeling. And microglia of Itgb2+ and Itgb2- were clustered by the scSTAR method. Finally, the functions of the subpopulations were defined by Matescape, and three different time points after tMCAO were found to exhibit specific functions. Results: Our analysis revealed a dynamic transcriptional profile of single cells in microglia after tMCAO and explored the important role of Itgb2 contributed to microglia by combined transcriptomics and proteomics analysis after modeling. Our further analysis revealed that the Itgb2+ microglia subcluster was mainly involved in energy metabolism, cell cycle, angiogenesis, neuronal myelin formation, and repair at 1, 3, and 7 days after tMCAO, respectively. Discussion: Our results suggested that Itgb2+ microglia act as a time-specific multifunctional immunomodulatory subcluster during CIRI, and the underlying mechanisms remain to be further investigated.

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.

Celastrol targeting Nedd4 reduces Nrf2-mediated oxidative stress in astrocytes after ischemic stroke.

Stroke is the second leading cause of death worldwide, and oxidative stress plays a crucial role. Celastrol exhibits strong antioxidant properties in several diseases; however, whether it can affect oxidation in cerebral ischemic-reperfusion injury (CIRI) remains unclear. This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms. Here, we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2 (Nrf2). Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry, we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4 (Nedd4) and then released Nrf2 from Nedd4 in astrocytes. Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI, which was significantly blocked by celastrol. Furthermore, by inhibiting oxidative stress and astrocyte activation, celastrol effectively rescued neurons from axon damage and apoptosis. Our study uncovered Nedd4 as a direct target of celastrol, and that celastrol exerts an antioxidative effect on astrocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.

Melatonin and Its Analogs for Prevention of Post-cardiac Surgery Delirium: A Systematic Review and Meta-Analysis.

Background: The effectiveness of melatonin and its analogs in preventing postoperative delirium (POD) following cardiac surgery is controversial. The purpose of this systematic review and meta-analysis was to confirm the benefits of melatonin and its analogs on delirium prevention in adults who underwent cardiac surgery. Methods: We systematically searched the PubMed, Cochrane Library, Web of Science, Embase, and EBSCOhost databases, the last search was performed in October 2021 and repeated before publication. The controlled studies were included if investigated the impact of melatonin and its analogs on POD in adults who underwent cardiac surgery. The primary outcome was the incidence of delirium. The Stata statistical software 17.0 was used to perform this study. Results: This meta-analysis included eight randomized controlled trials (RCTs) and two cohort studies with a total of 1,714 patients. The results showed that melatonin and ramelteon administration were associated with a significantly lower incidence of POD in adults who underwent cardiac surgery (odds ratio [OR], 0.46; 95% confidence interval [CI], 0.29-0.74; P = 0.001). The subgroup analyses confirmed that melatonin 3 mg (OR, 0.37; 95% CI, 0.18-0.76; P = 0.007) and 5 mg (OR, 0.34; 95% CI, 0.21-0.56; P < 0.001) significantly reduced the incidence of POD. Conclusion: Melatonin at dosages of 5 and 3 mg considerably decreased the risk of delirium in adults who underwent cardiac surgery, according to our results. Cautious interpretation of our results is important owing to the modest number of studies included in this meta-analysis and the heterogeneity among them. Systematic Review Registration: PROSPERO registration number: CRD42021246984.

HIF-1α Ameliorates Diabetic Neuropathic Pain via Parkin-Mediated Mitophagy in a Mouse Model.

Mitochondrial dysfunction, which can be regulated by mitophagy, plays a central role in diabetic neuropathic pain (DNP). Mitophagy that was involved in nerve damage-induced neuropathic pain has been reported. Hyperglycemia and cellular hypoxic were the two main characters of diabetes. Hypoxia-inducible factor 1α subunit (HIF-1α) plays a vital role in mitochondrial homeostasis under hypoxia. However, it remains unclear whether mitophagy was changed and could be regulated by HIF-1α in DNP. In this study, the results showed that mitophagy was activated and HIF-1α was upregulated in the spinal cord of diabetic mice. HIF-1α agonist dimethyloxalylglycine (DMOG) could further elevate HIF-1α and Parkin protein, enhance mitophagy, decrease mitochondrial dysfunction, and hyperalgesia. Furthermore, Park2 (encoding Parkin) knockout aggravated hyperalgesia and mitochondrial dysfunction in diabetic mice. Furthermore, mitophagy could not be activated and induced by HIF-1α agonist DMOG in Park2-/- diabetic mice. In this study, we first demonstrated that HIF-1α could upregulate mitophagy in the spinal cord of mice with DNP through modulating the Parkin signaling pathway, promoting new insights into the mechanisms and research of treatment strategies for patients with DNP.

