Ashwagandha and Its Active Ingredient, Withanolide A, Increase Phosphorylation of TrkB in Cultured Hippocampal Neurons

Aims: To primary rat embryonic hippocampal neurons in culture, ashwagandha or one of its active ingredients, withanolide A were added in the presence or absence of nutrient supplementation and then assayed for activity of the BDNF receptor, TrkB. Study Design: Primary hippocampal neurons were cultured and grown in nutrient-rich or nutrient-poor medium. Ashwagandha or withanolide A were then be added to both types of media with or without an inhibitor of TrkB or either the PI-3K or MAPK pathway. Place and Duration of Study: Department of Biological Sciences, California State University, Los Angeles, CA, USA, between July 2021 and August 2022. Methodology: Rat embryos were removed by cesarean section from mother rats at 18 days’ gestation and the hippocampi of the former dissected, plated into culture dishes, and treated with the appropriate drug(s) (see Study Design above). After 4 days, neurons were harvested for Western blotting. Optical density of Western blot bands were quantified and statistically analyzed in a 2-way ANOVA, using a level of statistical significance at P < .05. Results: Under normal conditions (with N2 supplement), ashwagandha, but not withanolide A, increased phospho-TrkB immunoreactivity when compared to the effects of vehicle (controls, F(11, 24) = 22.48, P < .001), although withanolide A did not quite reach statistical significance (P = .069) when compared to that of the controlled condition. Likewise, under nutrient-deprived conditions, both ashwagandha and withanolide A also increased phosphorylation of TrkB when compared to that of vehicle-nutrient-deprived conditions (P < .0001). The same results were obtained in the presence of inhibitors of TrkB itself and the PI-3K (ashwagandha, P < .001; withanolide A, P < .001) and MAPK (ashwagandha, P = .027; withanolide A, P = .045) pathways. Conclusion: Ashwagandha or withanolide A activates TrkB, in nutrient-deprived hippocampal neurons, underscoring its role in neuronal survival signaling.

Therapeutic effect and mechanism of Xiaoyao Kangai Jieyu Recipe on mice with breast cancer related depression through regulating COX pathway / 中国中药杂志

This study aimed to investigate the intervention effect and mechanism of Xiaoyao Kangai Jieyu Recipe(XKJR) on hip-pocampal microglia and neuronal damage in mice with breast cancer related depression. The mouse model of breast cancer related depression was established by inoculation of 4T1 breast cancer cells in axilla and subcutaneous injection of corticosterone(30 mg·kg~(-1)). The successfully modeled mice were randomly divided into a model group, a positive drug group(capecitabine 60 mg·kg~(-1)+fluoxetine 19.5 mg·kg~(-1)), and XKJR group(19.5 mg·kg~(-1) crude drug), with 6 in each group. Another 6 normal mice were taken as a normal group. The administration groups were given corresponding drugs by gavage, while the normal and model groups were given an equal volume of distilled water, once a day for 21 consecutive days. The depressive behavior of mice was assessed by glucose consumption test, open field test and novelty-suppressed feeding test. Hematoxylin and eosin(HE) staining and tumor suppression rate were used to evaluate the changes of axillary tumors. The mRNA expressions and the relative protein expressions of interleukin-1β(IL-1β), interleukin-18(IL-18), cyclooxyganese-2(COX-2) and glutamyl-prolyl-tRNA synthetase(EPRs) in the hippocampus of mice were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry, respectively. Immunofluorescence was performed to detect the mean fluorescence intensity of CD11b, a marker of hippocampal microglia activation. Nissler staining and transmission electron microscopy were employed to observe the morphological changes and the ultramorphological changes of hippocampal neurons, respectively. The experimental results indicated that compared with the normal group, the model group had reduced glucose consumption and lowered number of total activities in open field test(P<0.05, P<0.01), prolonged first feeding latency in no-velty-suppressed feeding test(P<0.01), and significant depression-like behavior; the contents of IL-1β, IL-18, COX-2, and EPRs in hippocampus were increased(P<0.05, P<0.01), with hippocampal microglia activation and obvious neuronal damage. Compared with the model group, the positive drug group and the XKJR group presented an improvement in depressive behaviors, a decrease in the contents of IL-1β, IL-18, COX-2 and EPRs in hippocampus, and an alleviation in the activation of hippocampal microglia and neuronal damage; the tumor suppression rates of positive drug and XKJR were 40.32% and 48.83%, respectively, suggesting a lower tumor growth rate than that of the model group. In summary, XKJR may improve hippocampal microglia activation and neuronal damage in mice with breast cancer related depression through activating COX signaling pathway.

Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain / 中国实验方剂学杂志

ObjectiveTo evaluate the intervention effect of dihydroartemisinin （DHA） on hippocampal nerve injury in L5 spinal nerve ligation （SNL） model and tumor necrosis factor-α （TNF-α） hippocampal continuous injection model. In primary cultured microglia-hippocampal neurons， the regulatory pattern of DHA on microglia-hippocampal neuronal interactions was confirmed. MethodThe experimental animals were divided into Sham group， SNL group， and DHA group （16 mg·kg-1）， with 3 mice in each group. The hippocampal CA3 glutamatergic neurons were labeled with adeno-associated virus ［Calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） dTomato AAV］， and their contributions to the hippocampal CA1， prefrontal cortex （Frc）， anterior cortex （ACC）， projections of nucleus accumbens （Nac）， and Basolateral Amygdala （BLA） were traced by immunofluorescence staining. The experimental animals were divided into a Sham group， a TNF-α hippocampus continuous injection model group， DHA-L， DHA-M， and DHA-H groups （4， 8， 16 mg·kg-1）， and pregabalin group （25 mg·kg-1）， with 4 mice in each group. The morphology of pyramidal neurons in the hippocampal CA1 and CA3 regions was counted by Golgi staining. The continuous activation of hippocampal primary neurons and microglia was induced， DHA intervention was given by co-culture， and the cell soma area and the expression of postsynaptic density protein 95 （PSD95） inside and outside the primary and secondary dendritic spines of neurons were counted by immunofluorescence. ResultCompared with the Sham group， the projection of CA3 glutamatergic neurons to CA1 region， Frc， and ACC in the SNL group was significantly reduced （P<0.01）， while the projection to Nac and BLA was significantly increased （P<0.01）. As compared with the SNL group， the projection of hippocampal CA3 glutamatergic neurons to CA1 region， Frc， and ACC was significantly increased in the DHA group （P<0.01）， while the projection to Nac and BLA was significantly reduced （P<0.01）. Golgi staining results showed that as compared with the Sham group， the density of dendritic spines and the number of dendritic branches in the CA1 and CA3 pyramidal neurons in the TNF-α hippocampal continuous injection model group were significantly reduced （P<0.01）. As compared with the TNF-α hippocampal continuous injection model， the density of dendritic spines and the number of dendritic branches in hippocampal CA1 and CA3 pyramidal neurons in the DHA-M and DHA-H groups were significantly increased （P<0.05， P<0.01）. Compared with DHA-M group， the total dendrite length of CA1 pyramidal neurons in hippocampus in DHA-H group was significantly increased （P<0.01）， while the total dendrite length of CA1 neurons and the total dendrite base length of CA3 neurons in DHA-L group was significantly decreased （P<0.01）. Compared with the blank control group， the cell soma area of the glycine group and glutamate group increased significantly （P<0.01）. As compared with the glycine group and glutamate group， the cell area of the glycine + glutamate group was significantly increased （P<0.01）， and as compared with the glutamate group， the cell soma area of the glutamate + DHA group was significantly reduced （P<0.01）. As compared with the glycine acid + glutamate group， the cell soma area of the glycine + glutamate + DHA group was significantly reduced （P<0.01）， and as compared with the glutamate + DHA group， the cell soma area of the glycine + glutamate + DHA group was also significantly reduced （P<0.05）. Compared with the blank control group， the cell soma area of the glutamate group was significantly increased （P<0.01）. As compared with the glutamate group， the cell soma area of the glutamate + DHA-L， glutamate + DHA-M， and glutamate + DHA-H groups was significantly reduced （P<0.01）. As compared with the blank control group， the expression of the resting primary microglia + glycine group in primary and secondary dendritic internal and external postsynaptic density protein 95 （PSD95） was significantly increased （P<0.01）. As compared with the resting primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons of the activated primary microglia + glycine group was significantly reduced （P<0.01）. As compared with the activated primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. As compared with the activated primary microglia + DHA group， the expression of PSD95 in the primary and secondary dendritic spines and outside neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. ConclusionDHA has a significant repair effect on vertebral neuronal damage caused by hippocampal microglia and TNF-α overexpression in NP pathology， and this repair is closely related to the dual inhibition of neuronal-microglia by DHA.

