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Aim To screen and study the expression of long non-coding RNA (IncRNA) in rats with middle cerebral artery occlusion (MCAO) with MCAO treated with Tao Hong Si Wu decoction (THSWD) and determine the possible molecular mechanism of THSWD in treating MCAO rats. Methods Three cerebral hemisphere tissue were obtained from the control group, MCAO group and MCAO + THSWD group. RNA sequencing technology was used to identify IncRNA gene expression in the three groups. THSWD-regulated IncRNA genes were identified, and then a THSWD-regu-lated IncRNA-mRNA network was constructed. MCODE plug-in units were used to identify the modules of IncRNA-mRNA networks. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the enriched biological functions and signaling pathways. Cis- and trans-regulatory genes for THSWD-regulated IncRNAs were identified. Reverse transcription real-time quantitative pol-ymerase chain reaction (RT-qPCR) was used to verify IncRNAs. Molecular docking was used to identify IncRNA-mRNA network targets and pathway-associated proteins. Results In MCAO rats, THSWD regulated a total of 302 IncRNAs. Bioinformatics analysis suggested that some core IncRNAs might play an important role in the treatment of MCAO rats with THSWD, and we further found that THSWD might also treat MCAO rats through multiple pathways such as IncRNA-mRNA network and network-enriched complement and coagulation cascades. The results of molecular docking showed that the active compounds gallic acid and a-mygdalin of THSWD had a certain binding ability to protein targets. Conclusions THSWD can protect the brain injury of MCAO rats through IncRNA, which may provide new insights for the treatment of ischemic stroke with THSWD.
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Aim To investigate the dynamic time-course changes in neuronal cytoskeleton after acute ischemia and reperfusion in rats. Methods Reperfusion was performedin rats by blocking the middle cerebralarteryfor 90 min, then therats wereobserved and collected at different time points. The brain damage wasobserved by Nissl staining,and neurobehavioural function was evaluated with neurological deficit score and forelimb placement test. The cellular changes in the alternations of cytoskeletal elements including microtubule associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) were observed by immunohistochemistry staining and Western blot. Impaired axons, dendrites and cytoskeletal alternations were detected by electron microscope. Results Brain damage and neurobehavioural function were gradually aggravated with the prolongation of reperfusion. Brain damage appeared earlier and more severe in striatum than in cortex. Moreover, decreased MAP2-related and increased NF-H-related immunoreactive intensities were found in the ischemic areas. Impaired cytoskeletal arrangement and reduced dense were indicated. Damaged cytoskeletal components such as microtubules and neurofilament arrangement, decreased axonal filament density, and swelled dendrites were observed after cerebral ischemia reperfusion by ultrastructural observations. Conclusions Different brain regions have diverse tolerance to ischemia-reperfusion injury. Major elements of neuronal cytoskeleton show dynamic responses to ischemia and reperfusion, which may further contribute to brain damage and neurological impairment following MCAO and reperfusion.
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OBJECTIVE@#The present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats.@*METHODS@#Twenty-six rats were randomly divided into four groups, including sham group, sham + LTC group, MCAO group, and MCAO + LTC group. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC treatment group were orally administrated with 300 mg/kg of LTC once daily for seven consecutive days. LTC therapy was validated in terms of neurobehavioral abnormality evaluation, cerebral infarct area, and histological assessments. The plasma metabolome comparisons amongst different groups were conducted by UHPLC-Q Exactive MS in combination with subsequent multivariate statistical analysis, aiming to finding the molecules in respond to the surgery or LTC treatment.@*RESULTS@#Intragastric administration of LTC significantly decreased not only the neurobehavioral abnormality scores but also the cerebral infarct area of MCAO rats. The interstitial edema, atrophy, and pyknosis of glial and neuronal cells occurred in the infarcted area, core area, and marginal area of cerebral cortex were improved after LTC treatment. A total of 13 potential biomarkers were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE were more than 0.7, which were involved in neuroprotective process. The correlation and pathway analysis showed that LTC was beneficial to ischemic stroke rats via regulating glycerophospholipid and sphingolipid metabolism, together with nicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC.@*CONCLUSION@#The present study could provide evidence that metabolomics, as systematic approach, revealed its capacity to evaluate the holistic efficacy of TCM, and investigate the molecular mechanism underlying the clinical treatment of LTC on ischemic stroke.
