ObjectiveTo investigate the effects of Dihuang Yinzi on the cognitive function in the mouse model of learning and memory impairments induced by scopolamine （SCOP） and explore the treatment mechanism. MethodsA mouse model of learning and memory impairment was induced by intraperitoneal injection of SCOP. Sixty male C57BL/6J mice were randomized into six groups： control （0.9% NaCl， n=10）， model （SCOP 1 mg·kg^-1·d^-1， n=10）， low-， medium-， and high-dose Dihuang Yinzi （SCOP 1 mg·kg^-1·d^-1 + Dihuang Yinzi 5.5， 11.0， and 22.0 g·kg^-1·d^-1， n=10）， and donepezil （SCOP 1 mg·kg^-1·d^-1 + donepezil 0.84 mg·kg^-1·d^-1， n=10）. Mice were administrated with corresponding drugs for 6 weeks. Modeling started in the 4th week， and mice in other groups except the control group were injected with SCOP intraperitoneally 40 min after daily gavage. Behavioral testing began in the 5th week， 30 min after modeling each day. The Morris water maze and novel object recognition tests were carried out to evaluate the spatial learning and memory function of mice. Nissl staining was employed to observe the survival of neurons and Nissl bodies in the hippocampal CA1 region. Western blot was employed to determine the protein levels of silent information regulator 2 （SIRT2）， α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid （AMPA） receptor 1 （GluA1）， protein kinase A （PKA）， cAMP response element-binding protein （CREB）， phosphorylated-CREB （p-CREB）， postsynaptic density protein 95 （PSD95）， growth-associated protein-43 （GAP-43）， and synaptophysin （SYN） in the hippocampus. Immunofluorescence was used to detect the expression of doublecortin （DCX） in the hippocampal dentate gyrus （DG） region. ResultsCompared with the control group， the model group showed impaired learning and memory （P<0.01）， obvious neuronal damage in the hippocampal CA1 region， a reduction in neuron survival （P<0.01）， a decrease in DCX expression in the hippocampal DG region （P<0.01）， down-regulated proteins levels of GluA1， PKA， p-CREB/CREB， PSD95， SYN， and GAP-43 in the hippocampal tissue （P<0.05， P<0.01）， and an up-regulated protein level of SIRT2 （P<0.01）. Compared with the model group， the medium- and high-dose Dihuang Yinzi groups and the donepezil group showed improvements in learning and memory （P<0.05， P<0.01）， while the low-， medium-， and high-dose Dihuang Yinzi groups and the donepezil group had increased neuron survival （P<0.05， P<0.01）. The medium-dose Dihuang Yinzi group and the donepezil group showed increased DCX expression （P<0.05， P<0.01）. The medium- and high-dose Dihuang Yinzi groups and the donepezil group showed up-regulation in the protein levels of GluA1， PKA， p-CREB/CREB， PSD95， SYN， and GAP-43 （P<0.05， P<0.01） and down-regulation in the protein level of SIRT2 （P<0.01）. ConclusionDihuang Yinzi can improve the cognitive function in the mouse model of learning and memory impairments induced by SCOP by inhibiting the upregulation of SIRT2， activating the PKA/CREB signaling pathway， improving synaptic plasticity， and reducing hippocampal neuronal damage.

