Dietary Supplementation with Nervonic Acid Ameliorates Cerebral Ischemia-Reperfusion Injury by Modulating of Gut Microbiota Composition-Fecal Metabolites Interaction.

SCOPE: Cerebral ischemia-reperfusion (IR) injury stands as a prominent global contributor to disability and mortality. Nervonic acid (NA), a bioactive elongated monounsaturated fatty acid, holds pivotal significance in human physiological well-being. This research aims to explore the prophylactic effects and fundamental mechanisms of NA in a rat model of cerebral IR injury. METHODS AND RESULTS: Through the induction of middle cerebral artery occlusion, this study establishes a rat model of cerebral IR injury and comprehensively assesses the pharmacodynamic impacts of NA pretreatment. This evaluation involves behavioral analyses, histopathological examinations, and quantification of serum markers. Detailed mechanisms of nervonic acid's prophylactic effects are revealed through fecal metabolomics and 16S rRNA sequencing analyses. Our findings robustly support nervonic acid's capacity to ameliorate neurological impairments in rats afflicted with cerebral IR injury. Beyond its neurological benefits, NA demonstrates its potential by rectifying metabolic perturbations across diverse pathways, particularly those pertinent to unsaturated fatty acid metabolism. Additionally, NA emerges as a modulator of gut microbiota composition, notably by selectively enhancing vital genera like Lactobacillus. CONCLUSION: These comprehensive findings highlight the potential of incorporating NA as a functional component in dietary interventions aimed at targeting cerebral IR injury.

LC-MS based metabonomics study on protective mechanism of ESWW in cerebral ischemia via CYTC/Apaf-1/NDRG4 pathway.

BACKGROUND: Ershiwuwei Zhenzhu pills was originally recorded in the Tibetan medical book Si Bu Yi Dian in the 8th century AD and is now included in the Pharmacopoeia of the People's Republic of China (2020). The pills can calm the nerves and open the mind as well as treat cerebral ischemia reperfusion injury, stroke, hemiplegia. However, its quality standards have not yet been established, and the therapeutic effect on cerebral ischemia by regulating the mitochondrial apoptosis pathway has not been elucidated. STUDY DESIGN AND METHODS: LC-MS was used to establish quality standards for Ershiwuwei Zhenzhu pills. Metabonomics, molecular docking, neuroethology, cerebral infarction ratio, pathological detection of diencephalon, cortex, and hippocampus, and molecular biology techniques were used to reveal the mechanism of the pills in regulating the mitochondrial apoptosis pathway to treat cerebral ischemia. RESULTS: The contents of 20 chemical components in Ershiwuwei Zhenzhu pills from 12 batches and 8 manufacturers was determined for the first time. Eleven differential metabolites and three metabolic pathways, namely, fructose and mannose metabolism, glycerophospholipid metabolism, and purine metabolism, were identified by metabonomics. The pills improved the neuroethology abnormalities of MCAO rats and the pathological damage in the diencephalon and decreased the ratio of cerebral infarction. It also significantly reduced the mRNA expression of AIF, Apaf-1, cleared caspase8, CytC, and P53 mRNA in the brain tissue and the protein expression of Apaf-1 and CYTC and increased the protein expression of NDRG4. CONCLUSION: In vitro quantitative analysis of the in vitro chemical components of Ershiwuwei Zhenzhu pills has laid the foundation for improving its quality control. The potential mechanism of the pills in treating cerebral ischemia may be related to the Apaf-1/CYTC/NDRG4 apoptosis pathway. This work provides guidance for clinical drug use for patients.

[Effect of Naotaifang on microglial polarization and glial scar following cerebral ischemia reperfusion injury].