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OBJECTIVES@#Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO).@*METHODS@#Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting.@*RESULTS@#The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO.@*CONCLUSIONS@#Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.
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Animals , Rats , Blood-Brain Barrier/metabolism , Brain Ischemia , Claudin-5/metabolism , Infarction, Middle Cerebral Artery/metabolism , Ischemic Stroke/metabolism , Occludin/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Reperfusion Injury/metabolismABSTRACT
To study the cognitive effects of diterpene ginkgolides (DG), transient middle cerebral artery occlusion (tMCAO)-induced rats were established. tMCAO-rats induced by suture method were divided into sham operation group, solvent control group, NBP group, DG group. The animal experiments in the present study were performed in accordance with the Ethical Guidelines of the Laboratory Animal Welfare Ethical Committee of Peking Union Medical College (00000646, 00000635). The effects of DG on tMCAO rats were evaluated by neurological severity score, cerebral infarction volume measurement, step-down and Morris water maze test. In the acute tMCAO rat model, 100 mg·kg-1 DG improved the neural score and infarction volume. In the chronic tMCAO rat model, DG 100 mg·kg-1 significantly improved the survival rate of tMCAO-induced rats. The Morris water maze results showed 100 mg·kg-1 DG decreased the latency of tMCAO-induced rats to find the platform, while the effect was weaker than the NBP. However, DG 30 mg·kg-1 did not show a significant effect. In conclusion, DG exerted a therapeutic effect on transient middle cerebral artery occlusion.
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ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.
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OBJECTIVE To study the intervention effect and mechanism of Zhongfeng yure decoction on ischemic stroke model rats. METHODS Totally 85 rats were randomly divided into sham operation group （normal saline， n＝15）， model control group （normal saline， n＝18）， Nimodipine tablet group （positive control， 10.8 mg/kg， n＝18）， high-dose group of Zhongfeng yure decoction （20.52 g/kg， n＝17） and low-dose group of Zhongfeng yure decoction （5.13 g/kg， n＝17）， respectively. After 7 days of preventive continuous administration （once a day）， except for the sham operation group， the rats’ middle cerebral artery occlusion （MCAO） model was established by the modified suture method in other groups. After modeling， the rats in each group continued to be administered for 3 days. During experiment， general condition of the rats was observed， and the neurological function score was performed. After the last administration， the organ index was calculated， the cerebral infarction area and pathological changes of brain tissue were observed. The levels of tumor necrosis factor-α （TNF-α） and interleukin 6 （IL-6） in brain tissue and serum， and the average optical density value of caspase-3 and phosphorylated protein kinase B（p-AKT） protein in brain tissue were detected. RESULTS Three days after modeling， compared with sham operation group， the neurological function score， in brain tissue index， spleen tissue index， proportion of cerebral infarction area， the levels of TNF-α and IL-6 in brain tissue and serum， and the average optical density value of caspase-3 protein in brain tissue were significantly increased in the model control group （P＜0.05 or P＜0.01）； karyopyknosis， diffuse edema and other lesions appeared in brain tissue. Compared with the model control group， the above indexes in each administration group were improved to varying degrees. Among them， there were significant regression in brain tissue index， spleen tissue index， proportion of cerebral infarction area， TNF-α level in brain tissue and serum， and the average optical density values of caspase-3 protein and p-AKT protein in brain tissue of rats in high-dose group of Zhongfeng yure decoction （P＜0.05 or P＜0.01）. CONCLUSIONS Zhongfeng yure decoction has a certain intervention and therapeutic effect on MCAO model rats. The mechanism may be to reduce the secretion of inflammatory factors TNF-α and IL-6， down-regulate the expression of caspase-3 protein in ischemic brain tissue， up-regulate the expression of p-AKT protein， so as to protect the neurons.
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To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.