Objective To establish and evaluate an integrated disease-syndrome rat model of chronic cerebral ischemia with Qi deficiency and blood stasis syndrome.Methods Thirty male Wistar rats were allocated randomly into three groups(n=10 per group):sham operation(sham),2-vessel occlusion(2-VO)group,and sleep deprivation combined with 2-VO(SD+2-VO)group.We comprehensively assessed Qi deficiency and blood stasis syndrome manifestations in the rats using a dual evaluation approach,combining exhaustive swimming tests with quantitative tongue chroma analysis.Cognitive function was evaluated using the Barnes maze,and cerebral blood flow was compared using laser speckle contrast imaging.The histopathology of the hippocampal cytoarchitecture and white matter were examined using hematoxylin-eosin(HE)and Luxol fast blue(LFB)staining,respectively,and ultrastructural alterations of neurons in the hippocampal CA1 region were observed by transmission electron microscopy(TEM).Protein expression levels of NeuN,vascular endothelial growth factor A(VEGFA)and CD31 were detected by Western Blot and immunofluorescence.Results Cerebral blood flow was significantly reduced in rats in the 2-VO group compared with the sham group,but they failed to recapitulate the key clinical hallmarks of Qi deficiency and blood stasis syndrome.In contrast,rats in the SD+2-VO group exhibited significantly reduced locomotor activity,exacerbated cerebral hypoperfusion,shortened swimming duration,and darkened tongue color compared with 2-VO rats.Rats in the SD+2-VO group demonstrated significantly impaired learning and memory abilities in the Barnes maze test.Consistent with these observations,HE staining,TEM,and LFB staining revealed substantial neuronal and white matter damage in the SD+2-VO group.NeuN expression was decreased and VEGFA and CD31 expression levels were increased in the 2-VO and SD+2-VO groups,as shown by Western Blot.Taken together,these findings indicated that the SD+2-VO model effectively recapitulated the clinical features of chronic cerebral ischemia with Qi deficiency and blood stasis pattern.Conclusions The combination of sleep deprivation and bilateral carotid artery occlusion successfully established a rat model of chronic cerebral ischemia with Qi deficiency and blood stasis syndrome.Compared with the 2-VO model,SD+2-VO model demonstrates more pronounced syndrome manifestations and better clinical relevance,thus providing a valuable animal model for traditional Chinese medicine research on chronic cerebral ischemia.

Objective To establish and evaluate an integrated disease-syndrome rat model of chronic cerebral ischemia with Qi deficiency and blood stasis syndrome.Methods Thirty male Wistar rats were allocated randomly into three groups(n=10 per group):sham operation(sham),2-vessel occlusion(2-VO)group,and sleep deprivation combined with 2-VO(SD+2-VO)group.We comprehensively assessed Qi deficiency and blood stasis syndrome manifestations in the rats using a dual evaluation approach,combining exhaustive swimming tests with quantitative tongue chroma analysis.Cognitive function was evaluated using the Barnes maze,and cerebral blood flow was compared using laser speckle contrast imaging.The histopathology of the hippocampal cytoarchitecture and white matter were examined using hematoxylin-eosin(HE)and Luxol fast blue(LFB)staining,respectively,and ultrastructural alterations of neurons in the hippocampal CA1 region were observed by transmission electron microscopy(TEM).Protein expression levels of NeuN,vascular endothelial growth factor A(VEGFA)and CD31 were detected by Western Blot and immunofluorescence.Results Cerebral blood flow was significantly reduced in rats in the 2-VO group compared with the sham group,but they failed to recapitulate the key clinical hallmarks of Qi deficiency and blood stasis syndrome.In contrast,rats in the SD+2-VO group exhibited significantly reduced locomotor activity,exacerbated cerebral hypoperfusion,shortened swimming duration,and darkened tongue color compared with 2-VO rats.Rats in the SD+2-VO group demonstrated significantly impaired learning and memory abilities in the Barnes maze test.Consistent with these observations,HE staining,TEM,and LFB staining revealed substantial neuronal and white matter damage in the SD+2-VO group.NeuN expression was decreased and VEGFA and CD31 expression levels were increased in the 2-VO and SD+2-VO groups,as shown by Western Blot.Taken together,these findings indicated that the SD+2-VO model effectively recapitulated the clinical features of chronic cerebral ischemia with Qi deficiency and blood stasis pattern.Conclusions The combination of sleep deprivation and bilateral carotid artery occlusion successfully established a rat model of chronic cerebral ischemia with Qi deficiency and blood stasis syndrome.Compared with the 2-VO model,SD+2-VO model demonstrates more pronounced syndrome manifestations and better clinical relevance,thus providing a valuable animal model for traditional Chinese medicine research on chronic cerebral ischemia.

