Protective Effect of Huazhuo Jiedu Huoxue Tongluo Prescription on Cerebral Ischemia-reperfusion Injury via Regulating Autophagy Based on JNK Signaling Pathway / 中国实验方剂学杂志

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.

Analysis of Crucial Transcription Factors of Berberine Against Cerebral Ischemia Based on Transcriptomics and Proteomics / 中国实验方剂学杂志

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.

Spectrum-effect relationship of the blood-activating efficacy of Notoginseng Radix et Rhizoma / 中国中药杂志

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.

Metabonomics study of MCAO rats based on microdialysis technique combined with UPLC-Q/TOF-MS / 中草药

Objective: To study the brain striatum metabolomics characteristics of middle cerebral artery occlusion (MCAO) rats. Methods: The middle cerebral artery was occluded by monofilament to establish the MCAO rat model. The microdialysis probe was implanted into the striatum, and the brain microdialysis samples were collected under awake and free activity condition and were measured by UPLC-Q/TOF-MS in positive and negative ion mode respectively. Multivariate statistics were used to establish the metabobomics model. Results: Brain-targeted metabolomics studies based on microdialysis samples were successfully performed. Metabolomics models could clearly distinguish normal and model rats, and seven differential metabolites were identified. Conclusion: The brain metabolism characteristics of MCAO rats had changed significantly. The metabolomics research based on microdialysis technology enriches the research methods of metabolomics.

Effect of Rosae Chinensis Flos Total Flavones on Focal Cerebral Ischemia-reperfusion in Rats / 中国实验方剂学杂志

Objective: To study the effect of Rosae Chinensis Flos total flavones(RCTF) on the focal cerebral ischemia-reperfusion model in rats, in order to preliminarily explore the mechanism of action. Method: Rats were randomly divided into sham-operated group, model group, large, medium, and low-dose RCTF group(200,100,50 mg ·kg-1) and positive group[Nimodipine group(20 mg ·kg-1) and Naoluotong group (500 mg ·kg-1)]. After 7 days of continuous administration, 1 hour later after the last administration, the middle cerebral artery middle cerebral artery occlusion (MCAO) model was duplicated. After 2 hours of modeling, perfusion was performed for 22 hours. Mortality and neurological deficits were scored. Serum S-100β was detected; brain tissue malondialdehyde(MDA), superoxide dismutase (SOD), nitric oxide (NO), nitric oxide synthase (NOS), tumour necrosis factor-α(TNF-α), interleukin-1β(IL-1β), intercellular adhesion molecule-1(ICAM-1), adenosine triphosphate (ATP)ase were measured. The brain tissue morphological changes were observed. Result: The rat model of focal cerebral ischemia and reperfusion was successfully replicated. Compared with the model group, RCTF in large, medium, and low-dose RCTF group significantly decreased the score of neurological deficit in rats (Pβ in serum (PPP+K+-ATPase, Mg2+-ATPase, and Ca2+ in brain tissue (Pα content, IL-1β, ICAM-1 content in brain tissue (PPConclusion: RCTF have a protective effect on cerebral ischemia-reperfusion injury in rats. The mechanism may be related to the resistance of anti-free radicals, the reduction of inflammation in brain tissue and the improvement of brain energy metabolism after cerebral ischemia reperfusion injury.

Improvements of motor and cognitive functions in the rats with permanent middle cerebral artery occlusion treated with acupuncture and rota-rod training / 中国针灸

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.