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Objective:To explore the possible mechanism of exacerbation of anxiety-like behavior in db/db mice after distal middle cerebral artery occlusion (dMCAO).Methods:The db/db mice was used to establish a type 2 diabetes mellitus model. Meanwhile, heterozygous db/+ mice and C57 wild-type (WT) mice were chosen as double control groups. Then a permanent distal middle cerebral artery occlusion model was employed as an acute ischemic stroke model. The blood glucose levels before and post-dMCAO surgery on day1, day3, and day5 were detected. The brain tissue loss at 35 days after stroke was measured by immunofluorescent staining of MAP2. The open-field test was performed to estimate anxiety-like behavior and general motor and exploring ability of the animals. Axons and myelin were immunostained with non-phosphorylated neurofilaments (SMI32) and myelin basic protein (MBP), respectively, to evaluate differences in white matter integrity in WT, db/+ and db/db mice 35 days after stroke. The correlation between SMI32/MBP and open field test parameters (time in center and corner) was analyzed. Flow cytometry was employed to detect the amount of T cells and B cells, including regulatory T cells (Tregs) in the brain tissue.Results:Blood glucose levels in db/db mice were significantly higher than db/+ mice and WT mice in both sham and dMCAO groups ( P<0.01). There was no significant difference in brain tissue loss 35 days post-stroke among db/db mice, db/+ mice, and WT mice. In the open field test, there were significant differences in the total distance of db/db mice, db/+ and WT mice in the sham and dMCAO groups. Db/db mice shorter than db/+ mice ( P<0.05), WT mice ( P<0.01), and db/+ mice shorter than WT mice ( P<0.05). There were significant time differences in the center among db/db, db/+, and WT mice in sham and dMCAO groups. In both the sham and dMCAO groups, db/db mice spent less time in the center area of the open field than WT mice ( P<0.01). In the sham group, db/+ mice spent less time in the center area than WT mice ( P<0.05). In dMCAO group, db/db mice spent less time in the center area than db/+mice ( P<0.05), and db/+ mice spent less time in the center area than WT mice ( P<0.01). For the time in the corner, in both the sham and dMCAO groups, db/db mice and db/+ mice consumed more time than WT mice ( P<0.01 or <0.05). In the dMCAO group, db/db mice spent more time in the corner than db/+ mice ( P<0.05). Referring to white matter injury, an increased SMI32/MBP ratio in EC area and CTX area (data was not shown in this article) after dMCAO in db/db, db/+ and WT mice were detected. In EC area, db/db mice have a higher SMI32 ratio than db/+ mice and WT mice: 4.24 ± 0.37 vs. 1.96 ± 0.37, 1.80 ± 0.36, both have significant differences ( P<0.01). For db/db mice and WT mice, the SMI32/MBP ratio negatively correlates with time in center and positive correlation with time in the corner. Three days after dMCAO, the total cells of CD 3+ T cells, CD 8+ cells, Tregs, in db/db mice group have significantly decreased compared to WT group: 4 079 ± 1 345 vs. 70 055 ± 3 374, 141.30 ± 28.36 vs. 2 714.00 ± 463.20, 148.00 ± 61.15 vs. 3 007.00 ± 639.90 ( P<0.01), while B cell has no change between two groups. Conclusions:By comparing the severity of anxiety-like behavior of db/db mice, the severity of white matter injury, and the number of T cells and B cells in brain tissue after dMCAO, immune-mediated brain white matter injury may aggravate db/db mice′s post-dMCAO anxiety-like behavior. Due to the gene dose effect, db/+ mice are not suitable as a control group for db/db mice in animal experiments involving anxiety-like behavior assessment.
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In this study, we performed high-throughput sequencing technology methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptome sequencing (RNA-seq) and bioinformatics to analyze the differentially m6A-methylated and differentially expressed profile of circular RNA (circRNA) in middle cerebral artery occlusion/reperfusion (MCAO/R) model, which provided some scientific evidences for revealing the relationship between RNA epigenetic modification and cerebral ischemia reperfusion injury. The neurological deficit scores of mice were evaluated by the Longa score standard. TTC staining was used to detect cerebral infarction volumes, and dot blot was used for the quantification of m
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[Abstract] Objective To investigate the role of dihydromyricetin (DHM) in the treatment of ischemic stroke in rats, and to explore the effect of DHM on the expression of inflammasome. Methods The middle cerebral artery occlusion (MCAO) model was induced by endovascular suture method. The therapeutic effect and mechanism of DHM were investigated by Longa score, TTC staining, Nissl staining, immunohistochemical staining and Western bloting. Results After DHM treatment, the motor capacity of MCAO rats was significantly improved, the infarct volume was significantly reduced, the brain structure and neuron morphology were improved, and the expressions of nod-like receptor protein-3 (NLRP3) and interleukin-1(IL-1) decreased significantly. Conclusion DHM can down-regulate the expression of NLRP3 and thus reduces the cerebral infarction volume and improves neurobehavioral performance in MCAO rats.