This study aims to investigate the effect of Naotaifang(NTF) on the proteins associated with microglial polarization and glial scar in the rat model of cerebral ischemia reperfusion injury(CIRI). The CIRI model was established by middle cerebral artery occlusion/reperfusion. The 48 successfully modeled rats were randomized into model 7 d, model 14 d, NTF 7 d, and NTF 14 d groups(n=12). In addition, 12 SD rats were selected as the sham group. The NTF group was administrated with NTF suspension at 27 g·kg~(-1)·d~(-1) by gavage, and the sham, model 7 d, and model 14 d groups were administrated with the same volume of normal saline every day by gavage for 7 and 14 days, respectively. After the intervention, Longa score was evaluated. The infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) staining. Morris water maze and open field tests were carried out to evaluate the spatial learning, memory, cognitive function, and anxiety degree of rats. Hematoxylin-eosin(HE) staining was employed to observe the morphological structure and damage of the brain tissue. The immunofluorescence assay was employed to measure the expression of glial fibrillary acidic protein(GFAP) and glial scar. Western blot was employed to determine the protein levels of GFAP, neurocan, phosphacan, CD206, arginase-1(Arg-1), interleukin(IL)-1ß, IL-6, and IL-4. Compared with the sham, model 7 d and model 14 d groups showed cerebral infarction of different degrees, severe pathological injury of cerebral cortex and hippocampus, neurological impairment, reduced spatial learning and memory, cognitive dysfunction, severe anxiety, astrocyte hyperplasia, thickening penumbra glial scar, and up-regulated protein levels of IL-1ß, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P<0.01). Compared with the model group, NTF 7 d and NTF 14 d groups improved spatial learning, memory, and cognitive function, reduced anxiety, improved nerve function, reduced cerebral infarction volume, reduced astrocyte hyperplasia, thinned penumbra glial scar, down-regulated the protein levels of GFAP, neurocan, phosphacan, IL-6, and IL-1ß, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P<0.05 or P<0.01). NTF exerts a neuroprotective effect on CIRI by inducing the M2 polarization of microglia, inhibiting inflammatory response, and reducing the formation of glial scar.

[Mechanism of astragaloside â £ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification].

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 µmol·L~(-1)) + PNS(30 µmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P<0.05) and the cell damage rate in the brain tissue(P<0.05), promoted the expression of vWF and VEGFA(P<0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P<0.05, P<0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.05, P<0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P<0.05,P<0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

[Interaction between central nervous system and immune system after ischemic stroke and its therapeutic significance of traditional Chinese medicine].

Ischemic stroke is divided into acute phase, subacute phase, and recovery phase, with different pathological and physiological characteristics manifested at each stage. Among them, immune and inflammatory reactions persist for several days and weeks after ischemia. Ischemic stroke not only triggers local inflammation in damaged brain regions but also induces a disorder in the immune system, thereby promoting neuroinflammation and exacerbating brain damage. Therefore, conducting an in-depth analysis of the interaction between the central nervous system and the immune system after ischemic stroke, intervening in the main factors of the interaction between them, blocking pathological cascades, and thereby reducing brain inflammation have become the treatment strategies for ischemic stroke. This study summarizes and sorts out the interaction pathways between the central nervous system and the immune system. The impact of the central nervous system on the immune system can be analyzed from the perspective of the autonomic nervous system, the hypothalamic-pituitary-adrenal axis(HPA), and local inflammatory stimulation. The impact of the immune system on the central nervous system can be analyzed from the dynamic changes of immune cells. At the same time, the relevant progress in the prevention and treatment of traditional Chinese medicine(TCM) is summarized, so as to provide new insights for the analysis of complex mechanisms of TCM in preventing and treating ischemic stroke.

Effect of electroacupuncture on proBDNF/mBDNF and synaptic plasticity in rats with learning and memory impairment after cerebral ischemia-reperfusion. / ç”µé’ˆå¯¹è„‘ç¼ºè¡€å†çŒæ³¨å­¦ä¹ è®°å¿†éšœç¢å¤§é¼ æµ·é©¬è„‘æºæ€§ç¥žç»è¥å »å› å­è½¬åŒ–å’Œçªè§¦å¯å¡‘æ€§çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the rats' behavior and the transforming precursor of brain-derived neurotrophic factor (proBDNF) into mature brain-derived neurotrophic factor (mBDNF) in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia-reperfusion (IR), so as to explore its mechanisms underlying improvement of learning and memory ability. METHODS: SD rats were randomly divided into blank, sham operation, model, and EA groups, with 6 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (1 Hz/20 Hz) was applied to GV24 and GV20 for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria 24 h after modeling and after intervention. Morris water maze test was used to detect the learning and memory function of the rats. TTC staining was used to evaluate the cerebral infarction volume on the affected side. The protein expression levels of proBDNF, mBDNF, tissue plasminogen activator (tPA), tyrosine kinase receptor B (TrkB) and p75 neurotrophin receptor (p75NTR) in hippocampal tissue were detected by Western blot. RESULTS: Compared with the sham operation group, the neurological function score, the percentage of cerebral infarction volume and the expression levels of proBDNF and p75NTR protein in hippocampus were increased (P<0.01), while the times of crossing the original platform and the total distance in the target quadrant, the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were decreased (P<0.01, P<0.05) in the model group. Compared with the model group, the neurological function score, the percentage of cerebral infarction volume, and the expression levels of proBDNF and p75NTR protein in hippocampus were decreased (P<0.01, P<0.05), while the times of crossing the original platform, the total distance in the target quadrant, and the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were increased (P<0.05, P<0.01) in the EA group. CONCLUSIONS: EA can alleviate learning and memory impairment in IR rats, which may be related to its function in up-regulating the expression of tPA protein and promoting the transformation of proBDNF to mBDNF, thus improving the synaptic plasticity.