Resveratrol modulates the Akt/GSK-3β signaling pathway in a middle cerebral artery occlusion animal model / 한국실험동물학회지

Cerebral ischemia is a major cause of neurodegenerative disease. It induces neuronal vulnerability and susceptibility, and leads to neuronal cell death. Resveratrol is a polyphenolic compound that acts as an anti-oxidant. It exerts a neuroprotective effect against focal cerebral ischemic injury. Akt signaling pathway is accepted as a representative cell survival pathway, including proliferation, growth, and glycogen synthesis. This study investigated whether resveratrol regulates Akt/glycogen synthase kinase-3β (GSK-3β) pathway in a middle cerebral artery occlusion (MCAO)-induced ischemic brain injury. Adult male rats were intraperitoneally injected with vehicle or resveratrol (30 mg/kg) and cerebral cortices were isolated 24 h after MCAO. Neurological behavior test, corner test, brain edema measurment, and 2,3,5-triphenyltetrazolium chloride staining were performed to elucidate the neuroprotective effects of resveratrol. Phospho-Akt and phospho-GSK-3β expression levels were measured using Western blot analysis. MCAO injury led to severe neurobehavioral deficit, infraction, and histopathological changes in cerebral cortex. However, resveratrol treatment alleviated these changes caused by MCAO injury. Moreover, MCAO injury induced decreases in phospho-Akt and phospho-GSK-3β protein levels, whereas resveratrol attenuated these decreases. Phosphorylations of Akt and GSK-3β act as a critical role for the suppression of apoptotic cell death. Thus, our finding suggests that resveratrol attenuates neuronal cell death in MCAO-induced cerebral ischemia and Akt/GSK-3β signaling pathway contributes to the neuroprotective effect of resveratrol.

Hyperglycemia aggravates decrease in alpha-synuclein expression in a middle cerebral artery occlusion model / 한국실험동물학회지

Hyperglycemia is one of the major risk factors for stroke. Hyperglycemia can lead to a more extensive infarct volume, aggravate neuronal damage after cerebral ischemia. α-Synuclein is especially abundant in neuronal tissue, where it underlies the etiopathology of several neurodegenerative diseases. This study investigated whether hyperglycemic conditions regulate the expression of α-synuclein in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury. Male Sprague-Dawley rats were treated with streptozotocin (40 mg/kg) via intraperitoneal injection to induce hyperglycemic conditions. MCAO were performed four weeks after streptozotocin injection to induce focal cerebral ischemia, and cerebral cortex tissues were obtained 24 hours after MCAO. We confirmed that MCAO induced neurological functional deficits and cerebral infarction, and these changes were more extensive in diabetic animals compared to non-diabetic animals. Moreover, we identified a decrease in α-synuclein after MCAO injury. Diabetic animals showed a more serious decrease in α-synuclein than non-diabetic animals. Western blot and reverse-transcription PCR analyses confirmed more extensive decreases in α-synuclein expression in MCAO-injured animals with diabetic condition than these of non-diabetic animals. It is accepted that α-synuclein modulates neuronal cell death and exerts a neuroprotective effect. Thus, the results of this study suggest that hyperglycemic conditions cause more serious brain damage in ischemic brain injuries by decreasing α-synuclein expression.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

The influence of ultra-shortwave irradiation on Ca2+-ATPase expression after cerebral ischemia and reperfusion / 中华物理医学与康复杂志

Objective To observe the influence of ultra-shortwave (USW) irradiation on infarct volume and Ca2+-ATPase (SPCA) secretion after brain ischemia and reperfusion.Methods Eighty Sprague-Dawley rats were randomly divided into a sham operation group (n=8),a model group (n=36) and a USW group (n=36).The animal model of middle cerebral artery ischemia and reperfusion (MCAO/R) was established using the suture method in the rats of the model and USW groups,while the sham operation group was given the same operation but without inserting the thread plug.One day,3 days and 7 days after the intervention,12 rats were sacrificed and the infarct volumes and SPCA1 protein expression were measured using 2,3,5-triphenyltetrazolium chloride staining and western blotting.Results No white infarcted tissue was found in the sham operation group.In the model and USW groups the volume of infarcted tissue decreased with time.Significantly less infarcted volume was observed in the USW group compared to the model group at each time point.The SPCA1 levels in the brain tissue were lower than in the sham operation group after one and 3 days of USW treatment,but they were significantly lower in the model group as well.As time went by,the average SPCA1 level increased significantly in the model and USW groups.A slightly higher SPCA1 level was observed in the USW group compared to the model group after one day of treatment,but with no significance.However,significant differences were found between them after 3 and 7 days of intervention.Conclusion Ultra-shortwave irradiation can protect against MCAO/R injury by decreasing the infarcted volume,which may be related to down-regulation of SPCA1,minimizing nerve cell apoptosis and promoting neural functional recovery,at least in rats.