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ObjectiveOn the basis of determining the protective effect of berberine （BBR） on cerebral ischemia， crucial transcription factors （TFs） of BBR against cerebral ischemia was identified by using transcriptome and proteome sequencing. MethodThe model of middle cerebral artery occlusion （MCAO） was established by thread embolization. The sham operation group， model group， low-dose group of BBR （dose of 37.5 mg·kg-1·d-1） and high-dose group of BBR （75 mg·kg-1·d-1） were set up. The rats were killed after continuous intragastric administration for 7 days. The pharmacodynamics was evaluated by Longa score and cerebral infarction rate， and the expressions of inflammatory cytokines， such as interleukin （IL）-1β， tumor necrosis factor （TNF）-α and monocyte chemotactic protein-1 （MCP-1） were measured by enzyme-linked immunosorbent assay （ELISA）. Then， RNA-Seq technique was used to detect the differentially expressed genes （DEGs） before and after BBR intervention， and DAVID 6.8 was used for enrichment analysis of DEGs. CatTFREs technique was used to detect differential TFs before and after BBR intervention， and DAVID 6.8 and STRING 11.0 were used for enrichment analysis and TFs association analysis. Finally， by integrating the activity of TFs and the changes of downstream target genes， crucial TFs were identified and the related regulatory network was constructed by Cytoscape 3.7.1. ResultCompared with the sham operation group， the neurological impairment was significant in the model group （P<0.01）， and compared with the model group， the low and high dose BBR groups could significantly reduce the neurological function damage （P<0.01） and decrease the rate of cerebral infarction （P<0.01）. Transcriptome data analysis showed that BBR was involved in the recovery process after cerebral ischemia mainly by affecting cell adhesion， brain development， neuron migration， calcium signaling pathway， cyclic adenosine monophosphate （cAMP） signaling pathway， inflammatory response and other related functions and signaling pathways. Proteomic data analysis showed that the differentially expressed TFs after BBR intervention interfered with cerebral ischemia mainly by regulating cell differentiation， immune system process， cell proliferation and other biological processes. In addition， integration analysis of TFs and DEGs revealed that transcription factor CP2-like 1 （TFCP2L1）， nuclear factor erythroid-2 like 1 （NFE2L1）， neurogenic differentiation protein 6 （NeuroD6） and POU domain， class 2， transcription factor 1 （POU2F1） were crucial TFs against cerebral ischemia-reperfusion injury mediated by BBR. ConclusionBBR has obvious protective effect on cerebral ischemia-reperfusion injury and its crucial TFs include TFCP2L1， NFE2L1， NeuroD6 and POU2F1.
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This study aims to establish the high-performance liquid chromatography(HPLC) fingerprints of different batches of Notoginseng Radix et Rhizoma, determine their pharmacodynamic indexes of promoting blood circulation, and explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the efficacy of promoting blood circulation. Firstly, the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma were established. Then, the pharmacodynamic indexes were determined after the capillary coagulation experiment and the cerebral ischemia-reperfusion in rats, including capillary coagulation time, percentage of cerebral ischemic area, cerebral water loss rate, and brain-body index. Afterward, the partial least-squares method was used to explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the pharmacodynamic indexes. The results showed that this study successfully established the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma, found 23 common peaks, and identified 12 of them, all of which were saponins. The method was proved stable and reliable. Both the capillary coagulation experiment and the middle cerebral artery occlusion(MCAO)-induced cerebral ischemia-reperfusion experiment on rats revealed that there were obvious differences in the pharmacodynamic indexes of different batches of Notoginseng Radix et Rhizoma. The relationships between 23 common components of Notoginseng Radix et Rhizoma in different batches and the pharmacodynamic indexes were discussed by means of spectrum-effect correlation analysis, of which 17 components had positive effects while 6 components had negative effects on the pharmacodynamic indexes. This study provides a certain reference basis for the clinical rational use and quality control of Notoginseng Radix et Rhizoma.