Effect of 2-Week Naringin Supplementation on Neurogenesis and BDNF Levels in Ischemia-Reperfusion Model of Rats.

BACKGROUND: Ischemic stroke is the leading cause of mortality and disability worldwide with more than half of survivors living with serious neurological sequelae; thus, it has recently attracted a lot of attention in the field of medical study. PURPOSE: The aim of this study was to determine the effect of naringin supplementation on neurogenesis and brain-derived neurotrophic factor (BDNF) levels in the brain in experimental brain ischemia-reperfusion. STUDY DESIGN: The research was carried out on 40 male Wistar-type rats (10-12 weeks old) obtained from the Experimental Animals Research and Application Center of Selçuk University. Experimental groups were as follows: (1) Control group, (2) Sham group, (3) Brain ischemia-reperfusion group, (4) Brain ischemia-reperfusion + vehicle group (administered for 14 days), and (5) Brain ischemia-reperfusion + Naringin group (100 mg/kg/day administered for 14 days). METHODS: In the ischemia-reperfusion groups, global ischemia was performed in the brain by ligation of the right and left carotid arteries for 30 min. Naringin was administered to experimental animals by intragastric route for 14 days following reperfusion. The training phase of the rotarod test was started 4 days before ischemia-reperfusion, and the test phase together with neurological scoring was performed the day before and 1, 7, and 14 days after the operation. At the end of the experiment, animals were sacrificed, and then hippocampus and frontal cortex tissues were taken from the brain. Double cortin marker (DCX), neuronal nuclear antigen marker (NeuN), and BDNF were evaluated in hippocampus and frontal cortex tissues by Real-Time qPCR analysis and immunohistochemistry methods. RESULTS: While ischemia-reperfusion increased the neurological score values, DCX, NeuN, and BDNF levels decreased significantly after ischemia in the hippocampus and frontal cortex tissues. However, naringin supplementation restored the deterioration to a certain extent. CONCLUSION: The results of the study show that 2 weeks of naringin supplementation may have protective effects on impaired neurogenesis and BDNF levels after brain ischemia and reperfusion in rats.

Effects of Banhabaekchulcheonma-Tang on Brain Injury and Cognitive Function Impairment Caused by Bilateral Common Carotid Artery Stenosis in a Mouse Model.

Vascular dementia (VD) is the second most prevalent dementia type, with no drugs approved for its treatment. Here, the effects of Banhabaekchulcheonma-Tang (BBCT) on ischemic brain injury and cognitive function impairment were investigated in a bilateral carotid artery stenosis (BCAS) mouse model. Mice were divided into sham-operated, BCAS control, L-BBCT (40 ml/kg), and H-BBCT (80 ml/kg) groups. BBCT's effects were characterized using the Y-maze test, novel object recognition test (NORT), immunofluorescence staining, RNA sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The NORT revealed cognitive function improvement in the H-BBCT group, while the Y-maze test revealed no significant difference among the four groups. The CD68+ microglia and GFAP+ astrocyte numbers were reduced in the H-BBCT group. Furthermore, H-BBCT treatment restored the dysregulation of gene expression caused by BCAS. The major BBCT targets were predicted to be cell division cycle protein 20 (CDC20), Epidermal growth factor (EGF), and tumor necrosis factor receptor-associated factor 1 (TRAF1). BBCT regulates the neuroactive ligand-receptor interaction and neuropeptide signaling pathways, as predicted by KEGG and GO analyses, respectively. BBCT significantly improved cognitive impairment in a BCAS mouse model by inhibiting microglial and astrocyte activation and regulating the expression of CDC20, EGF, TRAF1, and key proteins in the neuroactive ligand-receptor interaction and neuropeptide signaling pathways.

Mechanism of acupuncture in attenuating cerebral ischaemia-reperfusion injury based on nuclear receptor coactivator 4 mediated ferritinophagy.