Effects of circulating microvesicles containing microRNA-27 a on blood brain barrier tight junction injury of ischemia stroke mice / 中国药理学通报

Aim To study the effect of circulating mi-crovesicles containing miR-27 a on blood brain barrier tight junction injury of ischemia stroke mice and its mechanism. Methods The middle cerebral artery occlusion mouse model was established, and the mi-crovesicles in the supernatant of 2 h of ischemic stroke brain tissues were separated by centrifugal ultrafiltra-tion method. The transmission electron microscopy was used to observe microvesicle morphology, and the di-ameter of microvesicles was detected. Based on the 2 h ischemia stroke mouse model, mice were injected via femoral vein with microvesicles at 5 mg · kg-1 . TTC staining was applied to detect infarction volume of is-chemia brain, while HE staining was applied to detect the expression change of tight junction protein occludin and claudin-5 . Western blot was subjected to detect occludin, claudin-5, TLR4, NF-κB and p38 protein expression. ELISA method was used to measure the contents of cytokines IL-1β and TNF-α. Results The microvesicle shape was approximately circular bi-lateral membrane structure, with an average diameter of 160 nm, which conformed to the morphological char-acteristics of microvesicles. Compared with the ische-mic stroke group, injection of microvesicles could ag-gravate the damage of brain tissues in ischemic mice, further increase the infarct volume and reduce the posi-tive staining areas of occludin and claudin-5 in ische-mia brain tissues. Meanwhile, the protein expressions of occludin and claudin-5 further decreased, and fur-ther up-regulated TLR4 and phosphorylation of NF-κB and p38 compared with the ischemia stroke group. Al-so, more content of IL-1βand TNF-αwere detected in ischemia stroke group injected with microvesicles com-pared with those in ischemia stroke group with signifi-cant difference, while the injection of antagomir-27a could alleviate brain damage and reduce the activation of TLR4, NF-κB and p38 in ischemia stroke mice. Conclusions Microvesicles containing miR-27 a could significantly attenuate brain injury in ischemia stroke mice, while aggravate the tight junction damage of ischemia brain. The mechanism might be correlated with the up-regulation of the expression of TLR4 , the phosphorylation of NF-κB, p38, and the release of cy-tokines IL-1β and TNF-α.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Detection of hypothalamic infarction in intraluminal middle cerebral artery occlusion model rat by DWI scanning / 上海交通大学学报（医学版）

Objective: To detect and evaluate the hypothalamic infarction in middle cerebral artery occlusion (MCAO) model rat. Methods: For 15 Sprague-Dawley rats weighed 200-250 g, aged 6-8 months, their right middle cerebral artery was occluded for 90 min by a silicon-coated 4-0 nylon filament and reperfused. Sprague-Dawley rats underwent diffusion weighted MR imaging (DWI) scanning (at 1 h and 24 h after reperfusion) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining (at 24 h after reperfusion) to determine the hypothalamic and cerebral infarct volume. The relationship between hypothalamic infarct volume and cerebral infarction volume was analyzed by DWI scanning. The results of TTC staining were compared with those of 24 h DWI scanning. Results: Fifteen Sprague-Dawley rats successfully received intraluminal MCAO/reperfusion procedures. The incidences of hypothalamic infarction on brain DWI scanning and TTC staining were 100% and 40% at 24 h after reperfusion, respectively. Therefore, DWI scanning was more sensitive than TTC staining to detect hypothalamic injury (P=0.001). The hypothalamic infarct volume on DWI scanning was (8.59±2.89) mm3 and (11.65±3.19) mm3 at 1 h and 24 h after reperfusion, respectively. On DWI scanning, hypothalamic and cerebral infarct volume at 24 h after reperfusion were correlated with each other significantly (r=0.573, P=0.025), so were the increases of hypothalamic and cerebral infarct volume (r=0.554, P=0.032) from 1 h to 24 h. Conclusion: DWI scanning was more sensitive than TTC staining to detect hypothalamic injury in intraluminal transient MCAO model. Hypothalamic and cerebral infarct volume were correlated with each other.