ObjectiveTo observe the possible mechanism of Dihuang Yinzi Decoction （地黄饮子） for improving cognitive dysfunction in Alzheimer's disease （AD） from the perspective of retina. MethodsForty-five APP/PS1 mice （AD model mice） were randomly divided into model group， Dihuang Yinzi Decoction group， and memantine group， with 15 mice in each group， while 15 wild-type C57BL/6J mice from the same litter were used as blank group. Mice in Dihuang Yinzi Decoction group were given Dihuang Yinzi Decoction 30.03 g/（kg·d） by gavage， mice in the memantine group were given memantine hydrochloride 6.1 mg/（kg·d） by gavage， and mice in the blank group and the model group were given normal saline 2 ml/（kg·d） by gavage for 4 consecutive weeks. Fasting blood glucose was measured weekly. After 4 weeks of intervention， oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were performed； Morris water maze was used to detect the changes in spatial memory ability of mice； glucose oxidase method was used to detect retinal glucose content of mice； enzyme-linked immunosorbent assay （ELISA） was used to detect serum and retinal insulin content of mice， and Homeostatic Model Assessment of insulin resistance （HOMA-IR） was calculated. Hematoxylin-eosin （HE） staining was performed to observe the histopathological changes in the retina， and the retinal thickness and ganglion cell number were counted； protein immunoblotting was performed to detect the retinal pathway-associated proteins ［insulin receptor substrate 1 （IRS1）， phosphorylated insulin receptor substrate 1 （pIRS1）， phosphatidylinositol-3-hydroxykinase （PI3K）， protein kinase B （Akt1）， phosphorylated protein kinase B （pAkt1）］ expression； retinal glucose transporter protein 4 （GLUT4） expression was detected by immunohistochemistry. ResultsCompared with the blank group， fasting blood glucose of mice in the model group at weeks 1， 2， 3， and 4， blood glucose and area under the curve （AUC） at different time point of OGTT and ITT test， fasting serum insulin， and HOMA-IR increased （P＜0.05， P＜0.01）； in the Morris water maze experiment， the escape latency increased from day 3 to day 5， and the number of crossing platforms， the percentage of target quadrant distance， and the percentage of target quadrant time decreased （P＜0.05， P＜0.01）； the outer nuclear layer of the retina became sparse， thinner， and the number of ganglion cells decreases （P＜0.01）； the expression level of retinal glucose increased， while the expression levels of insulin， pIRS1/IRS1， PI3K/β-Actin， pAkt1/Akt1， and GLUT4 proteins decreased （P＜0.01）. Compared with the model group， fasting blood glucose at week 4， blood glucose at each time point of the OGTT and ITT tests AUC decreased （P＜0.05， P＜0.01）， and fasting serum insulin and HOMA-IR decreased （P＜0.05） in Dihuang Yinzi Decoction group； In the Morris water maze test， the escape latency shortened on day 4 and day 5， number of platform crossings， target quadrant distance as a proportion of total distance， and target quadrant movement time as a proportion of total time decreased （P＜0.05， P＜0.01）； retinal pathological changes alleviated， and retinal thickness and ganglion cell number increased （P＜0.01）； retinal glucose content decreased， and retinal pIRS1/IRS1， PI3K/β-Actin， and GLUT4 protein expression elevated （P＜0.05 or P＜0.01）. ConclusionsDihuang Yinzi Decoction can improve cognitive dysfunction of Alzheimer's disease， which may be related to regulating retinal insulin content and insulin signaling pathway.

OBJECTIVE To investigate the mechanism by which salvianolic acid A (Sal A) reduces the inflammatory response and oxidative stress of BV2 cells injured by oxygen and glucose deprivation/reperfusion (OGD/R).METHODS An OGD/R injury model of BV2 cells was established with sugar free Earle solution containing Na2S2O410 mmol·L-1.Na2S2O4 sugar free Earle solution was added and cultured in an incubator (37 ℃,5％CO2) for 1.5 h (oxygen glucose deprivation) before a normal medium was used for 24 h (reperfusion).Then,the cells were divided into the cell control group,OGD/R group,OGD/R+Sal A 1,5 and 10 μmol·L-1 group,OGD/R+ML385 group,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 group and OGD/R+edaravone (Eda,50μmol·L-1) group.After twenty-four hours of culture,the cell survival rate was measured by CCK8 kit.The contents of Interleukin-1β (IL-1β),IL-6,tumor necrosis factor-α(TNF-α),IL-10,IL-4 and transforming growth factor-β(TGF-β) in the cell supernatant were detected by ELISA.Reactive oxygen species (ROS) in cells was detected using the chemical fluo-rescence method.The contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-PX) and chloramphenicol acetyltransferase (CAT) in cells were determined with the colorimetric method.Protein expressions of Kelch like ECH-associated protein 1 (Keap1),nuclear factor erythroid-2 related factor 2 (Nrf2),Heme oxygenase-1 (HO-1),NADPH:quinone oxidoreductase-1 (NQO1) and p-nuclear factor kappa-B p65 (p-NF-κB p65) were detected by Western blotting.RESULTS ①Compared with the cell control group,the cell survival rate of the OGD/R group was significantly decreased (P＜0.01).Compared with the OGD/R group,the survival rates of OGD/R+Sal A 1,5 and 10μmol·L-1 groups were significantly increased (P＜0.05,P＜0.01).②Compared with the cell control group,the contents of IL-1β,IL-6 and TNF-α were significantly increased,the contents of IL-10,IL-4 and TGF-β were significantly decreased,the contents of ROS and MDA were significantly increased,and the activities of SOD,CAT and GSH-Px were significantly decreased in the OGD/R group (P＜0.01).Compared with the OGD/R group,the content of IL-6 was significantly decreased,the contents of IL-10,IL-4 and TGF-β were significantly increased,the contents of ROS and MDA were significantly decreased,and the activities of SOD,CAT and GSH-Px were significantly increased in OGD/R+Sal A 1,5 and 10μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).③Compared with the cell control group,the protein expression of p-NF-κB P65 in the OGD/R group was significantly increased (P＜0.01).Compared with the OGD/R group,the protein expressions of Keap1 and cytoplasmic Nrf2 were significantly decreased,the expressions of nuclear Nrf2,HO-1 and NQO1 proteins were significantly increased,and the expression of p-NF-κB p65 protein was significantly decreased in OGD/R+Sal A 5 and 10 μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).In OGD/R+ML385,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 groups,the protein expression of Keap1 was significantly increased,the protein expressions of cytoplasmic Nrf2,nuclear Nrf2,HO-1 and NQO1 protein were significantly decreased,and the protein expression of p-NF-κB P65 was significantly increased (P＜0.01).CONCLU-SION Sal A reduces the inflammatory response and oxidative stress of OGD/R injured BV2 cells possi-bly by activating the Keap1/Nrf2 pathway and inhibiting the NF-κB pathway.