OBJECTIVE: To explore the effect of acupuncture treatment on cerebral ischaemia-reperfusion injury (CIRI) and reveal the underlying mechanism of the effect based on nuclear receptor coactivator 4 (NCOA4) mediated ferritinophagy. METHODS: Sprague-Dawley male rats were divided into four groups: the sham group, model group, acupuncture group, and sham acupuncture group. After 2 h of middle cerebral artery occlusion (MCAO), reperfusion was performed for 24 h to induce CIRI. The rats were treated with acupuncture at the Neiguan (PC6) and Shuigou (GV26) acupoints. Their neurological function was evaluated by taking their Bederson scores at 2 h after ischaemia and 24 h after reperfusion. Triphenyltetrazolium chloride staining was applied to assess the cerebral infarct volume at 24 h after reperfusion. The malondialdehyde (MDA) and ferrous iron (Fe2+) levels were observed after 24 h of reperfusion using an assay kit. Western blotting was performed to detect the expression of NCOA4 and ferritin heavy chain 1 (FTH1) at 24 h after reperfusion. Moreover, the colocalization of ferritin with neurons, NCOA4 with microtubule-associated protein 1 light chain 3 (LC3), and NCOA4 with ferritin was visualized using immunofluorescence staining. RESULTS: Acupuncture significantly improved neurological function and decreased cerebral infarct volume in the acupuncture group. Following CIRI, the expression of NCOA4, LC3 and FTH1 was increased, which enhanced ferritinophagy and induced an inappropriate accumulation of Fe2+ and MDA in the ischaemic brain. However, acupuncture dramatically downregulated the expression of NCOA4, LC3 and FTH1, inhibited the overactivation of ferritinophagy, and decreased the levels of MDA and Fe2+. CONCLUSIONS: Acupuncture can inhibit NCOA4-mediated ferritinophagy and protect neurons against CIRI in a rat model.

Chuanzhitongluo capsule improves cognitive impairment in mice with chronic cerebral hypoperfusion via the cholinergic anti-inflammatory pathway.

Vascular cognitive impairment (VCI) has become a common disease-causing cognitive deficit in humans, second only to Alzheimer's Disease (AD). Chuanzhitongluo capsule (CZTL) is a Traditional Chinese Medicine (TCM) preparation known for its effective protection against cerebral ischemia. However, its potential to ameliorate VCI remains unclear. This study aimed to investigate the cognitive improvement effects of CZTL in a mouse model of VCI. Chronic cerebral hypoperfusion (CCH) was induced in mice by bilateral common carotid artery stenosis (BCAS) to simulate the pathological changes associated with VCI. Spatial learning and memory abilities were assessed using the Morris Water Maze (MWM). RNA sequencing (RNA-Seq) was employed to identify differentially expressed genes (DEGs) in the hippocampus. Levels of inflammatory factors were measured through enzyme-linked immunosorbent assay (ELISA), while immunofluorescence (IF) determined the expression intensity of target proteins. Western Blot (WB) confirmed the final action pathway. Results indicated that CZTL significantly improved the spatial learning and memory abilities of CCH mice, along with alterations in gene expression profiles in the hippocampus. It also reduced neuroinflammation in the hippocampus and upregulated the choline acetyltransferase (ChAT) and α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR), which are in synaptic plasticity and neuronal development. Moreover, CZTL inhibited the NF-κB signaling pathway. In conclusion, CZTL may alleviate neuroinflammation induced by CCH and improve cognitive impairment in CCH mice by regulating the cholinergic anti-inflammatory pathway (CAIP) involving ChAT/α7nAChR/NF-κB.