Effects of harpagide on nerve cell and caspase-independent apoptotic signaling pathway in MCAO mice / 中国药理学通报

Aim To study the effects of harpagide on hippocampal neurons, mitochondrial function and caspase-independent apoptosis pathway after cerebral ischemia in mice. Methods The middle cerebral ar-tery occlusion (MCAO ) was employed to establish MCAO model. After that,the mice were given harp-agide (4,8,12 mg·kg - 1 )and edaravone (3. 2 mg ·kg - 1 )by tail vein injection after MCAO immediate-ly,and the model and control mice were given equal a-mounts of saline by the same way. After MCAO for 6 h,the apoptosis rate of hippocampal neuron and the mitochondrial membrane potential (MMP)of MCAO mice were detected by flow cytometry. We observed the clarity of inner and outer membrane of the hipp-ocampal neuronal mitochondrial,the integrity of the mitochondrial cristae,the changes of matrix electron density of mitochondria,and mitochondria swelling by transmission electron microscopy. Western blot was employed to determine the expression of apoptosis in-duced factor (AIF)and endonuclease G (Endo G)in mitochondrion and pro-caspase-3 in endochylema. qPCR was employed to determine the expression of AIF and Endo G. Results Compared with control group, the apoptosis rate of hippocampal neuron of MCAO mice significantly increased(P < 0. 01),the MMP of hippocampal neuron significantly decreased and the mi-tochondrial ultrastructure of cerebral ischemic area was severely damaged, loosely arranged and obviously swollen;the expression of AIF and Endo G in mito-chondria of cerebral tissue of MCAO mice significantly decreased,and the releases of AIF and Endo G signifi-cantly increased(P < 0. 01);the expressions of AIF, Endo G mRNA were evidently up-regulated (P <0. 01). Compared with model group,each dose of harpagide could significantly decrease the apoptosis rate of hippocampal neurons of mice brain (P < 0 . 05 ,P < 0. 01);MMP markedly increased in hippocampal nerve cells(P < 0. 01);the ultrastructure of neuronal mitochondria was obviously improved. Compared with model group,harpagide (8,12 mg·kg - 1 )could sig-nificantly increase the expression of AIF and Endo G protein in mitochondria of mouse brain,and the release of mitochondrial AIF and Endo G protein decreased(P< 0. 05,P < 0. 01);harpagide (4 mg·kg - 1 )could significantly increase the expression of Endo G protein in mitochondria of mouse brain,and the release of En-do G protein markedly decreased (P < 0. 05);harp-agide (8,12 mg·kg - 1 )could significantly decrease the expression of AIF and Endo G mRNA in hippocam-pus of mice(P < 0. 05,P < 0. 01). Conclusion The protective effect of harpagide on MCAO may be related to the protective effects on cerebral nerve cells,the ac-tivity of the mitochondria and the inhibition of caspase-independent apoptotic signaling pathways.

