WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common complication of acute and severe neurosurgery. Remodeling of N6-methyladenosine (m6A) stabilization may be an attractive treatment option for neurological dysfunction after TBI. In the present study, we explored the epigenetic methylation of RNA-mediated NLRP3 inflammasome activation after TBI. METHODS: Neurological dysfunction, histopathology, and associated molecules were examined in conditional knockout (CKO) WTAP[flox/flox, Camk2a-cre], WTAPflox/flox, and pAAV-U6-shRNA-YTHDF1-transfected mice. Primary neurons were used in vitro to further explore the molecular mechanisms of action of WTAP/YTHDF1 following neural damage. RESULTS: We found that WTAP and m6A levels were upregulated at an early stage after TBI, and conditional deletion of WTAP in neurons did not affect neurological function but promoted functional recovery after TBI. Conditional deletion of WTAP in neurons suppressed neuroinflammation at the TBI early phase: WTAP could directly act on NLRP3 mRNA, regulate NLRP3 mRNA m6A level, and promote NLRP3 expression after neuronal injury. Further investigation found that YTH domain of YTHDF1 could directly bind to NLRP3 mRNA and regulate NLRP3 protein expression. YTHDF1 mutation or silencing improved neuronal injury, inhibited Caspase-1 activation, and decreased IL-1ß levels. This effect was mediated via suppression of NLRP3 protein translation, which also reversed the stimulative effect of WTAP overexpression on NLRP3 expression and inflammation. CONCLUSION: Our results indicate that WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after TBI and that WTAP/m6A/YTHDF1 downregulation therapeutics is a viable and promising approach for preserving neuronal function after TBI, which can provide support for targeted drug development.

Akkermansia muciniphila-Nlrp3 is involved in the neuroprotection of phosphoglycerate mutase 5 deficiency in traumatic brain injury mice.

Introduction: Gut-microbiota-brain axis is a potential treatment to decrease the risk of chronic traumatic encephalopathy following traumatic brain injury (TBI). Phosphoglycerate mutase 5 (PGAM5), a mitochondrial serine/threonine protein phosphatase, resides in mitochondrial membrane and regulates mitochondrial homeostasis and metabolism. Mitochondria mediates intestinal barrier and gut microbiome. Objectives: This study investigated the association between PGAM5 and gut microbiota in mice with TBI. Methods: The controlled cortical impact injury was established in mice with genetically-ablated Pgam5 (Pgam5-/-) or wild type, and WT male mice were treated with fecal microbiota transplantation (FMT) from male Pgam5-/- mice or Akkermansia muciniphila (A. muciniphila). Then the gut microbiota abundance, blood metabolites, neurological function, and nerve injury were detected. Results: Treated with antibiotics for suppressing gut microbiota in Pgam5-/- mice partially relieved the role of Pgam5 deficiency in the improvement of initial inflammatory factors and motor dysfunction post-TBI. Pgam5 knockout exhibited an increased abundance of A. muciniphila in mice. FMT from male Pgam5-/- mice enabled better maintenance of amino acid metabolism and peripherial environment than that in TBI-vehicle mice, which suppressed neuroinflammation and improved neurological deficits, and A. muciniphila was negatively associated with intestinal mucosal injury and neuroinflammation post-TBI. Moreover, A. muciniphila treatment ameliorated neuroinflammation and nerve injury by regulating Nlrp3 inflammasome activation in cerebral cortex with TBI. Conclusion: Thus, the present study provides evidence that Pgam5 is involved in gut microbiota-mediated neuroinflammation and nerve injury, with A. muciniphila-Nlrp3 contributing to peripheral effects.

Srs11-92, a ferrostatin-1 analog, improves oxidative stress and neuroinflammation via Nrf2 signal following cerebral ischemia/reperfusion injury.

AIM: Ferroptosis is increasingly becoming to be considered as an important mechanism of pathological cell death during stroke, and specific exogenous ferroptosis inhibitors have the ability to reverse cerebral ischemia/reperfusion injury. However, research on Srs11-92 (AA9), a ferrostatin-1 (Fer-1) analog, in preclinical studies is limited. METHODS: In the middle cerebral artery occlusion-reperfusion (MCAO/R) mice model or oxygen-glucose deprivation/reperfusion (OGD/R) cell model, Fer-1, AA9, and/or ML385 were administered, and brain infarct size, neurological deficits, neuronal damage, oxidative stress, and neuroinflammation were determined after the damage, in vitro and in vivo. RESULTS: Fer-1 and AA9 improved brain infarct size, neuronal damage, and neurological deficits in mice model of MCAO/R, and inhibited the overloaded iron deposition, ROS accumulation, and neuroinflammation response: it also increased the expression of GPx4, Nrf2, and HO-1 and suppressed the expression of HMGB1 and NF-κB p65 in the epicenter of injured hippocampal formation. However, Nrf2 inhibitor ML385 reversed the neuroprotective effect of AA9, including the oxidative stress and neuroinflammation. In vitro studies showed that AA9 relieved OGD/R-induced neuronal oxidative stress and neuroinflammation via the Nrf2 pathway, which was impaired by ML385 in primary neurons. CONCLUSION: The findings imply that Fer-1 analog AA9 may be suitable for further translational studies for the protection of neuronal damage via Nrf2 signal pathway-mediated oxidative stress and neuroinflammation in stroke and others neurological diseases.