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Animals , Rats , Blood Coagulation , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Quality Control , Rhizome , SaponinsABSTRACT
Objective:To explore the function of epidermal growth factor 8 in the polarization of microglia after ischemic brain injury and its mechanism.Methods:Forty C57BL/6 mice were randomly divided into a sham operation group, an ischemia group, a recombinant mouse milk fat globule epidermal growth factor 8 (rmMFG-E8) group and an rmMFG-E8 + colivelin TFA group, each of 10. The middle cerebral artery occlusion and reperfusion model (tMCAO) was established in all except the sham operation group. Right after the modelling the mice in the rmMFG-E8 group were immediately injected with 2μL of 0.4μg/μL of rmMFG-E8 into the ventricle contralateral to the cerebral infarction. The rmMFG-E8+ Colivelin TFA group was injected with the same dose of rmMFG-E8 plus 2μL of 5pmol/μL colivelin TFA. On the 1st, 3rd, 5th and 7th day after the modelling, neurological functioning was documented using behavioral tests. The volume proportion of the cerebral infarction was observed after tissue staining on the 7th day after the operation. The gene expression levels of M1 polarization marker-induced nitric oxide synthase (iNOS), M2 polarization marker arginase-1 (Arg1) and mouse chitinase-like molecule 3 (YM1) in the microglia were detected using real-time fluorescence quantitative polymerase chain reactions. The protein expression levels of MFG-E8, phosphorylated signal transduction and transcription factor 3 (p-STAT3) and cytokine signal transduction inhibitor-3 (SOCS3) were determined using western blotting.Results:The behavior tests revealed significant differences between the sham operation group and the other groups on all four days. Compared with the sham operation group, the average expression of MFG-E8 gene and its protein, Arg1 and Ym1, and the SOCS3/GAPDH protein ratio had decreased significantly in the ischemic group, while the average expression of iNOS and the p-STAT3/STAT3 protein ratio had increased significantly. On the 7th day after the modelling, compared with the ischemic group, the infarct volume was significantly smaller in the rmMFG-E8 group. The average expression of iNOS and the average p-STAT3/STAT3 ratio in the rmMFG-E8+ colivelin TFA group had increased significantly compared with the rmMFG-E8 group, while the average expression of Arg1 and Ym1, and the SOCS3/GAPDH ratio were significantly lower.Conclusion:MFG-E8 promotes the polarization of M2-type microglia after cerebral ischemia through STAT3 signaling, promoting the recovery of neurological functioning.
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Serum metabonomic profiles of the model of focal cerebral ischemia reperfusion is established with the suture-occluded method by Longa to study the effect of ginsenosides. In this study, 48 rats were randomly divided into six groups: sham-operated group, pathological model group, positive drug group(6 mg·kg~(-1)·d~(-1)) and high, medium, low-dose ginsenosides groups(200, 100, 50 mg·kg~(-1)·d~(-1)). They are given intragastric administration respectively with same amount of 0.5% CMC-Na,nimodipine and ginsenoside for 5 days. At 2 h after the final administration, the model was established with the suture-occluded method, and free radical-scavenging activity changes of ginsenoside were observed by maillard reaction, and Longa was possible used as a renoprotective agent-occluded method. At the end of 24 h after the reperfusion, the hemolymph of rats in each group was collected, and the ~1H-NMR spectrum was collected after being treated by certain methods, and analyzed by principal component analysis(PCA). Compared with sham-operated group, pathological model group showed significant increases in the levels of lactate, glutamate, taurine, choline, glucose and methionine, but decreases in the levels of 3-hydroxybutyrate and phosphocreatine/creatine in serum. After treatment with ginsenosides, lipid, 3-hydroxybutyrate and phosphocreatine/creatine were increased in the serum of ginsenosides group rats, but with decreases in lactate and glutamate. The results showed that ginsenosides could regulate metabolic disorders in rats with focal cerebral ischemia reperfusion, and promote a recovery in the process of metabolism. It's helpful to promote the metabolic changes in rats with focal cerebral ischemia reperfusion via ~1H-NMR, and lay a foundation to develop ginsenosides as a new drug to treat ischemic cerebral paralysis.
