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.

Electroacupuncture Alleviates Post-stroke Cognitive Impairment Through Inhibiting miR-135a-5p/mTOR/NLRP3 Axis-mediated Autophagy.

Post-stroke cognitive impairment is a significant challenge with limited treatment options. Electroacupuncture (EA) has shown promise in improving cognitive function after stroke. Our study explores the underlying mechanism of EA in alleviating cognitive impairment through the inhibition of autophagy. We utilized a rat model of stroke induced by middle cerebral artery occlusion (MCAO) to evaluate the efficacy of EA. Treatment with EA was observed to markedly improve cognitive function and reduce inflammation in MCAO rats, as evidenced by decreased neurological deficit scores, shorter latencies in the water maze test, and diminished infarct volumes. EA also attenuated tissue damage in the hippocampus and lowered the levels of pro-inflammatory cytokines and oxidative stress markers. Although autophagy was upregulated in MCAO rats, EA treatment suppressed this process, indicated by a reduction in autophagosome formation and alteration of autophagy-related protein expression. The protective effects of EA were reversed by the autophagy activator rapamycin. EA treatment elevated the levels of microRNA (miR)-135a-5p expression, and suppression of this elevation attenuated the remedial efficacy of EA in addressing cognitive impairment and inflammation. MiR-135a-5p targeted mammalian target of rapamycin (mTOR)/NOD-like receptor protein 3 (NLRP3) signaling to repress autophagy. EA treatment inhibits autophagy and alleviates cognitive impairment in post-stroke rats. It exerts its beneficial effects by upregulating miR-135a-5p and targeting the mTOR/NLRP3 axis.

Electroacupuncture ameliorates neuroinflammation by inhibiting TRPV4 channel in ischemic stroke.

AIMS: We investigated the potential mechanisms underlying the therapeutic efficacy of electroacupuncture (EA) at the Shuigou (GV26) and Baihui (GV20) acupoints in the treatment of ischemic stroke. METHODS: We assessed the therapeutic effects of EA on MCAO mice through behavioral studies and TTC staining. Various techniques, such as RT-PCR, immunofluorescence, and Western blots, were employed to evaluate the activation and polarization of microglia/macrophages, and changes in the TRPV4 ion channel. We used the TRPV4 antagonist GSK2193874 (GSK219) to verify the involvement of TRPV4 in the therapeutic effects of EA. RESULTS: EA effectively improved neurological impairments and reduced cerebral infarction volume in MCAO mice. It suppressed activated microglia/macrophages and inhibited their polarization toward the M1 phenotype post-MCAO. EA also downregulated the expression of pro-inflammatory cytokines, including Tnf-α, Il-6, Il-1ß, and Ccl-2 mRNA. Furthermore, EA reduced the elevated expression of TRPV4 following MCAO. Treatment with the TRPV4 antagonist GSK219 mirrored the effects of EA in MCAO mice. Notably, the combination of EA and GSK219 did not demonstrate an additive or synergistic effect. CONCLUSION: EA may inhibit neuroinflammation and exhibit a protective effect against ischemic brain injury by suppressing TRPV4 and the subsequent M1 polarization of microglia/macrophages.