OBJECTIVE To investigate the mechanism by which salvianolic acid A (Sal A) reduces the inflammatory response and oxidative stress of BV2 cells injured by oxygen and glucose deprivation/reperfusion (OGD/R).METHODS An OGD/R injury model of BV2 cells was established with sugar free Earle solution containing Na2S2O410 mmol·L-1.Na2S2O4 sugar free Earle solution was added and cultured in an incubator (37 ℃,5％CO2) for 1.5 h (oxygen glucose deprivation) before a normal medium was used for 24 h (reperfusion).Then,the cells were divided into the cell control group,OGD/R group,OGD/R+Sal A 1,5 and 10 μmol·L-1 group,OGD/R+ML385 group,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 group and OGD/R+edaravone (Eda,50μmol·L-1) group.After twenty-four hours of culture,the cell survival rate was measured by CCK8 kit.The contents of Interleukin-1β (IL-1β),IL-6,tumor necrosis factor-α(TNF-α),IL-10,IL-4 and transforming growth factor-β(TGF-β) in the cell supernatant were detected by ELISA.Reactive oxygen species (ROS) in cells was detected using the chemical fluo-rescence method.The contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-PX) and chloramphenicol acetyltransferase (CAT) in cells were determined with the colorimetric method.Protein expressions of Kelch like ECH-associated protein 1 (Keap1),nuclear factor erythroid-2 related factor 2 (Nrf2),Heme oxygenase-1 (HO-1),NADPH:quinone oxidoreductase-1 (NQO1) and p-nuclear factor kappa-B p65 (p-NF-κB p65) were detected by Western blotting.RESULTS ①Compared with the cell control group,the cell survival rate of the OGD/R group was significantly decreased (P＜0.01).Compared with the OGD/R group,the survival rates of OGD/R+Sal A 1,5 and 10μmol·L-1 groups were significantly increased (P＜0.05,P＜0.01).②Compared with the cell control group,the contents of IL-1β,IL-6 and TNF-α were significantly increased,the contents of IL-10,IL-4 and TGF-β were significantly decreased,the contents of ROS and MDA were significantly increased,and the activities of SOD,CAT and GSH-Px were significantly decreased in the OGD/R group (P＜0.01).Compared with the OGD/R group,the content of IL-6 was significantly decreased,the contents of IL-10,IL-4 and TGF-β were significantly increased,the contents of ROS and MDA were significantly decreased,and the activities of SOD,CAT and GSH-Px were significantly increased in OGD/R+Sal A 1,5 and 10μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).③Compared with the cell control group,the protein expression of p-NF-κB P65 in the OGD/R group was significantly increased (P＜0.01).Compared with the OGD/R group,the protein expressions of Keap1 and cytoplasmic Nrf2 were significantly decreased,the expressions of nuclear Nrf2,HO-1 and NQO1 proteins were significantly increased,and the expression of p-NF-κB p65 protein was significantly decreased in OGD/R+Sal A 5 and 10 μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).In OGD/R+ML385,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 groups,the protein expression of Keap1 was significantly increased,the protein expressions of cytoplasmic Nrf2,nuclear Nrf2,HO-1 and NQO1 protein were significantly decreased,and the protein expression of p-NF-κB P65 was significantly increased (P＜0.01).CONCLU-SION Sal A reduces the inflammatory response and oxidative stress of OGD/R injured BV2 cells possi-bly by activating the Keap1/Nrf2 pathway and inhibiting the NF-κB pathway.