Improvement Effects of β-boswellic Acid on Hippocampal Neurons Cells Injury Induced by Oxygen-glucose Deprivation / 中国药房

OBJECTIVE：To study the imp rovement effects of β-boswellic acid on hippocampal neurons cells injury of rats induced by oxygen-glucose deprivation. METHODS ：The hippocampal neurons cell of rats were divided into normal control group ， model group and β-boswellic acid low-concentration ，medium-concentration and high-concentration groups （1，10，100 μmol/L）. Except for normal control group ，other groups were cultured with relevant medium and given oxygen glucose deprivation to induce oxygen-glucose deprivation induced injury model. MTT assay was adopted to detect cell viability. Chemical colorimetry was used to detect LDH activity in cell culture supernatant. Hoechst-PI staining was used to detect the morphology change of cells. Flow cytometry was used to detect early apoptosis rate of cells. The expression of apoptosis-related protein （Bcl-2，Bax and cleaved caspase-3） were detected by Western blot. RESULTS ：Compared with model group ，the survival rate of cells and protein expression of Bcl- 2 were increased significantly in β-boswellic acid medium-concentration and high-concentration groups （P＜ 0.01），while LDH activity ，early apoptosis rate ，protein expression of cleaved caspase- 3 and Bax were all decreased significantly （P＜0.05 or P＜0.01）. The densely stained nuclei and fragmentation decreased significantly. CONCLUSIONS ：β-boswellic acid can relieve oxygen-glucose deprivation induced injury of hippocampal neurons cells ，the mechanism of which may be associated with down-regulating the protein expression of cleaved caspase- 3 and Bax and up-regulating the protein expression of Bcl- 2.

Mechanism of Huangjingwan in Inhibiting Tau Hyperphosphorylation in Hippocampal Neurons of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid Resulting in Learning and Memory Disorders / 中国实验方剂学杂志