E. globulus leaf EO exhibits anti-inflammatory effects by regulating GSDMD-mediated pyroptosis, thereby alleviating neurological impairment and neuroinflammation in experimental stroke mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aromatic and medicinal plants continue to be a major component of alternative and traditional medicine in the developing countries. Eucalyptus globulus (Labill.) is being employed to cultivation and production in China. However, few studies have reported the chemical composition and anti-inflammatory activity of Eucalyptus globulus (Labill.) leaf essential oil (E. globulus leaf EO) extracted from Eucalyptus globulus. AIM OF THE STUDY: This study aimed to assess the composition of E. globulus leaf EO and identify its bacteriostatic action as well as anti-inflammatory activity. Importantly, we evaluated the effect of E. globulus leaf EO on neurological impairment and neuroinflammation in experimental stroke mice. MATERIALS AND METHODS: Gas Chromatography-Mass Spectrometer (GC-MS) analyses was employed to evaluate the chemical components of E. globulus leaf EO, and the relative content of each component was determined by area normalization method. The antimicrobial activity of E. globulus leaf EO was determined by Oxford cup method and microbroth dilution assay. Cytotoxic activity of E. globulus leaf EO on THP-1 cells or BV2 cells in vitro was determined by CCK8 assay. In addition, the lipopolysaccharide (LPS)/ATP-induced inflammation model in THP-1 cells or BV2 cells were established, and the relative expression of TNF-α, IL-1ß, MCP-1and IL-6 were confirmed by RT-PCR. Furthermore, the expression of protein GSDMD, IL-lß, NLRP3 and NFκB signaling pathway were assessed by immunoblotting. In vivo,the experimental stroke model constructed by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice was employed and subsequently treated with E. globulus leaf EO (50,100 mg/kg, subcutaneous injection) for 3 days to assess neurological impairment and neuroinflammation. Behavioral and neuronal damage were assessed using grip strength test, rod trarod test, and Nissl staining. Pro-inflammatory factors in serum or ischemic brain tissue was detected by ELISA kits. RESULTS: GC-MS analyses revealed that the major compound in E. globulus leaf EO was eudesmol (71.967%). E. globulus leaf EO has antimicrobial activity against Staphylococcus aureus (gram positive bacteria, MIC = 0.0625 mg/mL), Escherichia coli (gram negative bacteria, MIC = 1 mg/mL), and Candida albicans (MIC = 4 mg/mL). E. globulus leaf EO (0.5312, 1.0625, and 2.15 mg/mL) significantly decreased the expression of inflammation-related genes, including IL-1ß, TNF-α, MCP-1, and IL-6. Furthermore, reduced levels of TLR4, Myd88, phosphorylated NF-κB P65, and IκBα were found in the E. globulus leaf EO group for BV2 cells (1.025, and 2.125 mg/mL). In addition, the expression levels of GSDMD, NLRP3, IL-1ß and AIM2 were significantly decreased in the E. globulus leaf EO group when compared with the LPS -stimulated group, regulating GSDMD-mediated pyroptosis. In vivo, E. globulus leaf EO improved neurological functional deficits, inhibited excessive activation of microglia, and reduced the secretion of pro-inflammatory factors IL-1ß, TNF-α in the ischemic tissue and serum after MCAO/R. CONCLUSION: E. globulus leaf EO has strong antibacterial and anti-inflammatory activity, which has been implicated in blocking GSDMD-mediated pyroptosis. Moreover, E. globulus leaf EO could exert neuroprotective effect on cerebral ischemia-reperfusion injury.

METTL3-dependent N6-methyladenosine modification is involved in berberine-mediated neuroprotection in ischemic stroke by enhancing the stability of NEAT1 in astrocytes.

Ischemic stroke (IS) is one of the principal causes of disability and death worldwide. Berberine (BBR), derived from the traditional Chinese herbal medicine Huang Lian, has been reported to inhibit the progression of stroke, but the specific mechanism whereby BBR modulates the progression of ischemic stroke remains unclear. N6-methyladenosine (m6A) modification is the most typical epigenetic modification of mRNA post-transcriptional modifications, among which METTL3 is the most common methylation transferase. During the study, the middle cerebral artery occlusion/reperfusion (MCAO/R) was established in mice, and the mice primary astrocytes and neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was simulated in vitro. Level of LncNEAT1, miR-377-3p was detected via RT-qPCR. The levels of Nampt and METTL3 were measured by Western blot. CCK8 and LDH assay was performed to detect cell viability. Here, we found that berberine alleviates MCAO/R-induced ischemic injury and up-regulates the expression of Nampt in astrocytes, miR-377-3p inhibits the expression of Nampt in astrocytes after OGD/R, thus promoting neuronal injury. NEAT1 binds to miR-377-3p in OGD/R astrocytes and plays a neuronal protective role as a ceRNA. METTL3 can enhance NEAT1 stability in OGD/R astrocytes by modulating m6A modification of NEAT1. Taken together, our results demonstrate that berberine exerts neuroprotective effects via the m6A methyltransferase METTL3, which regulates the NEAT1/miR-377-3p/Nampt axis in mouse astrocytes to ameliorate cerebral ischemia/reperfusion (I/R) injury.

Gypenoside Pretreatment Alleviates the Cerebral Ischemia Injury via Inhibiting the Microglia-Mediated Neuroinflammation.

Neuroinflammation is closely related to prognosis in ischemic stroke. Microglia are the main immune cells in the nervous system. Under physiological conditions, microglia participate in clearance of dead cells, synapse pruning and regulation of neuronal circuits to maintain the overall health of the nervous system. Once ischemic stroke occurs, microglia function in the occurrence and progression of neuroinflammation. Therefore, the regulation of microglia-mediated neuroinflammation is a potential therapeutic strategy for ischemic stroke. The anti-inflammatory activity of gypenosides (GPs) has been confirmed to be related to the activity of microglia in other neurological diseases. However, the role of GPs in neuroinflammation after ischemic stroke has not been studied. In this study, we investigated whether GPs could reduce neuroinflammation by regulating microglia and the underlying mechanism through qRT-PCR and western blot. Results showed that GPs pretreatment mitigated blood-brain barrier (BBB) damage in the mice subjected to middle cerebral artery occlusion (MCAO) and improved motor function. According to the results of immunofluorescence staining, GPs pretreatment alleviated neuroinflammation in MCAO mice by reducing the number of microglia and promoting their phenotypic transformation from M1 to M2. Furthermore, GPs pretreatment reduced the number of astrocytes in the penumbra and inhibited their polarization into the A1 type. We applied oxygen and glucose deprivation (OGD) on BV2 cells to mimic ischemic conditions in vitro and found similar effect as that in vivo. At the molecular level, the STAT-3/HIF1-α and TLR-4/NF-κB/HIF1-α pathways were involved in the anti-inflammatory effects of GPs in vitro and in vivo. Overall, this research indicates that GPs are potential therapeutic agents for ischemic stroke and has important reference significance to further explore the possibility of GPs application in ischemic stroke.