In recent years,increasing attention has been paid to the study on the pathogenesis of cerebral ischemia in terms of the overall structure and function of neurovascular unit(NVU),which has become one of the hot spots in the field of brain sciences and major brain diseases.This paper is intended to outline the roles of the four main NVU cells(neurons,astrocytes,microglia and cerebral microvascular endothelial cells)in brain function and pathogenesis of cerebral ischemia,which are closely related in structure,work together to maintain cerebral homeostasis in function,and play an important role in brain function and cerebral ischemic injuries.NVU injury leads to microvascular and blood brain barrier integrity impairment,neuronal cell death,glial reaction and immune cell infiltration,and even tissue injury and brain edema.This paper also aims to elucidate the roles of NVU structure and function in the pathogenesis of cerebral ischemia,and offer new ideas and strategies for the research related to drugs for prevention and treatment of cerebral ischemia based on NVU structure and function.

OBJECTIVE:To formulate the quality standard of Cnidium monnieri liniment (called "liniment" for short)preliminarily. METHODS:The property of liniment was observed. TLC was used for qualitative identification of osthole in liniment;relative density,alcohol content and pH value were also determined. The osthole and imperatorin in liniment were analyzed qualitatively by HPLC. RESULTS:3 batch of samples were reddish brown liquid and fragrant smelling. In TLC of test sample,the same color fluorescent spots were found in corresponding position as chromatogram of control sample. The linear range of osthole and imperatorin were 0.101 2-0.910 8(r=0.999 6)and 0.006 2-0.124 4 mg/L(r=0.999 6). RSDs of precision tests were 1.38% and 0.79%(n=6). RSDs of stability tests were 0.33% and 0.41%(n=6). RSDs of reproducibility tests were 0.83% and 1.98%(n=6),respectively. Average recoveries rate were 98.73%(RSD=1.29%,n=6)and 99.25%(RSD=1.22%,n=6).Results of content determination showed that the content of osthole and imperatorin were 2.20-2.35 mg/mL and 0.310-0.340 mg/mL,respectively. CONCLUSIONS:Established method is simple,rapid,accurate and can be used for quality control of C. monnieri liniment.

Breast background parenchymal enhancement (BPE) is a special form of breast dynamic contrast enhanced MRI.The diagnosis and research of breast diseases which shows BPE is becoming more and more important.The relationship between BPE and the risk factors of breast cancer is one of the focuses in breast MRI.In this review,the definition and typical manifestations of BPE,the relationship between BPE and fibroglandular tissue,and the influence factors of BPE in the breast MRI were reviewed.

Objective To explore the value of manganese-enhanced MRI in locating the rat visual nuclei.Methods The visual nuclei of thirty-six rats were located by 3 different ways including individual MEMRI locating (group A,n= 1 6),anatomical atlas locating (group B,n=1 6)and direct puncture by using the data obtained in MEMRI (group C,n=4).After unilateral intra-vitreal injection of MnCL2 (30 mmol/L×3 μL)in group A,the brain MRI was performed 24 h later.The location coordinate of lateral geniculate nucleus (LGN) and superior colliculus (SC)were recorded individually.The nuclei injections (3% fluorogold solution,1 μL)were performed by using different location coordinate in groups A and B.The rat’s retinas were examined under fluorescence microscope 5 days later,and the results were compared between the two groups.After brain nucleus puncture injection (30 mmol/L MnCL2 solution,0.5 μL),MRI was performed 1 h later in group C.Results The success rate was 93.8% (1 5/1 6)in group A,and 65.5% (10/1 6)in group B.The difference between groups was statistically significant (P<0.05).All the injection locations of C group were agreed with atlas.Conclusion MEMRI in the visual nucleus stereotactic can improve the accuracy of location.