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.

Propofol Exposure Disturbs the Differentiation of Rodent Neural Stem Cells via an miR-124-3p/Sp1/Cdkn1b Axis.

Accumulating studies have indicated that propofol may lead to neurotoxicity and its effect on neural stem cells (NSCs) may play pivotal role in propofol-related neurotoxicity. Previously, we found that propofol could promote NSCs proliferation and could regulate several microRNA expressions. However, the underlying mechanism between microRNAs and NSCs development after propofol exposure is still unclear. Our data first observed that rat primary neural stem cells exposed to propofol exhibited a cell cycle arrest status and an inclination to differentiate into GFAP+ or S100ß+ cells. This phenomenon was accompanying with a lower miR-124-3p expression and could be reversed via overexpression miR-124-3p in NSCs. Using bioinformatic predictions and luciferase assay we confirmed that Sp1 (Specificity Protein 1) is the target gene of miR-124-3p, indicating that miR-124-3p may regulate NSCs development through Sp1. Further, knockdown of Sp1 rescue the effect of propofol on NSCs differentiation. Finally, we demonstrated that Sp1 could bind cdkn1b promoter region through chromatin immunoprecipitation assay, indicating that Sp1 affect NSC's cell cycle through cdkn1b directly. Overall, our study highlights the miR-124-3p/Sp1/cdkn1b axis to be important in propofol interfering the differentiation of NSCs.

Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation.

Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq). A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO) analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Validation and bioinformatic analysis of propofol-induced differentially expressed microRNAs in primary cultured neural stem cells.

Propofol, a widely used intravenous anesthetic, was previously considered as a neuroprotective agent. Recently, however, accumulating evidence suggests that it may cause neurotoxicity, especially in the development of neural stem cells (NSCs). The potential mechanisms contributing to propofol-induced neurotoxicity during neurogenesis, such as those involving microRNAs (miRNAs), are still unknown. In this study, a total of 27 differentially expressed miRNAs were identified in our initial screen and 6 miRNAs were validated by qRT-PCR. Three miRNAs were up-regulated (miR-377-5p, miR-194-3p and miR-143-5p), and three were down-regulated (miR-3583-3p, miR-466b-5p and miR-410-5p). Following gene ontology and KEGG pathway enrichment analysis, Gabbr1, Canca1b and Gabbr2, which are enriched in the GABAergic synapse pathway, were selected as genes potentially playing a role in propofol-induced neurotoxicity. Gabbr1 and Cacna1b, which are targeted by miRNAs that are up-regulated following propofol exposure, showed decreased expression at the mRNA and protein levels. Gabbr2, targeted by miRNAs that were down-regulated following treatment with propofol, was up-regulated at both the levels of mRNA and protein expression. The two clusters of miRNAs that show differential expression following propofol exposure may act in a synergistic manner to regulate several genes simultaneously during the development of NSCs. Our results may contribute to clarify the molecular mechanism and provide potential therapeutic targets for propofol induced neurotoxicity.

[RhoA/Rho-kinase contributes to chronic pain following thoracotomy by up-regulating glutaminase 1 expression in rat spinal dorsal cord].

OBJECTIVE: To investigate whether RhoA/Rho-kinase contributes to the occurrence of chronic post-thoracotomy pain (CPSP) by up regulation of glutaminase 1 (GLS1) expression in the spinal dorsal cord. METHODS: Twenty five male Sprague Dawley (SD) rats were divided into control group (n=5) and model group (n=20). The rats in the model group were randomized into two sub groups (n=10) for observation on day 10 and day 21 after thoracotomy, and each group was further divided into CPSP and non CPSP groups according to the behavioral test results. All the rats were sacrificed after behavioral test for examination of GLS1 and RhoA expressions in the spinal cord using Western blotting and RT PCR. We also compared the effect of the Rho kinase inhibitor fasudil and saline, both injected intraperitoneally daily at 10 mg/kg for 7 consecutive days following thoracotomy, on CPSP and GLS1 expression in 30 male SD rats on day 21 after thoracotomy. RESULTS: Compared with the control group, the rats with CPSP showed significantly increased expressions of GLS1 and RhoA mRNA in the spinal cord on both day 10 and day 21 following thoracotomy (P<0.01), but the rats without CPSP did not show obvious changes in GLS1 and RhoA expressions. In fasudil treated rats, the mechanical pain threshold was obviously increased and the expressions of GLS1 and RhoA were significantly reduced as compared with those in saline treated rats (P<0.01). CONCLUSION: RhoA plays an important role in the occurence of CPSP by up-regulating the expression of GLS1 in the spinal dorsal cord of rats.