Electroacupuncture Ameliorates Neuronal Injury by NLRP3/ASC/Caspase-1 Mediated Pyroptosis in Cerebral Ischemia-Reperfusion.

NLRP3/ASC/Caspase-1 mediated pyroptosis is one of the important causes of cerebral ischemia-reperfusion (I/R) injury. Electroacupuncture (EA) is widely used in clinical treatment of ischemic stroke. However, mechanism of EA on ischemic stroke remains unclear. Therefore, on basis of a previous work, this study used middle cerebral artery occlusion (MCAO) 2 h and then reperfusion 7 days in rats to simulate brain I/R process. EA with Bahui (GV20) and Zusanli (ST36) and VX-765 (a specific inhibitor of Caspase-1) was performed. In this study, we found that EA improved cerebral infarct size and neuronal damage, including ultrastructural injury, and ameliorated nitro/oxidative stress in cerebral I/R. Additionally, EA treatment significantly decreased ASC, Caspase-1, GSDMD, and IL-1ß expression and VX-765 treatment significantly decreased NLRP3, Caspase-1, and IL-1ß expression. This proved that EA can regulate NLRP3/ASC/Caspase-1 mediated pyroptosis, improve neuronal injury during cerebral I/R, and provide basic experimental data for clinical treatment.

Shexiang Tongxin dropping pills protect against ischemic stroke-induced cerebral microvascular dysfunction via suppressing TXNIP/NLRP3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Patients with ischemic stroke (IS) often continue to exhibit cerebral microcirculatory dysfunction even after receiving thrombolytic therapy. Enhancing the function of cerebral microvascular endothelia represents a pivotal advancement in the therapeutic strategy for ischemic microcirculatory disturbances. A traditional Chinese medicinal formulation named Shexiang Tongxin Dropping Pills (STDP), has been clinically employed to ameliorate microcirculatory abnormalities. Existing literature attests to the beneficial role of STDP on endothelial cells (ECs). Nevertheless, specific impacts and underlying mechanisms of STDP in rectifying IS-induced cerebral microvascular dysfunction warrant further exploration. AIM OF THE STUDY: This investigation seeks to delineate the effects of STDP on cerebral microvascular endothelial damage induced by ischemic stroke and to elucidate the underlying mechanism involved. MATERIALS AND METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) technique was employed to established ischemic stroke model in mice. The therapeutic efficacy of STDP on cerebral microvascular function was assessed through laser speckle contrast imaging, behavioral assays, and histological evaluations. Biochemical markers in the brain tissue, including GSH, SOD, MDA, and ROS, were quantified using specific assay kits. In vitro study, oxygen-glucose deprivation and reperfusion (OGD/R) was performed in bEnd.3 cells. The cytoprotective potential of STDP was then evaluated by measuring cell viability, LDH activity, endothelial permeability, and oxidative stress parameters. Important targets in critical pathway were verified by immunoblotting and immunofluorescence both in mice brain slices and bEnd.3 cells. RESULTS: STDP decrease brain infarct size, repaired microvascular cerebral blood flow and attenuated neurological deficiency in MCAO/R mice. Moreover, STDP abolished MCAO/R-induced oxidative stress which was reflected by rescuing GSH content, restoration of SOD activity and T-AOC, reduction of MDA and ROS. Ex vivo, STDP increased cerebral microvascular endothelial cells viability, abolished oxidative stress and decreased their permeability after ODG/R. Mechanistically, STDP significantly suppressed endothelial ROS-TXNIP mediated the activation of NLRP3 inflammasome in vivo and in vitro. CONCLUSION: STDP improves ischemic stroke-induced cerebral microcirculatory deficits by regulating cerebral microvascular endothelial ROS/TXNIP/NLRP3 signaling pathway.