Synergistic amelioration between Ligusticum striatum DC and borneol against cerebral ischemia by promoting astrocytes-mediated neurogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort (LCH), with the accepted name of Ligusticum striatum DC in "The Plant List" database, is a widely used ethnomedicine in treating ischemic stroke, and borneol (BO) is usually prescribed with LCH for better therapy. Our previous study confirmed their synergistic effect on neurogenesis against cerebral ischemia. However, the underlying mechanism is still unclear. AIM OF THE STUDY: More and more evidence indicated that astrocytes (ACs) might be involved in the modulation of neurogenesis via polarization reaction. The study was designed to explore the synergic mechanism between LCH and BO in promoting astrocyte-mediated neurogenesis. MATERIALS AND METHODS: After primary cultures and identifications of ACs and neural stem cells (NSCs), the oxygen-glucose deprivation (OGD) model and the concentrations of LCH and BO were optimized. After the OGD-injured ACs were treated by LCH, BO, and their combination, the conditioned mediums were used to culture the OGD-injured NSCs. The proliferation, migration, and differentiation of NSCs were assessed, and the secretions of BDNF, CNTF, and VEGF from ACs were measured. Then the expressions of C3 and PTX3 were detected. Moreover, the mice were performed a global cerebral ischemia/reperfusion model and treated with LCH and (or) BO. After the assessments of Nissl staining, the expressions of Nestin, DCX, GFAP, C3, PTX3, p65 and p-p65 were probed. RESULTS: The most appropriate duration of OGD for the injury of both NSCs and ACs was 6 h, and the optimized concentrations of LCH and BO were 1.30 µg/mL and 0.03 µg/mL, respectively. The moderate OGD environment induced NSCs proliferation, migration, astrogenesis, and neurogenesis, increased the secretions of CNTF and VEGF from ACs, and upregulated the expressions of C3 and PTX3. For the ACs, LCH further increased the secretions of BDNF and CNTF, enhanced PTX3 expression, and reduced C3 expression. Additionally, the conditioned medium from LCH-treated ACs further enhanced NSC proliferation, migration, and neurogenesis. The in vivo study showed that LCH markedly enhanced the Nissl score and neurogenesis, and decreased astrogenesis which was accompanied by downregulations of C3, p-p65, and p-p65/p65 and upregulation of PTX3. BO not only decreased the expression of C3 in ACs both in vitro and in vivo but also downregulated p-p65 and p-p65/p65 in vivo. Additionally, BO promoted the therapeutic effect of LCH for most indices. CONCLUSION: A certain degree of OGD might induce ACs to stimulate the proliferation, astrogenesis, and neurogenesis of NSCs. LCH and BO exhibited a marked synergy in promoting ACs-mediated neurogenesis and reducing astrogenesis, in which LCH played a dominant role and BO boosted the effect of LCH. The mechanism of LCH might be involved in switching the polarization of ACs from A1 to A2, while BO preferred to inhibit the formation of A1 phenotype via downregulating NF-κB pathway.

Moxibustion preconditioning reduces inflammatory response in rats with cerebral ischemia-reperfusion injury by regulating PI3K / AKT / mTOR signaling pathway. / 艾灸预处理通过调控PI3K/AKT/mTORä¿¡å·é€šè·¯å‡è½»è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤å¤§é¼ ç‚Žæ€§ååº”.

OBJECTIVES: To observe the effect of moxibustion preconditioning on inflammatory response in rats with cerebral ischemia reperfusion injury (CIRI), so as to explore its mechanisms underlying improving CIRI. METHODS: Seventy-five male SD rats were randomly divided into sham operation, model, moxibustion preconditioning 3 days (Moxi 1), moxibustion preconditioning 5 days (Moxi 2) and moxibustion preconditioning 7 days (Moxi 3) groups, with 15 rats in each group. Moxibustion was applied at "Baihui"(GV20), "Dazhui"(GV14) and "Zusanli"(ST36) for 20 min once a day, totally for 3, 5 or 7 days. Thirty minutes after the last moxibustion treatment, the CIRI model was established by occlusion of the middle cerebral artery. The neurological deficit score was assessed by using Longa's method. The infarct size of the brain assessed after staining with 2% triphenyltetrazolium chloride (TTC). The morphological changes of cortical neurons were observed by HE staining. The contents of inflammatory factors interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), S-100ß protein (S-100ß) and neuron-specific enolase (NSE) were detected by ELISA. The expression of phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and mammalian target of rapamycin (mTOR) proteins in the ischemic cortex tissues were detected by immunohistochemistry and Western blot. RESULTS: Compared with the sham operation group, the neurological function score and the percentage of cerebral ischemic volume were increased (P<0.01). The contents of serum IL-1ß, TNF-α, S-100ß and NSE were significantly increased (P<0.01), while the protein expressions of PI3K, p-PI3K, AKT and mTOR in the cerebral cortex were significantly decreased (P<0.01) in the model group. Compared with the model group, the neurological function score and the percentage of cerebral ischemic volume were significantly decreased (P<0.01). The contents of serum IL-1ß, TNF-α, S-100ß and NSE were significantly decreased (P<0.01), and the expressions of PI3K, p-PI3K, AKT and mTOR proteins in the cerebral cortex were significantly increased (P<0.01) in three moxibustion groups. Compared with the Moxi 1 and Moxi 2 groups, the above indicators were significantly improved in rats of the Moxi 3 group (P<0.01, P<0.05). CONCLUSIONS: Moxibustion preconditioning can significantly improve the neurological function of rats after ischemia-reperfusion, inhibit serum inflammatory factors IL-1 ß and TNF-α, inhibit brain tissue injury markers S-100ß and NSE, which may be related to the activation of PI3K/AKT/mTOR signaling pathway. The protective effect of moxibustion preconditioning for 7 days on CIRI was better than that of 5 days and 3 days.

Progress of researches on mechanisms of acupuncture underlying improvement of cerebral ischemia-reperfusion injury by regulating calcium overload. / é’ˆåˆºè°ƒæŽ§è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤åŽé’™è¶ è½½æœºåˆ¶ç ”ç©¶è¿›å±•.