A Review of the Ethical Use of Animals in Functional Experimental Research in China Based on the "Four R" Principles of Reduction, Replacement, Refinement, and Responsibility.

Use of live laboratory animals is essential in the process of functional experimentation teaching. There are ethical problems, such as poor experimental environment, non-standard operation, and neglect of animal rights in experimental teaching. As an important basic course in life science education, functional experimentation should establish the correct ethics of use of laboratory animals. The welfare of laboratory animals has become one of the frontier directions of medical ethics research. The "4R" principle of animal welfare is based on the principles of reduction, replacement, refinement, and responsibility, which may provide a way to solve ethical problems in the teaching and research activities of functional experimentation. In addition to receiving relevant knowledge and education, laboratory animal practitioners and students in functional experimentation teaching should consciously abide by relevant regulations and rules and actively follow the "4R" principles. Animal ethics education is reflected in all teaching and research activities. Based on the principle of "4R" and the premise of guaranteeing teaching objectives, virtual simulation experiment teaching is a great supplement to functional experimentation. In teaching, medical ethics education should be strengthened to cultivate the consciousness of respecting the life of experimental animals, and awareness of laboratory animal ethics should be improved among teachers and students of functional experimentation to further promote ideological and political education in colleges and universities. This brief summary analyzes the general situation of animal ethics in functional experimentation in China based on the principle of "4R" and provides certain references and support for course teaching and training.

Interventional treatment of basilar trunk artery aneurysms associated with situs inversus totalis: A case report.

Basilar trunk artery aneurysm (BTAA) has an overall low incidence in intracranial aneurysm, but its rupture is associated with high morbidity and mortality in older people. Situs inversus totalis (SIT) is a rare congenital abnormality characterized by visceral rotation and vascular abnormalities. It has been described in several uncommonly clinical cases, along with middle cerebral artery aneurysms and large carotid cavernous aneurysms. However, the association between interventional embolization for BTAA and SIT has not been reported. We described the angiography findings and interventional treatment of the association of BTAA with SIT.

Bibliometric Visualization Analysis of Microbiome-Gut-Brain Axis from 2004 to 2020.

BACKGROUND The microbiome-gut-brain axis (MGBA) is the biochemical signal of the digestive tract and central nervous system. MGBA disorders have been increasingly involved in the pathological process of neurological diseases. This study aimed to investigate the research hot spots of MGBA from 2004 to 2020. MATERIAL AND METHODS Using bibliometric analysis from the Web of Science Core Collection (WOSCC) database, 3993 documents on the MGBA were retrieved and visual analysis was conducted. RESULTS The MGBA has received attention worldwide and will continue to be a research hot spot. Emerging research organizations and scholars of the MGBA and the research of John F. Cryan and colleagues from Ireland in the MGBA have been recognized by many scholars. However, the research of Chinese scholars and organizations appeared to have less impact due to lack of research innovation and collaboration with other countries/regions. Keyword analysis showed that neuroinflammation was a hot spot and that eminent scholars had begun to work in the field of MGBA. CONCLUSIONS This work provided an overall view of the literature on the MGBA worldwide, and the analysis provided a comprehensive overview of MGBA research. It further revealed the interaction between the gut microbiota (eg, Akkermansia, Parabacteroides) and the specific regulatory network of the gut microbiota and metabolites, neuroinflammation, and neural networks, which can facilitate the development of effective treatment strategies using microbiota for targeting neuroinflammation and conducting large-scale clinical trials of neurological diseases.

Phosphoglycerate Mutase 5 Knockdown Alleviates Neuronal Injury After Traumatic Brain Injury Through Drp1-Mediated Mitochondrial Dysfunction.