Danlou tablet attenuates ischemic stroke injury and blood‒brain barrier damage by inhibiting ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danlou tablet (DLT) is a traditional Chinese medicinal formulation known for replenishing Qi, promoting blood circulation, and resolving stasis. Its pharmacological actions primarily involve anti-inflammatory, antioxidant stress reduction, antiapoptotic, proangiogenic, and improved energy metabolism. DLT has been confirmed to have favorable therapeutic effects on ischemic stroke (IS). However, the underlying mechanism through which DLT affects IS-induced brain injury remains unknown. AIM OF THE STUDY: This study aims to investigate the effects and underlying mechanisms of danlou tablet on ischemic stroke based on network pharmacology and experimental verification. MATERIALS AND METHODS: Using a transient middle cerebral artery occlusion (tMCAO) mouse model, the impact of DLT on the blood‒brain barrier (BBB) and brain injury in mice was assessed. Network pharmacology and bioinformatics analyses were utilized to explore the potential mechanisms of DLT in treating IS. Endothelial cells were cultured to observe the effects of DLT on vascular endothelial cells after oxygen-glucose deprivation/reperfusion, and these findings were validated in the brains of tMCAO mice. RESULTS: DLT alleviated oxidative stress and brain damage in tMCAO mice, mitigating BBB damage. A total of 185 potential targets through which DLT regulates IS were identified, including COX2, a known critical marker of ferroptosis, which identified as a key target. In vitro and in vivo experiments demonstrated that DLT significantly (p < 0.05) improved cell death and vascular barrier damage in IS, reducing intracellular oxidative stress and COX2 protein levels while increasing SLC7A11 and GPX4 protein levels. CONCLUSIONS: This study demonstrated that DLT maintained BBB integrity and alleviated brain injury of tMCAO mice by inhibiting ferroptosis. The study partially unraveled the mechanism through which DLT functioned in treating IS and further clarified the pivotal active components of DLT, thereby providing a theoretical scientific basis for treating IS with DLT.

Electroacupuncture at GB20 improves cognitive ability and synaptic plasticity via the CaM-CaMKII-CREB signaling pathway following cerebral ischemia-reperfusion injury in rats.

BACKGROUND: This study aimed to investigate the effects of electroacupuncture (EA) on cognitive recovery and synaptic remodeling in a rat model of middle cerebral artery occlusion (MCAO) followed by reperfusion and explore the possible mechanism. METHOD: Focal cerebral ischemia was modeled in healthy adult Sprague-Dawley rats by MCAO. The MCAO rats were classified into four groups: sham, MCAO, MCAO + GB20 (receiving EA at GB20) and MCAO + NA (receiving EA at a "non-acupoint" location not corresponding to any traditional acupuncture point location about 10 mm above the iliac crest). Neurological deficit scores and behavior were assessed before and during the treatment. After intervention for 7 days, the hippocampus was dissected to analyze growth-associated protein (GAP)-43, synaptophysin (SYN) and postsynaptic density protein (PSD)-95 expression levels by Western blotting. Bioinformatic analysis and primary hippocampal neurons with calcium-voltage gated channel subunit alpha 1B (CACNA1B) gene overexpression were used to screen the target genes for EA against MCAO. RESULTS: Significant amelioration of neurological deficits and learning/memory were found in MCAO + GB20 rats compared with MCAO or MCAO + NA rats. Protein levels of GAP-43, SYN and PSD-95 were significantly improved in MCAO + GB20-treated rats together with an increase in the number of synapses in the hippocampal CA1 region. CACNA1B appeared to be a target gene of EA in MCAO. There were increased mRNA levels of CACNA1B, calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and cyclic adenosine monophosphate response element binding (CREB) and increased phosphorylation of CaM, CaMKII and CREB in the hippocampal region in MCAO + GB20 versus MCAO and MCAO + NA groups. CACNA1B overexpression modulated expression of the CaM-CaMKII-CREB axis. CONCLUSION: EA treatment at GB20 may ameliorate the negative effects of MCAO on cognitive function in rats by enhancing synaptic plasticity. EA treatment at GB20 may exert this neuroprotective effect by regulating the CACNA1B-CaM-CaMKII-CREB axis.

Electroacupuncture pretreatment alleviates spasticity after stroke in rats by inducing the NF-κB/NLRP3 signaling pathway and the gut-brain axis.