Ischemic stroke is currently the most common type of stroke, and the key pathological link is cerebral ischemia-reperfusion injury (CIRI), while the key factor leading to apoptosis and necrosis of ischemic nerve cells is calcium overload. Current studies have confirmed that acupuncture therapy has a good modulating effect on calcium homeostasis and can reduce cerebral ischemia-reperfusion induced damage of neuronal cells by inhibiting calcium overload. After reviewing the relevant literature published in the past 15 years, we find that acupuncture plays a role in regulating the pathological mechanism of calcium overload after CIRI by inhibiting the opening of connexin 43 hemichannels, regulating the intracellular free calcium ion concentration, suppressing the expression of calmodulin, and blocking the function of L-type voltage-gated calcium channels, thereby inhibiting calcium overload, regulating calcium homeostasis and antagonizing neuronal damage resulted from cerebral ischemia-reperfusion, which may provide ideas for future research.

FDA compound library screening Baicalin upregulates TREM2 for the treatment of cerebral ischemia-reperfusion injury.

Acute ischemic stroke (AIS) is a leading cause of global incidence and mortality rates. Oxidative stress and inflammation are key factors in the pathogenesis of AIS neuroinjury. Therefore, it is necessary to develop drugs that target neuroinflammation and oxidative stress in AIS. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), primarily expressed on microglial cell membranes, plays a critical role in reducing inflammation and oxidative stress in AIS. In this study, we employed a high-throughput screening (HTS) strategy to evaluate 2625 compounds from the (Food and Drug Administration) FDA library in vitro to identify compounds that upregulate the TREM2 receptor on microglia. Through this screening, we identified Baicalin as a potential drug for AIS treatment. Baicalin, a flavonoid compound extracted and isolated from the root of Scutellaria baicalensis, demonstrated promising results. Next, we established an in vivo mouse model of cerebral ischemia-reperfusion injury (MCAO/R) and an in vitro microglia cell of oxygen-glucose deprivation reperfusion (OGD/R) to investigate the role of Baicalin in inflammation injury, oxidative stress, and neuronal apoptosis. Our results showed that baicalin effectively inhibited microglia activation, reactive oxygen species (ROS) production, and inflammatory responses in vitro. Additionally, baicalin suppressed neuronal cell apoptosis. In the in vivo experiments, baicalin not only improved neurological functional deficits and reduced infarct volume but also inhibited microglia activation and inflammatory responses. Overall, our findings demonstrate the efficacy of Baicalin in treating MCAO/R by upregulating TREM2 to reduce inflammatory responses and inhibit neuronal apoptosis.

Cathodal bilateral transcranial direct-current stimulation regulates selenium to confer neuroprotection after rat cerebral ischaemia-reperfusion injury.

Non-invasive transcranial direct-current stimulation (tDCS) is a safe ischaemic stroke therapy. Cathodal bilateral tDCS (BtDCS) is a modified tDCS approach established by us recently. Because selenium (Se) plays a crucial role in cerebral ischaemic injury, we investigated whether cathodal BtDCS conferred neuroprotection via regulating Se-dependent signalling in rat cerebral ischaemia-reperfusion (I/R) injury. We first showed that the levels of Se and its transport protein selenoprotein P (SEPP1) were reduced in the rat cortical penumbra following I/R, whereas cathodal BtDCS prevented the reduction of Se and SEPP1. Interestingly, direct-current stimulation (DCS) increased SEPP1 level in cultured astrocytes subjected to oxygen-glucose deprivation reoxygenation (OGD/R) but had no effect on SEPP1 level in OGD/R-insulted neurons, indicating that DCS may increase Se in ischaemic neurons by enhancing the synthesis and secretion of SEPP1 in astrocytes. We then revealed that DCS reduced the number of injured mitochondria in OGD/R-insulted neurons cocultured with astrocytes. DCS and BtDCS prevented the reduction of the mitochondrial quality-control signalling, vesicle-associated membrane protein 2 (VAMP2) and syntaxin-4 (STX4), in OGD/R-insulted neurons cocultured with astrocytes and the ischaemic brain respectively. Under the same experimental conditions, downregulation of SEPP1 blocked DCS- and BtDCS-induced upregulation of VAMP2 and STX4. Finally, we demonstrated that cathodal BtDCS increased Se to reduce infract volume following I/R. Together, the present study uncovered a molecular mechanism by which cathodal BtDCS confers neuroprotection through increasing SEPP1 in astrocytes and subsequent upregulation of SEPP1/VAMP2/STX4 signalling in ischaemic neurons after rat cerebral I/R injury. KEY POINTS: Cathodal bilateral transcranial direct-current stimulation (BtDCS) prevents the reduction of selenium (Se) and selenoprotein P in the ischaemic penumbra. Se plays a crucial role in cerebral ischaemia injury. Direct-current stimulation reduces mitochondria injury and blocks the reduction of vesicle-associated membrane protein 2 (VAMP2) and syntaxin-4 (STX4) in oxygen-glucose deprivation reoxygenation-insulted neurons following coculturing with astrocytes. Cathodal BtDCS regulates Se/VAMP2/STX4 signalling to confer neuroprotection after ischaemia.