UHPLC-LTQ-Orbitrap-based metabolomics coupled with metabolomics pathway analysis method for exploring the protection mechanism of Kudiezi injection in a rat anti-ischemic cerebral reperfusion damage model / 中国天然药物

Kudiezi injection has been used extensively in the treatment of cerebrovascular and cardiovascular diseases. However, its therapeutic effects and underlying mechanism of action are not fully understood. The aim of the present study was to clarify the protective mechanisms of Kudiezi injection on cerebral ischemic injury, using metabolomics methods. Middle cerebral artery occlusion (MCAO) was introduced in rats to build the cerebral ischemic damage. UHPLC-LTQ-Orbitrap-based analytical method was established for analysis of the metabolites. The raw mass data of all samples were normalized with Sieve 2.2 software and then introduced to orthogonal partial least squares discriminant analysis (OPLS-DA) model. Finally, 23 metabolites in plasma (15 were tentatively identified) were chosen as potential biomarkers, according to accurate mass measurements (< 5 ppm), MS/MS fragmentation patterns, and diagnostic product ions. Furthermore, on the basis of metabolic pathway analysis via metabolomics pathway analysis (MetPA), we first discovered that the protection mechanism in anti-ischemic cerebral reperfusion damage of Kudiezi injection was possibly related to the biosynthesis of phenylalanine, tyrosine, and tryptophan. The present study provided a useful approach for exploring the mechanism of ischemic stroke and evaluating the efficacy of Kudiezi injection or other traditional medicines.

Hyperglycemia decreases preoxiredoxin-2 expression in a middle cerebral artery occlusion model / 한국실험동물학회지

Diabetes is a major risk factor for stroke and is also associated with worsened outcomes following a stroke. Peroxiredoxin-2 exerts potent neuroprotective effects against oxidative stress. In the present study, we identified altered peroxiredoxin-2 expression in an ischemic stroke model under hyperglycemic conditions. Adult male rats were administrated streptozotocin (40 mg/kg) via intraperitoneal injection to induce diabetes. Middle cerebral artery occlusion (MCAO) was induced surgically 4 weeks after streptozotocin treatment and cerebral cortex tissues were isolated 24 hours after MCAO. Peroxiredoxin-2 expression was evaluated in the cerebral cortex of MCAO-operated animals using a proteomics approach, and was found to be decreased. In addition, the reduction in peroxiredoxin-2 levels was more severe in cerebral ischemia with diabetes compared to animals without diabetes. Reverse-transcriptase PCR and Western blot analyses confirmed the significantly reduced peroxiredoxin-2 expression in MCAO-operated animals under hyperglycemic conditions. It is an accepted fact that peroxiredoxin-2 has antioxidative activity against ischemic injury. Thus, the findings of this study suggest that a more severe reduction in peroxiredoxin-2 under hyperglycemic conditions leads to worsened brain damage during cerebral ischemia with diabetes.

Regulation of thin recipe of Buyang Huanwudecoction on Cdk5 expression of rats after cerebral ischemia / 中国药理学通报

Aim To evaluate the regulation of thin recipe of Buyang Huanwu decoction on cyclin-dependent kinase 5(Cdk5)expressions in hippocampus tissue of rats after cerebral ischemia.Methods Male SD rats were divided into sham-operation group,MCAO group,Buyang Huanwu decoction group(ig.3.15 g·kg-1)and its thin recipe composition group(ig.2.41 g·kg-1).Each group was then divided into five subgroups based on the time after administration for 1,3,7,14,28 d respectively.Cdk5 protein and mRNA levels in each group were examined by using immunohistochemistry,Western blot and real-time PCR respectively.Results The up-regulation of Cdk5 was observed in model rat hippocampus after cerebral ischemia 1 day,and kept increasing with the aggravation of ischemia injury,the peaked expression was observed after 7～14 d,while the downtrend was observed after 28 days compared with the corresponding sham-operation groups(P0.05).Conclusion The thin recipe of Buyang Huanwu decoction could exert the protective effect by regulating Cdk5 after cerebral ischemia.