Objective:To investigate the effect of Huangjingwan （HW） on the activities of glycogen synthase kinase-3β （GSK-3β）， protein phosphatase 2A （PP2A） and the mechanism in inhibiting tau protein hyperphosphorylation in the hippocampal neurons of mice with Alzheimer's disease. Method:After subcutaneous injection with 1.0% D-galactose （0.14 g·kg-1·d-1） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL （75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10-3 g·kg-1·d-1） and HW group （2.5 g·kg-1·d-1）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group for modeling control. Two weeks after modeling， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. The mice in normal control group were only given daily feed. At the end of gavage， all the mice were tested by the open field experiment and jumping platform experiment to evaluate the differences in exploratory activity ability， anxiety level and learning and memory ability. The number of neurons in CA1 and CA3 areas of hippocampus in all the mice was detected by Nissl staining. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to detect mRNA expressions of GSK-3β and PP2A in hippocampus of mice in each group. Protein expressions of GSK-3β， PP2A， phosphorylated tau （p-tau） and total tau protein （t-tau） in hippocampus of mice in each group were detected by Western blot. Result:Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by a lower spontaneous activity， lower exploration behavior ability， higher anxiety level， less movement and easier to stay and hide， longer learning response time， significantly increased number of learning and memory errors， and decreased numbers of hippocampal neuron in CA1 and CA3 areas， and reduced mRNA and protein expressions of PP2A， mRNA and protein expressions of GSK-3β， p-tau protein and the ratio of p-tau/t-tau were all increased significantly （P<0.01）， while expression of t-tau protein was decreased， with no significant difference. Compared with the AD model group， mice in the HW group showed a higher spontaneous activity， higher exploration ability， lower anxiety level， higher learning and memory performance， and the numbers of hippocampal neuron in CA1 and CA3 areas increased， while mRNA and protein expressions of PP2A increased， and the mRNA and protein expressions of GSK-3β， the expression of p-tau protein and the ratio of p-tau/t-tau were all decreased significantly （P<0.01）， but with no significant difference in the protein expression of t-tau. Conclusion:HW can inhibit tau hyperphosphorylation in hippocampal neurons of AD mice， restore tau protein function， protect hippocampal neurons， and exert an anti-AD effect， which may be related to the regulatory mechanism in the activity balance between GSK-3β and PP2A in hippocampal neurons.

Effect of Huangjingwan on Expressions of Wnt/β-catenin Signal Pathway-associated Proteins in Hippocampus of Mice with Alzheimer's Disease Induced by D-galactose and Okadaic Acid with Learning and Memory Disorders / 中国实验方剂学杂志

Objective:To explore the effect of Huangjingwan （HW） on the expressions of Wnt/β-catenin signal pathway-associated proteins in the hippocampus of mice with Alzheimer's disease （AD） induced by D-galactose and okadaic acid with learning and memory disorders， as well as its mechanism. Method:After subcutaneous injection with 1.0% D-galactose （0.14 g·kg-1·d-1） into the back and neck of mice for 4 weeks， the right ventricle of mice was injected with 2 μL（75 ng） of okadaic acid for one time to make AD model， and the successfully modeled AD mice were selected by Morris water maze. Then， the selected AD mice were randomly divided into AD model group， memantine group （1.3×10-3 g·kg-1·d-1） and HW group （2.5 g·kg-1·d-1）. In addition， the sham model control group and the normal control group were set up. At the same time， 2 μL normal saline was injected into the right ventricle of mouse in the sham model control group as the modeling control. Two weeks after molding， the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition， after 2 weeks of AD modeling， mice in sham model control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. There was no special treatment in the normal control group. At the end of gavage， the shuttle experiment was performed to detect the differences in learning and memory levels of mice in each group. The changes of β-catenin and GSK-3β positive neurons in CA1 area of hippocampus in each group were tested by immunohistochemistry. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expressions of GSK-3β， β-catenin and CyclinD1 in hippocampus of mice in each group. The Western blot was used to detect the expressions of total GSK-3β （t-GSK-3β）， phosphorylation of GSK-3β at Ser9 （p-Ser9-GSK-3β）， phosphorylation of GSK-3β at Tyr216 （p-Tyr216-GSK-3β）， total β-catenin （t-β-catenin）， phosphorylation of β-catenin （p-β-catenin） and CyclinD1 proteins in hippocampus of mice in each group. Result:Compared with the normal control group， mice in AD model group showed an obvious dementia state， which was characterized by significant declines in learning and memory ability， the number of β-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-β-catenin and CyclinD1， the protein expressions of p-Ser9-GSK-3β， and the ratio of p-Ser9-GSK-3β/t-GSK-3β and p-Tyr216-GSK-3β/t-GSK-3β in hippocampal region （P<0.01）， and significant increases in the number of GSK-3β immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3β， the protein expressions of p-Tyr216-GSK-3β and p-β-catenin， the ratio of p-β-catenin/t-β-catenin in hippocampal region （P<0.01 respectively）. Compared with the AD model group， the dementia symptoms of mice in HW group were significantly alleviated， and the number of β-catenin immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-β-catenin and CyclinD1， the protein level of p-Ser9-GSK-3β， the ratio of p-Ser9-GSK-3β/t-GSK-3β in hippocampal region were all significantly increased （P<0.01 respectively）， whereas the number of GSK-3β immunoreactive neurons in hippocampal CA1 area， the mRNA and protein expressions of t-GSK-3β， the proteins expressions of p-Tyr216-GSK-3β and p-β-catenin， the ratio of p-β-catenin/t-β-catenin in hippocampal region were all significantly decreased （P<0.01 respectively）， but the ratio of p-Tyr216-GSK-3β/t-GSK-3β has no significant statistical difference. Conclusion:HW shows the role of AD treatment， which can down-regulate the expression of GSK-3β in the hippocampus of AD mice and reduce its protein activity， and up-regulate the expression of β-catenin as well as increase its protein activity， so as to enhance the expression of downstream CyclinD1 and promote the transcription of the target genes. Its mechanism may be related to the activation of Wnt/β-catenin signal pathway.