OBJECTIVE: Spasticity is one of the most prevalent ischemic stroke sequelae and the leading cause of disability after stroke. Although electroacupuncture pretreatment has been shown to be effective in the treatment of ischemic stroke, its therapeutic effect and mechanism on post-stroke spasm remain unknown. The purpose of this study was to look into the potential mechanism of electroacupuncture pretreatment in inducing the NF-κB/NLRP3 signaling pathway and the gut-brain axis in the therapy of spasm after stroke. METHODS: After electroacupuncture treatment at Baihui (DU20) and Qubin (G87), the rat model of middle cerebral artery occlusion (MCAO) was first established. HE, Nissl, and TUNEL staining were used to detect pathological alterations in the rat brain. The relative levels of IL-4, IL-6, TNF-α, and TMAO were determined by ELISA. qRT-PCR and Western blot were used to evaluate the mRNA and protein levels of NF-κB p65, NLRP3, caspase3 and caspase9. Gas chromatography-mass spectrometry (GC-MS) was used to determine the levels of short-chain fatty acids (SCFAs) in rat gut. RESULTS: Hippocampal cells from rats with spasticity following stroke in the MCAO group were chaotic and loosely distributed with an unclear border, a blurred nucleolus, and vanished cytoplasm when compared to those from the sham operation group. Furthermore, the number of surviving neurons decreased while the number of apoptotic cells increased. In the I/R group, relative levels of IL-6, TNF-α, and TMAO increased considerably, while NF-κB p65, NLRP3, caspase3, and caspase9 were dramatically downregulated. The intestinal contents of n-propyl acetate and propyl butyrate were lowered in rats with spasticity following stroke. Electroacupuncture treatments miraculously remedied all of the foregoing pathogenic alterations. CONCLUSION: Pretreatment with electroacupuncture relieves spasticity after stroke by decreasing the inflammatory response, suppressing the NF-κB/NLRP3 signaling pathway, and modulating the gut-brain axis by increasing n-propyl acetate and propyl butyrate levels in the bowel. Our findings establish a new molecular mechanism and theoretical foundation for electroacupuncture therapy of ischemic stroke.

Characteristics of Electroacupuncture Preconditioning and Treatment on Regulation of the Brain Transcriptome in Rats with Ischemic Stroke.

BACKGROUND: Accumulating evidence suggests that acupuncture may serve as a potent strategy to mitigate the deleterious effects of ischemic stroke on neural tissue. The present investigation delineated the neuroprotective potential of electroacupuncture (EA) administered pre-and post-stroke, with a focus on determining the commonalities and disparities between these two therapeutic approaches in ameliorating ischemic stroke-induced brain injury. The ultimate objective is to inform optimal timing for acupuncture intervention in the clinical management and prevention of stroke. METHODS: The extent of cerebral infarction was quantified with 2,3,5-triphenyltetrazolium chloride staining. The integrity of the blood-brain barrier was assessed by evaluating the extravasation of Evans blue (EB) dye, while neurological function was appraised using the Longa neurological scoring system. RNA sequencing was employed to examine the transcriptomic landscape of ischemic brain tissue, with subsequent bioinformatics annotation of the sequencing data facilitated by Metascape. RESULTS: (1) A notable decrease in the ischemic infarct volume was observed in both the EA-preconditioned plus middle cerebral artery occlusion (MCAO), EA-preconditioned plus middle cerebral artery occlusion (EAM) and MCAO plus EA-treated (MEA) groups, compared to the MCAO group. Furthermore, the decreased leakage of EB and reduction in neurological function impairment scores were evident in the EAM and MEA groups compared with the MCAO group. (2) Relative to the Sham group, the MCAO group exhibited a total of 4798 differentially expressed genes (DEGs), with 67.84% demonstrating an expression fold change (FC) greater than 1.5, and 34.16% exceeding a FC of 2. The EAM and MEA groups displayed 4020 and 1956 DEGs, respectively, compared to the MCAO group. In both groups, more than 55% of DEGs showed an expression FC surpassing 1.5, whereas only approximately 10% exhibited a change greater than 2-fold. Remarkably, EA preconditioning and EA treatment resulted in the reversal of 18.72% and 28.91% of DEGs, respectively, in the MCAO group. (3) The DEGs upregulated in response to ischemic stroke were predominantly implicated in immune inflammatory processes and cellular apoptosis, whereas the downregulated DEGs were associated with neurogenesis and neuronal signal transduction. The MEA-induced upregulated DEGs were primarily involved in neural transmission and metabolic processes, whereas the downregulated DEGs were linked to excessive inflammatory responses to physical and chemical stimuli, as well as cell matrix adhesion chemotaxis. In the context of EAM, the upregulated DEGs were chiefly related to protein biosynthesis, and energy and metabolic processes, whereas the downregulated genes were connected to gene transcriptional activity, synaptic function, and neuronal architecture. CONCLUSIONS: Both preconditioning and post-event treatment with acupuncture demonstrated efficacy in mitigating pathological damage to brain tissue in a rat model of ischemic stroke, albeit with some divergences in their gene targets. The integration of EA preconditioning and treatment may potentially confer enhanced neuroprotection in the clinical management of stroke patients.

A preliminary investigation of the regulatory effect of acupuncture on hippocampal circHDAC2/miR-3065/SLC30A3 axis in CIRI rats. / å¤§é¼ æµ·é©¬ç»„ç»‡circHDAC2/miR-3065/SLC30A3è½´å‚ä¸Žé’ˆåˆºè°ƒèŠ‚è„‘ç¼ºè¡€å†çŒæ³¨æŸä¼¤çš„åˆæ­¥ç ”ç©¶.