Study on in Vitro Screening and in Vivo Validation of Optimized Buyang Huanwu Decoction / 中国中医药信息杂志

Objective To screen the optimized Buyang Huanwu Decoction (BYHWD);To verify it. Methods H2O2 was used to induce PC12 cell oxidative stress models. MTT method was used to determine the prevention effects of BYHWD at different concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 3.5 mg/mL) on in vitro oxidative stress cell models to define the optimized concentration. Orthogonal design was used to divide BYHWD single medicine into decomposed BYHWD groups, control group (only with DMEM), normal group (without H2O2 and medicine processing), and model group, to investigate the protective effects on PC12 cells. Optimized BYHWD was screened to decide the compatibility ratio of each medicine. MTT was used to detect the cell survival rate in each group. Middle cerebral artery occlusion was used to replicate MACO rat models. SD rats were randomly divided into sham-operation group, model group, BYHWD group and optimized BYHWD high-, medium-and low-dose groups. Each medication group was given relevant medicine for gavage. The screened results were verified. Results Compared with other decomposed BYHWD groups, the protective effects of the compatibility of Astragali Radix+Chuanxiong Rhizoma+Pheretima on PC12 cells was the best (P<0.05), which was nearly equaled to BYHWD. Compared with the model group, BYHWD and the optimized one could evidently reduce cerebral cortex infarction area and improve the impaired brain edema (P<0.05), and the medium-dose group was the best. Conclusion The optimized BYHWD ratio is:Astragali Radix:Chuanxiong Rhizoma:Pheretima=10:3:1.

Repeated Oral Administration of Human Serum Albumin Protects from the Cerebral Ischemia in Rat Brain Following MCAO

Albumin is known to have neuroprotective effects. The protein has a long half-life circulation, and its effects can therefore persist for a long time to aid in the recovery of brain ischemia. In the present study, we investigated the neuroprotective effects of human serum albumin (HSA) on brain hemodynamics. Albumin is administrated using repeated oral gavage to the rodents. Sprague-Dawley rats underwent middle cerebral artery occlusion procedures and served as a stroke model. Afterwards, 25% human serum albumin (1.25 g/kg) or saline (5 ml/kg) was orally administrated for 2 weeks in alternating days. After 2 weeks, the rodents were assessed for levels of brain ischemia. Our testing battery consists of behavioral tests and in vivo optical imaging sessions. Modified neurological severity scores (mNSS) were obtained to assess the levels of ischemia and the effects of HSA oral administration. We found that the experimental group demonstrated larger hemodynamic responses following sensory stimulation than controls that were administered with saline. HSA administration resulted in more significant changes in cerebral blood volume following direct cortical electric stimulation. In addition, the mNSS of the treatment group was lower than the control group. In particular, brain tissue staining revealed that the infarct size was also much smaller with HSA administration. This study provides support for the efficacy of HSA, and that long-term oral administration of HSA may induce neuroprotective effects against brain ischemia.

Hyperglycemia exacerbates downregulation of dynamin-like protein 1 in ischemic cerebral injury / 한국실험동물학회지

Ischemic stroke is one of the leading causes of adult disability and death. Hyperglycemia is associated with an increased risk of stroke and poor outcomes after brain injury. Dynamin-like protein I (DLP-1) regulates mitochondrial fission and promotes mitochondrial dynamics. Neurodegenerative diseases are associated with mitochondrial dysfunction, and the downregulation of DLP-1 has been previously identified in a stroke animal model. Here, we investigated the changes in DLP-1 protein expression in an animal model of focal cerebral ischemia with induced hyperglycemia. Streptozotocin (40 mg/kg) was intraperitoneally injected into male rats to induce hyperglycemia, and middle cerebral artery occlusion (MCAO) was surgically induced 4 weeks after streptozotocin treatment. Brain tissue was isolated 24 hours after MCAO, and cerebral cortex samples were used for this study. Proteomics revealed a decrease in DLP-1 expression in MCAO animals when compared with controls, and this downregulation was more prominent in MCAO animals with hyperglycemia. Reverse-transcription polymerase chain reaction and Western blot analyses confirmed that DLP-1 was significantly downregulated in MCAO-injured animals with hyperglycemia compared to those without hyperglycemia. The decrease in DLP-1 indicates mitochondrial morphological changes and dysfunction. Together, these results suggest that the severe decrease of DLP-1 seen after brain injury under hyperglycemic conditions may exacerbate the damage to the brain.