Aims: Traumatic brain injury (TBI) is a major cause of disability and death, and a better understanding of the underlying mechanisms of mitochondrial dysfunction will provide important targets for preventing damage from neuronal insults. Phosphoglycerate mutase 5 (PGAM5) is localized to the mitochondrial outer-inner membrane contact sites, and the PGAM5-Drp1 pathway is involved in mitochondrial dysfunction and cell death. The purpose of this project was to evaluate the effects of PGAM5 on neuronal injury and mitochondrial dysfunction. Results: PGAM5 was overexpressed in mice subjected to TBI and in primary cortical neurons injured by mechanical equiaxial stretching. PGAM5 deficiency alleviated neuroinflammation, blocked Parkin, PINK1, and Drp1 translocation to mitochondria and abnormal phosphorylation of Drp1, mitochondrial ultrastructural changes, and nerve malfunction in TBI mouse model. PGAM5-shRNA (short hairpin RNA) reduced Drp1 translocation and activation, including dephosphorylation of p-Drp1 on Ser622 (human Drp1 Ser616) and phosphorylation of Drp1 on Ser643 (human Drp1 Ser637). The levels of inflammatory cytokines, the degree of mitochondrial impairment (mitochondrial membrane potential, ADP/ATP, AMP/ADP, antioxidant capacity), and neuronal injury in stretch-induced primary cortical neurons were reduced by blocking expression of PGAM5. The inhibition of PGAM5 is neuroprotective via attenuation of Drp1 activation, similar to that achieved by mitochondrial division inhibitor-1 (Mdivi1)-mediated Drp1 inhibition. Innovation and Conclusion: Our findings demonstrate the critical role of PGAM5 in progression of neuronal injury from TBI via Drp1 activation (dephosphorylation of p-Drp1 on Ser622 and phosphorylation of Drp1 on Ser643)-mediated mitochondrial dysfunction. The data may open a window for developing new drugs to prevent the neuropathology of TBI.

Bibliometric Analysis of Ferroptosis in Stroke From 2013 to 2021.

Background: Stroke is a major cause of long-term disability and death, but the clinical therapeutic strategy for stroke is limited and more research must be conducted to explore the possible avenues for stroke treatment and recovery. Since ferroptosis is defined, its role in the body has become the focus of attention and discussion, including in stroke. Methods: In this work, we aim to systematically discuss the "ferroptosis in stroke" research by bibliometric analysis. Documents were retrieved from the Web of Science Core Collection database on October 30, 2021. Statistical analysis and visualization analysis were conducted by the VOSviewer 1.6.15. Results: Ninety-nine documents were identified for bibliometric analysis. Research on "ferroptosis in stroke" has been rapidly developing and has remained the focus of many scholars and organizations in the last few years, but the Chinese groups in this field still lacked collaboration with others. Documents and citation analysis suggested that Rajiv R. Ratan and Brent R. Stockwell are active researchers, and the research by Qingzhang Tuo, Ishraq Alim, and Qian Li are more important drivers in the development of the field. Keywords associated with lipid peroxidation, ferroptosis, iron, oxidative stress, and cell death had high frequency, but apoptosis, necroptosis, pyroptosis, and autophagy had scant research, and there may be more research ideas in the future by scholars. Conclusion: Further exploration of the mechanisms of crosstalk between ferroptosis and other programmed cell death may improve clinical applications and therapeutic effects against stroke. Scholars will also continue to pay attention to and be interested in the hot topic "ferroptosis in stroke", to produce more exciting results and provide new insights into the bottleneck of stroke treatment.

LncRNA-Meg3 promotes Nlrp3-mediated microglial inflammation by targeting miR-7a-5p.

Recent studies have identified neuroinflammation as a significant contributor to the pathological process of traumatic brain injury (TBI) and as a potentially effective target for treatment. LncRNA maternally expressed gene 3 (Meg3) has further been observed to play a critical role in diverse biological processes, including microglial activation and the inflammatory response. However, its target gene and associated signaling pathway require further elucidation. This study found that lipopolysaccharide + ATP upregulated Meg3, promoted microglia activation, Nlrp3/caspase1 activation and inflammation, and markedly reduced miR-7a-5p. Overexpression of miR-7a-5p attenuated Meg3-induced microglial activation, but not Meg3 expression. Bioinformatic analysis and dual-luciferase assays indicated that Meg3 was a direct target of miR-7a-5p that negatively regulates miR-7a-5p expression. Further, we showed that Meg3 acted as a competing endogenous RNA for miR-7a-5p and induced microglial inflammation by regulating nod-like receptor protein 3 (Nlrp3) expression. Our study thus demonstrates Meg3 regulates microglia inflammation by targeting the miR-7a-5p /Nlrp3 pathway.

Rapamycin improves the neuroprotection effect of inhibition of NLRP3 inflammasome activation after TBI.