OBJECTIVES: To explore the mechanisms of acupuncture against cerebral ischemia/reperfusion injury (CIRI) through observing the expression of circular RNA HDAC2 (circHDAC2) in the hippocampus of rats. METHODS: SD rats were randomly divided into sham-operation, model and acupuncture groups, with 13 rats in each group. The rat model of CIRI was established by middle cerebral artery occlusion. In the acupuncture group, acupuncture was delivvered at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20), and the needles were retained for 30 min each time and acupuncture was conducted once every 12 h for a total of 7 sessions. Before and after intervention, using modified Garcia scale, the neurological function of the rats were evaluated, and TTC staining was employed to determine the cerebral infarct area. Gene chip technology was used to screen the circRNAs with differential expressions in the ischemic hippocampus, and the circRNAs with co-differential expression (co-DE circRNAs) in the model group/sham-operation group, and the acupuncture group/model group separately. Among those circRNAs, the core circRNAs were screened according to P value, fold change (FC) and gene ontology (GO) analysis；and their expressions in the ischemic hippocampus were determined using quantitative real-time PCR (qPCR). Based on the verification results, a competing endogenous RNA (ceRNA) prediction network was constructed. The expression levels of microRNA (miRNA) and mRNA with high node centrality in the prediction network were validated by qPCR. RESULTS: Before intervention, compared with the sham-operation group, the modified Garcia score of each modeling group decreased (P<0.01). After intervention, the modified Garcia score was reduced and the cerebral infarct area ratio increased (P<0.01) in the model group when compared with the sham-operation group. In the acupuncture group, the modified Garcia score was higher and the cerebral infarct area ratio lower (P<0.01) than those of the model group. The microarray results of gene chip showed that 16 co-DE circRNAs were down-regulated in the model group and up-regulated in the acupuncture group, and 7 co-DE circRNAs up-regulated in the model group and down-regulated in the acupuncture group. The core circHDAC2 and circNTRK2 were screened according to P value, FC and the enrichment number of GO entries. QPCR results showed that, compared with the sham-operation group, the expression of circHDAC2 and circNTRK2 of the ischemic hippocampal tissue was down-regulated in the model group (P<0.01)；and that of circHDAC2 and circNTRK2 up-regulated in the acupuncture group when compared with the model group (P<0.01). The relevant ceRNA regulatory network was constructed for circHDAC2 and the prediction results showed that the regulatory networks contained 12 miRNAs and 31 mRNAs. Results of verifying miRNA with high network node centrality and mRNA relevant with nerve regulation showed that, when compared with the sham-operation group, the expression levels of miR-29a, miR-29b and the solute carrier family 30 member 3 (SLC30A3) mRNA in the ischemic hippocampus were down-regulated (P<0.01)；and those of miR-3065 and mercaptopyruvate sulfurtransferase (MPST) up-regulated (P<0.01) in the model group. Compared with the model group, the expressions of miR-29a, miR-29b and SLC30A3 mRNA in the ischemic hippocampus were up-regulated (P<0.01, P<0.05), while that of miR-3065 down-regulated (P<0.05) in the acupuncture group. CONCLUSIONS: Acupuncture significantly improves the neurological function and reduces the cerebral infarct area in CIRI rats, which may be related to the regulation of hippocampal circHDAC2/miR-3065/SLC30A3 axis.

The effects of protein-rich extract from Rhizoma Gastrodiae against cerebral ischemia/reperfusion injury via regulating MAPK and PI3K/AKT signaling pathway.

BACKGROUND: Rhizoma Gastrodiae is a highly valuable traditional Chinese medicine and functional health food that has been used in China to treat neurological disorders for thousands of years. Rhizoma Gastrodiae contains various of biological activities, such as antioxidative, neuroprotective, learning improvement, anxiolytic, and antidepressant effects. However, no studies have been conducted to explore the effects of the protein components in Rhizoma Gastrodiae (GEPS) and its potential protective effects against ischemic stroke.Our main goal was to investigate the effects of GEPS on ischemia/reperfusion (I/R) injury and its possible mechanisms. METHODS: A middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia mouse model and an oxygen-glucose deprivation (OGD/R) injury model in HT22 cells were established. A neurobehavioral test was performed 24 h after MCAO, and brain infarction was measured. A Morris water maze experiment was conducted on Day 14 after reperfusion in mice. Hematoxylin and eosin (HE) and TUNEL staining were performed to assess apoptotic neuronal death. Immunohistochemical analysis was used to detect BDNF and GAP43 expression. The content of SOD, MDA, GSH-PX and ROS were detected. The protein expression was analyzed using Western blotting. Cell viability was determined by MTT assay. Cell apoptosis was examined by flow cytometry. RESULTS: GEPS reduced apoptosis, decreased cerebral infarction, improved neurological defects, and ameliorated oxidative stress in the ischemic penumbra. In addition, GEPS increased the expression of BDNF and GA43 in the penumbra. Mechanistically, GEPS counteracted MCAO-induced PI3K/AKT inhibition and activation of MAPK signaling pathways. CONCLUSION: GEPS has a clear neuroprotective effect on I/R injury, and its mechanism may be linked to the PI3K/AKT and MAPK signaling pathways.