Subject(s)
Animals , Rats , 3-Hydroxybutyric Acid , Brain Ischemia/metabolism , Creatine , Ginsenosides/pharmacology , Hemolymph , Metabolome , Phosphocreatine , Proton Magnetic Resonance Spectroscopy , Random Allocation , Reperfusion Injury/metabolismABSTRACT
Objective To observe the effect of hyperbaric oxygenation (HBO) on apoptosis-inducing factor (AIF) and Caspase-3 levels in rats with permanent middle cerebral artery occlusion (MCAO),and to elucidate the apoptosis pathways.Methods Sixty Sprague-Dawley rats were subjected to permanent MCAO and then randomly divided into a control group and an HBO group,each of 30.Three hours later the rats of the HBO group were put into a hyperbaric cabin held at a pressure of 0.2 MPa for 9 hours.They inhaled supplementary oxygen at the 1st,3rd,5th,7th and 9th hour while the rats in the control group inhaled air at normal pressure.The neurological outcome was measured at the 3rd,13th and 72nd hour after the MCAO using Garcia scores.Apoptosis in the tissue of the ischemic penumbra,nuclear and mitochondrial AIF and Caspase-3 levels were measured at the 13th and 72nd hours after the modeling.Results The scores were significantly higher at the 13th hour than after the 3rd hour in both groups,and then even higher at the 72nd hour.Apoptosis was evident in the ischemic penumbra at the 13th and 72nd hours in both groups,but the number of cells was less at the 72nd hour than at the 13th hour in the control group.There was significantly less apoptosis in the HBO group than in the control group at the 13th hour.The average AIF level had significantly decreased in the nuclei and increased in the mitochondria by the 72nd hour compared with the 13th hour in both groups.The average levels of nuclear AIF at the 13th hour and the 72nd hour were lower than those in the mitochondria.But they were significantly higher in the HBO group than in the control group at the same time points.The levels of Caspase-3,normally zero,had increased by the 13th hour in both groups.The average level of Caspase-3 was significantly lower in both groups at the 72nd hour than at the 13th hour.Conclusions HBO can improve neurological function,inhibit the transfer of AIF from the mitochondria to the nucleus and reduce Caspase-3 levels.The mechanism may involve reducing apoptosis through caspase-dependent and caspase-independent pathways in the mitochondria.
ABSTRACT
Endovascular recanalization therapy (ERT) has been a standard of care for patients with acute ischemic stroke due to large artery occlusion (LAO) within 6 hours after onset, since five landmark ERT trials conducted by 2015 demonstrated its clinical benefit. Recently, two randomized clinical trials demonstrated that ERT, even in the late time window of up to 16 hours or 24 hours after last known normal time, improved the outcome of patients who had a target mismatch, defined as either clinical-core mismatch or perfusion-core mismatch, which prompted the update of national guidelines in several countries. Accordingly, to provide evidence-based and up-to-date recommendations for ERT in patients with acute LAO in Korea, the Clinical Practice Guidelines Committee of the Korean Stroke Society decided to revise the previous Korean Clinical Practice Guidelines of Stroke for ERT. For this update, the members of the writing group were appointed by the Korean Stroke Society and the Korean Society of Interventional Neuroradiology. After thoroughly reviewing the updated evidence from two recent trials and relevant literature, the writing members revised recommendations, for which formal consensus was achieved by convening an expert panel composed of 45 experts from the participating academic societies. The current guidelines are intended to help healthcare providers, patients, and their caregivers make well-informed decisions and to improve the quality of care regarding ERT. The ultimate decision for ERT in a particular patient must be made in light of circumstances specific to that patient.
Subject(s)
Humans , Arteries , Caregivers , Cerebral Infarction , Consensus , Health Personnel , Korea , Mechanical Thrombolysis , Reperfusion , Standard of Care , Stroke , WritingABSTRACT
M1/M2 polarization of immune cells including microglia has been well characterized. It mediates detrimental or beneficial roles in neuroinflammatory disorders including cerebral ischemia. We have previously found that sphingosine 1-phospate receptor subtype 1 (S1P₁) in post-ischemic brain following transient middle cerebral artery occlusion (tMCAO) can trigger microglial activation, leading to brain damage. Although the link between S1P₁ and microglial activation as a pathogenesis in cerebral ischemia had been clearly demonstrated, whether the pathogenic role of S1P₁ is associated with its regulation of M1/M2 polarization remains unclear. Thus, this study aimed to determine whether S1P₁ was associated with regulation of M1/M2 polarization in post-ischemic brain. Suppressing S1P₁ activity with its functional antagonist, AUY954 (5 mg/kg, p.o.), attenuated mRNA upregulation of M1 polarization markers in post-ischemic brain at 1 day and 3 days after tMCAO challenge. Similarly, suppressing S1P₁ activity with AUY954 administration inhibited M1-polarizatioin-relevant NF-κB activation in post-ischemic brain. Particularly, NF-κB activation was observed in activated microglia of post-ischemic brain and markedly attenuated by AUY954, indicating that M1 polarization through S1P₁ in post-ischemic brain mainly occurred in activated microglia. Suppressing S1P₁ activity with AUY954 also increased mRNA expression levels of M2 polarization markers in post-ischemic brain, further indicating that S1P₁ could also influence M2 polarization in post-ischemic brain. Finally, suppressing S1P₁ activity decreased phosphorylation of M1-relevant ERK1/2, p38, and JNK MAPKs, but increased phosphorylation of M2-relevant Akt, all of which were downstream pathways following S1P₁ activation. Overall, these results revealed S1P₁-regulated M1/M2 polarization toward brain damage as a pathogenesis of cerebral ischemia.