Inflammation is the focus of many studies on traumatic brain injury (TBI) treatment and outcomes improvement. Some studies have demonstrated that the inhibition of NOD-like receptor protein-3 (NLRP3) inflammasome activation is a potential strategy for TBI therapy. Mitophagy is thought to play a crucial role in pathological conditions of TBI. We hypothesize simultaneous mitophagy activation and NLRP3 inflammsome inhibition, plays preferable role in delaying the progression and nerve damage post-TBI. In this study, TBI-mice and oxygen and glucose deprivation (OGD)-induced primary cortical neurons were treated with MCC950 (a NLRP3 selective inhibitor) or Rapamycin (Rap, a mTOR inhibitor, stimulated autophagy and mitophagy). We evaluated the effects of Rap and NLRP3 inhibition on the neurological deficits, neurological damage, and inflammatory response, to determine if Rap further induced the neuroprotection of suppression of NLRP3 inflammasome activation in vivo and in vitro TBI-model. TBI induced NLRP3 inflammasome activation and mitochondrial dysfunction, including increased caspase-1 p20 expression, exacerbated the secretion of LDH, IL-1ß and IL-18, and disorder of ATP, MMP, ROS and mitophagy (Pink1 and LC3 expression in mitochondria). NLRP3 inhibition and Rap attenuated the neurological damage and mitochondrial dysfunction, while combined treatment showed better neuroprotection compared with single treatment. Collectively, the data demonstrate that mitophagy and NLRP3 inflammasome have the interactivity, and Rap-induced mitophagy further enhances the neuroprotection of inhibition of NLRP3 inflammasome activation post-TBI. Our findings suggest that Rap-activated mitophagy combined with MCC950-induced NLRP3 inflammasome repression may be a potential strategy for TBI therapy.

The MKK7 inhibitor peptide GADD45ß-I attenuates ER stress-induced mitochondrial dysfunction in HT22 cells: Involvement of JNK-Wnt pathway.

JNK, a member of the mitogen activated protein kinases (MAPKs) superfamily, plays a key role in cell death in many neurological disorders, but systemic inhibition of JNK has detrimental side effects. JNK can be regulated by two direct upstream kinases: MAPK kinase 4 (MKK4) and MAPK kinase 7 (MKK7). Here, we investigated the effect of GADD45ß-I, a recently designed cell-permeable inhibitor peptide for MKK7, on endoplasmic reticulum (ER) stress-induced cytotoxicity in neuronal HT22 cells. We found that treatment with the ER stress inducer tunicamycin (TM) increased the phosphorylation of JNK and MKK7 in HT22 cells, which was nullified by GADD45ß-I. GADD45ß-I significantly attenuated TM-induced toxicity via inhibiting apoptotic cell death, as evidenced by decreased number of TUNEL-positive cells and reduced caspase-3 activity. GADD45ß-I treatment also decreased expression of ER stress associated pro-apoptotic proteins and prevented morphological changes of the ER after TM exposure. In addition, inhibition of mitochondrial oxidative stress and preservation of intracellular ATP levels were observed in GADD45ß-I-treated cells. The experiments using siRNA transfection and Topflash reporter assay revealed a possible involvement of Wnt/ß-catenin pathway in GADD45ß-I-induced protection in HT22 cells. In summary, our results demonstrated that GADD45ß-I exerted protective effects against TM-induced cytotoxicity via regulating JNK-Wnt pathway. Targeting MKK7 could represent a new therapeutic strategy for the treatment of neurological diseases where ER stress associated neuronal injury are involved.

Systems pharmacology dissection of the anti-stroke mechanism for the Chinese traditional medicine Xing-Nao-Jing.

Xing-Nao-Jing (XNJ) is a well-known injection that has been extensively applied in clinical treatment of stroke in China. However, the underlying mechanism of clinical administration of XNJ in stroke remains unclear. In this study, a systems pharmacology strategy based on pharmacokinetic and pharmacodynamics data was applied to analyze the pharmacological effect of XNJ on stroke. Sixteen active compounds were filtered from XNJ through Drug-likeness (DL) and Brain-blood-barrier (BBB) evaluations. Ninety-four potential targets of these active components were identified by SysDT and SEA. Biological process and pathway enrichment analyses of these targets demonstrated that XNJ exerted anti-stroke effects by biological processes and pathways, such as the response to oxidative stress, regulation of blood pressure, calcium signaling pathway, and apoptosis. Integrating the compound-target network and stroke-related PPI network, we found that Akt1, HIF-1α and ITGB2 may play key roles in the treatment of stroke. The experiments demonstrated that oxycurcumenol may prevent PC12 cells from oxidative stress-induced cell damage. Our study indicates that XNJ has an effect on stroke by protecting neuro cells from oxidative stress-induced cell damage via HIF1α, and the research strategy at the systems pharmacology level is feasible to reveal the mechanisms of novel lead compounds from natural products.