Electroacupuncture Alleviates Neuroinflammation by Regulating Microglia Polarization via STAT6/PPARγ in Ischemic Stroke Rats.

Previous study showed that electroacupuncture (EA) produced a protective effect on cerebral ischemia-reperfusion injury (CIRI) in rats and may correlate with the anti-inflammatory effects of microglia. This study aimed to investigate further whether EA could modulate neuroinflammation by targeting the Signal Transducer and Activator of Transcription 6 (STAT6) and Peroxisome Proliferator-Activated Receptor γ (PPARγ) pathway, the key regulator of microglia. Middle cerebral artery occlusion (MCAO) rats were used, and 6 h after reperfusion, EA interventions were performed in Chize (LU 5), Hegu (LI 4), Sanyinjiao (SP 6), and Zusanli (ST 36) on the affected side of the rats, the group that received EA + STAT6 phosphorylation inhibitor AS1517499 was used as a parallel control. The degree of neurological impairment, infarct volume, microglia polarization, inflammation levels and activity of STAT6/PPARγ pathway were then assessed by neurological deficit score, triphenyl tetrazolium chloride (TTC) staining, immunofluorescence, western blotting (WB), quantitative real-time PCR (qPCR) and Enzyme linked immunosorbent assay (ELISA). The data showed that EA significantly alleviated nerve injury, reduced infarct volume, enhanced the expression and activity of STAT6/PPARγ pathway, inhibited NF-κB activity, increased M2 microglia numbers and anti-inflammatory factor release, and inhibited microglia M1-type polarization and pro-inflammatory factor expression. In contrast, inhibition of STAT6 phosphorylation exacerbated neural damage, inhibited STAT6/PPARγ pathway activity, promoted microglia M1-type polarization and exacerbated neuroinflammation, resulting in an attenuated positive effect of EA intervention. Therefore, we concluded that EA intervention could attenuate microglia-associated neuroinflammation by enhancing the expression and activity of STAT6/PPARγ pathway, thereby reducing CIRI in MCAO rats.

Glycosides of Buyang Huanwu decoction inhibits pyroptosis associated with cerebral ischemia-reperfusion through Nrf2-mediated antioxidant signaling pathway both in vivo and in vitro.

BACKGROUND: Glycosides are the pharmacodynamic substances of Buyang Huanwu Decoction (BYHWD) and they exert a protective effect in the brain by inhibiting neuronal pyroptosis of cerebral ischemia-reperfusion (CIR). However, the mechanism by which glycosides regulate neuronal pyroptosis of CIR is still unclear. PURPOSE: A significant part of this study aimed to demonstrate whether glycosides have an anti-pyroptotic effect on CIR by nuclear factor erythroid 2-related factor (Nrf2)-mediated antioxidative mechanism. METHODS: Rats were used in vivo models of middle cerebral artery occlusion and reperfusion (MCAO/R). Neuroprotective effect of glycosides after Nrf2 inhibiting was observed by nerve function score, Nissl staining, Nrf2 fluorescence staining and pyroptotic proteins detection. SH-SY5Y cells were used in vitro models of oxygen-glucose deprivation/reperfusion (OGD/R). Glycosides was evaluated for their effects by measuring cell morphology, survival rate, lactate dehydrogenase (LDH) rate and expression of pyroptotic proteins. Nrf2 si-RNA 54-1 interference with lentivirus was used to create silenced Nrf2 SH-SY5Y cells (si-Nrf2-SH-SY5Y). Glycosides were evaluated on si-Con-SH-SY5Y and si-Nrf2-SH-SY5Y cells based on the expression of Nrf2 signaling pathway, pyroptotic proteins and cell damage manifestation. RESULTS: In vivo, glycosides significantly promoted the fluorescence level of nuclear Nrf2, added more Nissl bodies, reduced neurological function scores and inhibited the pyroptotic proteins level. In vitro, glycosides significantly repaired the morphological damage of cells, promoted the survival rate, reduced the LDH rate, inhibited the pyroptosis. Moreover, antioxidant activity of glycosides was enhanced via Nrf2 activation. Both Nrf2 blocking in vivo and Nrf2 silencing in vitro significantly weakened the pyroptosis inhibitory and neuroprotective effects of glycosides. CONCLUSION: These results suggested for the first time that glycosides inhibited neuronal pyroptosis by regulating the Nrf2-mediated antioxidant stress pathway, thereby exerting brain protection of CIR. As a result of this study, This study improved understanding of the pharmacodynamics and mechanism of BYHWD, as well as providing a Traditional Chinese Medicine (TCM) treatment strategy for CIR .