Subject(s)
Brain Injuries , Brain Ischemia , Brain , Infarction, Middle Cerebral Artery , Microglia , Phosphorylation , RNA, Messenger , Sphingosine , Up-RegulationABSTRACT
Endovascular recanalization therapy (ERT) has been a standard of care for patients with acute ischemic stroke due to large artery occlusion (LAO) within 6 hours after onset, since five landmark ERT trials conducted by 2015 demonstrated its clinical benefit. Recently, two randomized clinical trials demonstrated that ERT, even in the late time window of up to 16 hours or 24 hours after last known normal time, improved the outcome of patients who had a target mismatch, defined as either clinical-core mismatch or perfusion-core mismatch, which prompted the update of national guidelines in several countries. Accordingly, to provide evidence-based and up-to-date recommendations for ERT in patients with acute LAO in Korea, the Clinical Practice Guidelines Committee of the Korean Stroke Society decided to revise the previous Korean Clinical Practice Guidelines of Stroke for ERT. For this update, the members of the writing group were appointed by the Korean Stroke Society and the Korean Society of Interventional Neuroradiology. After thoroughly reviewing the updated evidence from two recent trials and relevant literature, the writing members revised recommendations, for which formal consensus was achieved by convening an expert panel composed of 45 experts from the participating academic societies. The current guidelines are intended to help healthcare providers, patients, and their caregivers make well-informed decisions and to improve the quality of care regarding ERT. The ultimate decision for ERT in a particular patient must be made in light of circumstances specific to that patient.
Subject(s)
Humans , Arteries , Caregivers , Cerebral Infarction , Consensus , Health Personnel , Korea , Mechanical Thrombolysis , Reperfusion , Standard of Care , Stroke , WritingABSTRACT
OBJECTIVE@#To explore the effects on the recovery of the motor and cognitive functions of the rats with permanent middle cerebral artery occlusion (pMCAO) after treated with 's three-needle acupuncture at head acupoints combined with rota-rod training.@*METHODS@#A total of 38 male SD rats were randomized into 3 groups, named a sham-operation group (11 rats), a model group (13 rats) and a treatment group (14 rats). The electrocoagulation method was adopted to establish the model of pMCAO on the right cerebrum. Starting from the 1st day after successful modeling, acupuncture was applied to the "three points of intelligence", the "three points of temporal area" and the "three points of brain". Additionally, the rota-rod training was used. Acupuncture was given once a day and the training was three times a day. In the sham-operation group and the model group, empty grasp fixation was performed when acupuncture was applied in the treatment group, and there was no intervention at the rest of the time. There was 1 day of interval after consecutive 6 days of intervention. Totally, the intervention was for 3 weeks. After modeling, the brain section was collected from 3 rats of each group on the 1st day and was stained with TTC to observe the condition of cerebral ischemia. From day 1 to 7, the neurological function score was evaluated. The footprint analysis and rota-rod test were performed on day 1, 7, 14 and 21. The Morris water maze test was performed from day 22 to 26.@*RESULTS@#Compared with the sham-operation group, cerebral ischemia presented obviously, the score of neurological function was increased, the back front distances on the left were increased on day 1, 7 and 14 separately, the revolutions per minute (RPM) of the rota-rod were reduced at each of the above 4 time points, the latency of navigation trial was increased and the movement time percentage in Q3 quadrant of spatial probe trial was reduced in the model group (0.05), the score of neurological function was reduced on day 6, the back front distance on the left was reduced on day 14, RPM of the rota-rod were increased on day 14 and 21, the latency of navigation trial were reduced from day 23 to 25 and the movement time percentage in Q3 quadrant of spatial probe trial was increased in the treatment group (<0.01, <0.05).@*CONCLUSION@#'s three-needle acupuncture at head acupoints combined with rota-rod training improve the behavioral performance of pMCAO rats and promote the recovery of motor and cognitive functions.