Multiomics profiling of the therapeutic effect of Dan-deng-tong-nao capsule on cerebral ischemia-reperfusion injury.

BACKGROUND: Stroke is a complex physiological process associated with intestinal flora dysbiosis and metabolic disorders. Dan-deng-tong-nao capsule (DDTN) is a traditional Chinese medicine used clinically to treat cerebral ischemia-reperfusion injury (CIRI) for many years. However, little is known about the effects of DDTN in the treatment of CIRI from the perspective of gut microbiota and metabolites. PURPOSE: This study aimed to investigate the regulatory roles of DDTN in endogenous metabolism and gut microbiota in CIRI rats, thus providing a basis for clinical rational drug use and discovering natural products with potential physiological activities in DDTN for the treatment of CIRI. METHODS: The chemical composition of DDTN in vitro and in vivo was investigated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLCHRMS), followed by target prediction using reverse molecular docking. Secondly, a biological evaluation of DDTN ameliorating neural damage in CIRI was performed at the whole animal level. Then, an integrated omics approach based on UHPLCHRMS and 16S rRNA sequencing was proposed to reveal the anti-CIRI effect and possible mechanism of DDTN. Finally, exploring the intrinsic link between changes in metabolite profiles, changes in the intestinal flora, and targets of components to reveal DDTN for the treatment of CIRI. RESULTS: A total of 112 chemical components of DDTN were identified in vitro and 10 absorbed constituents in vivo. The efficacy of DDTN in the treatment of CIRI was confirmed by alleviating cerebral infarction and neurological deficits. After the DDTN intervention, 21 and 26 metabolites were significantly altered in plasma and fecal, respectively. Based on the fecal microbiome, a total of 36 genera were enriched among the different groups. Finally, the results of the network integration analysis showed that the 10 potential active ingredients of DDTN could mediate the differential expression of 24 metabolites and 6 gut microbes by targeting 25 target proteins. CONCLUSION: This study was the first to outline the landscapes of metabolites as well as gut microbiota regulated by DDTN in CIRI rats using multi-omics data, and comprehensively revealed the systematic relationships among ingredients, targets, metabolites, and gut microbiota, thus providing new perspectives on the mechanism of DDTN in the treatment of CIRI.

Notoginsenoside R1 alleviates cerebral ischemia/reperfusion injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway through microbiota-gut-brain axis.

BACKGROUND: Ischemic stroke (IS) ranks as the second common cause of death worldwide. However, a narrow thrombolysis timeframe and ischemia-reperfusion (I/R) injury limits patient recovery. Moreover, anticoagulation and antithrombotic drugs do not meet the clinical requirements. Studies have demonstrated close communication between the brain and gut microbiota in IS. Notoginsenoside R1 (NG-R1), a significant component of the total saponins from Panax notoginseng, has been demonstrated to be effective against cerebral I/R injury. Total saponins have been used to treat IS in Chinese pharmacopoeia. Furthermore, previous research has indicated that the absorption of NG-R1 was controlled by gut microbiota. STUDY DESIGN: This study aimed to access the impact of NG-R1 treatment on neuroinflammation and investigate the microbiota-related mechanisms. RESULTS: NG-R1 significantly reduced neuronal death and neuroinflammation in middle cerebral artery occlusion/reperfusion (MCAO/R) models. 16S rRNA sequencing revealed that NG-R1 treatment displayed the reversal of microbiota related with MCAO/R models. Additionally, NG-R1 administration attenuated intestinal inflammation, gut barrier destruction, and systemic inflammation. Furthermore, microbiota transplantation from NG-R1 exhibited a similar effect in the MCAO/R models. CONCLUSION: In summary, NG-R1 treatment resulted in the restoration of the structure of the blood-brain barrier (BBB) and reduction in neuroinflammation via suppressing the stimulation of astrocytes and microglia in the cerebral ischemic area. Mechanistic research demonstrated that NG-R1 treatment suppressed the toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor kappa B (TLR4/MyD88/NF-κB) signaling pathway in both the ischemic brain and colon. NG-R1 treatment enhanced microbiota dysbiosis by inhibiting the TLR4 signaling pathway to protect MCAO/R models. These findings elucidate the mechanisms by which NG-R1 improve stroke outcomes and provide some basis for Panax notoginseng saponins in clinical treatment.

Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Metabolomics-Based Effects of a Natural Product on Remyelination After Cerebral Ischemia Injury Via GABABR-pCREB-BDNF Pathway.

BACKGROUND: Yi-Qi-Tong-Luo Granules (YQTLs) is a natural compound of Traditional Chinese Medicine authorized by China Food and Drug Administration (CFDA). These granules are employed in the convalescent stage of cerebral infarction and render notable clinical efficacy. This study aims to uncover the underlying mechanisms of YQTLs on remyelination after cerebral ischemia injury. MATERIALS AND METHODS: We established cerebral ischemia model in rats using microsphere-induced multiple cerebral infarction (MCI). We evaluated the pharmacological effects of YQTLs on MCI rats, through Morri's water maze test, open field test, hematoxylin and eosin staining, and glycine silver immersion. We employed liquid chromatography mass spectrometry metabolomics to identify differential metabolites. Enzyme-linked immunosorbent assay was utilized to measure the release of neurotrophins, while immunofluorescence staining was used to assess oligodendrocyte precursor cells differences and myelin regeneration. We used Western blotting to validate the protein expression of remyelination-associated signaling pathways. RESULTS: YQTLs significantly improves cognitive function following cerebral ischemia injury. Pathological tissue staining revealed that YQTLs administration inhibits neuronal denaturation and neurofibrillary tangles. We identified 141 differential metabolites among the sham, MCI, and YQTLs-treated MCI groups. Among these metabolites, neurotransmitters were identified, and notably, gamma-aminobutyric acid (GABA) showed marked improvement in the YQTLs group. The induction of neurotrophins, such as brain-derived neurotrophic factor (BDNF) and PDGFAA, upregulation of olig2 and MBP expression, and promotion of remyelination were evident in YQTLs-treated MCI groups. Gamma-aminobutyric acid B receptors (GABABR), pERK/extracellular regulated MAP kinase, pAKT/protein kinase B, and pCREB/cAMP response element-binding were upregulated following YQTLs treatment. CONCLUSION: YQTLs enhance the binding of GABA to GABABR, thereby activating the pCREB/BDNF signaling pathway, which in turn increases the expression of downstream myelin-associated proteins and promotes remyelination and cognitive function.

Effect of acupuncture on ischaemic stroke in patients with rheumatoid arthritis: a nationwide propensity score-matched study.

OBJECTIVE: To demonstrate that acupuncture is beneficial for decreasing the risk of ischaemic stroke in patients with rheumatoid arthritis (RA). DESIGN: A propensity score-matched cohort study. SETTING: A nationwide population-based study. PARTICIPANTS: Patients with RA diagnosed between 1 January 1997 and 31 December 2010, through the National Health Insurance Research Database in Taiwan. INTERVENTIONS: Patients who were administered acupuncture therapy from the initial date of RA diagnosis to 31 December 2010 were included in the acupuncture cohort. Patients who did not receive acupuncture treatment during the same time interval constituted the no-acupuncture cohort. PRIMARY OUTCOME MEASURES: A Cox regression model was used to adjust for age, sex, comorbidities, and types of drugs used. We compared the subhazard ratios (SHRs) of ischaemic stroke between these two cohorts through competing-risks regression models. RESULTS: After 1:1 propensity score matching, a total of 23 226 patients with newly diagnosed RA were equally subgrouped into acupuncture cohort or no-acupuncture cohort according to their use of acupuncture. The basic characteristics of these patients were similar. A lower cumulative incidence of ischaemic stroke was found in the acupuncture cohort (log-rank test, p<0.001; immortal time (period from initial diagnosis of RA to index date) 1065 days; mean number of acupuncture visits 9.83. In the end, 341 patients in the acupuncture cohort (5.95 per 1000 person-years) and 605 patients in the no-acupuncture cohort (12.4 per 1000 person-years) experienced ischaemic stroke (adjusted SHR 0.57, 95% CI 0.50 to 0.65). The advantage of lowering ischaemic stroke incidence through acupuncture therapy in RA patients was independent of sex, age, types of drugs used, and comorbidities. CONCLUSIONS: This study showed the beneficial effect of acupuncture in reducing the incidence of ischaemic stroke in patients with RA.