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia.

BACKGROUND: The urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. METHODS: The effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model. Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. RESULTS: We initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1ß, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. CONCLUSION: Crebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

Electroacupuncture Pretreatment Alleviates Cerebral Ischemia-reperfusion Injury by Down-regulating Mir-155-5p.

BACKGROUND: Increasing evidence shows that electroacupuncture pretreatment (EP) plays a crucial role in cerebral ischemia-reperfusion (I/R) injury, and cerebral I/R injury is the most serious complication of ischemic stroke treatment. The role of miR-155-5p in cerebral I/R injury has been studied, but the regulation of EP on miR-155-5p has not been reported. METHODS: The middle cerebral artery occlusion (MCAO) mice were used to investigate the role of EP in cerebral I/R injury. Longa and modified neurological severity scores (mNSS) were used to evaluate neurological impairment. HE staining and TUNEL staining were used to evaluate brain injury. The expressions of miR-155-5p, Yin Yang 1 (YY1) and p53 were detected by qRT-PCR. The expressions of related proteins were detected by western blot. The binding of YY1 to miR- 155-5p was verified by dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. Mice brain microvascular endothelial cells (BMECs) were isolated and cultured for in vitro experiments. Oxygen-glucose deprivation/reoxygenation (OGD/R) was used to verify the role of YY1, p53 and miR-155-5p in cerebral I/R injury in vitro. RESULTS: MCAO modeling induced brain injury, apoptosis, and increased levels of miR-155-5p, YY1, and p53. EP markedly alleviated brain injury and reduced levels of miR-155-5p, p53, and YY1. miR-155 agomir markedly increased the expression of miR-155-5p and p53. miR-155 antagomir decreased the levels of miR-155-5p and p53. Dual-luciferase reporter and ChIP assay verified that YY1 regulated miR-155-5p expression. YY1 shNRA greatly decreased miR-155-5p and p53. Inhibition of p53 decreased miR-155-5p expression. Both miR-155-5p inhibitor and YY1 shRNA promoted proliferation, inhibited apoptosis, and decreased levels of ICAM-1 and Eselectin of OGD/R-treated BMECs. Inhibition of p53 strengthened the effect of miR-155-5p inhibitor and YY1 shNRA on BMECs. CONCLUSION: Electroacupuncture pretreatment alleviates cerebral ischemia-reperfusion injury by regulating the YY1/p53/miR-155-5p axis.

Transplantation of hESCs-Derived Neural Progenitor Cells Alleviates Secondary Damage of Thalamus After Focal Cerebral Infarction in Rats.

Human embryonic stem cells-derived neural progenitor cells (hESCs-NPCs) transplantation holds great potential to treat stroke. We previously reported that delayed secondary degeneration occurs in the ventroposterior nucleus (VPN) of ipsilateral thalamus after distal branch of middle cerebral artery occlusion (dMCAO) in adult male Sprague-Dawley (SD) rats. In this study, we investigate whether hESCs-NPCs would benefit the neural recovery of the secondary damage in the VPN after focal cerebral infarction. Permanent dMCAO was performed with electrocoagulation. Rats were randomized into Sham, dMCAO groups with or without hESCs-NPCs treatment. HESCs-NPCs were engrafted into the peri-infarct regions of rats at 48 h after dMCAO. The transplanted hESCs-NPCs survive and partially differentiate into mature neurons after dMCAO. Notably, hESCs-NPCs transplantation attenuated secondary damage of ipsilateral VPN and improved neurological functions of rats after dMCAO. Moreover, hESCs-NPCs transplantation significantly enhanced the expression of BDNF and TrkB and their interaction in ipsilateral VPN after dMCAO, which was reversed by the knockdown of TrkB. Transplantated hESCs-NPCs reconstituted thalamocortical connection and promoted the formation of synapses in ipsilateral VPN post-dMCAO. These results suggest that hESCs-NPCs transplantation attenuates secondary damage of ipsilateral thalamus after cortical infarction, possibly through activating BDNF/TrkB pathway, enhancing thalamocortical projection, and promoting synaptic formation. It provides a promising therapeutic strategy for secondary degeneration in the ipsilateral thalamus post-dMCAO.

Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.

BACKGROUND: A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized. OBJECTIVE: To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels. METHODS: Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro. RESULTS: Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo. CONCLUSIONS: DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.