Neuroprotective Effect of Isobenzofuranones on Hydrogen Peroxide-Mediated Redox Imbalance in Primary Cultures of Hippocampal Neurons

Abstract In live organisms, there is a balance between the production of reactive oxygen species (ROS) and their neutralization. The increased level of these species leads to a condition called redox imbalance. The aim of this study was to evaluate the protective action of isobenzofuranones in primary cultures of hippocampal neurons subjected to redox imbalance. To accomplish this, MTT and LIVE/DEAD assays were initially performed. In the cultures pretreated with isobenzofuranones 1 and 2, there was a higher number of live cells when compared to that in the untreated ones. Regarding redox imbalance, there was a significant increase in the intracellular levels of ROS. The cultures pretreated with isobenzofuranones showed a reduction in ROS levels. Lipid peroxidation caused by oxidative damage was significantly reduced in the cultures pretreated with isobenzofuranones 1 and 2. Taken together, these data show the ability of isobenzofuranones 1 and 2 to significantly minimize cytotoxicity, cell death, intracellular levels of ROS and lipid peroxidation induced by redox imbalance. These results suggest that isobenzofuranones 1 and 2 represent a possible alternative therapy for the neurodegenerative disturbances that are triggered by ROS production increases.

Effects and Mechanism of Buyang Huanwu Decoction and the like on Oxygen Deprivation and Reoxygenation of Hippocampal Neurons in Vitro / 中国药学杂志

OBJECTIVE: To investigate the effects and mechanism of Buyang Huanwu Decoction and the like on primary cultured neonate hippocampal neurons induced by oxygen deprivation and reoxygenation. METHODS: The primary cultured hippocampal neurons cell model was established, oxygen sugar deprivation/reoxygenation (OGD/R) model was established in vitro. CCK-8 was used to determine the concentration of Buyang Huanwu Decoction and the like drug-containing serum to protect hippocampal neurons cell. The experimental groups were divided into control group, model group, Buyang Huanwu Decoction drug-containing serum group(10%), Naoxintong Capsule drug-containing serum group(10%), Yangyin Tongnao Granules drug-containing serum group(10%), Buyang Huanwu Decoction drug-containing serum+TAK-242 group(10%,1 μmol•L-1), Naoxintong Capsule drug-containing serum+TAK-242 group(10%,1 μmol•L-1), Yangyin Tongnao Granules drug-containing serum+TAK-242 group(10%,1 μmol•L-1). The morphology of hippocampal neuron was observed under inverted microscope. The expression of tumor necrosis factor-α(TNF-α) and interleukin 1(IL-1β) in the cell supernatant was detected by ELISA kit. The neuronal cell apoptosis was observed by Hoechst 33358 staining fluorescence microscope and the expression of TLR4/NF-κB signaling pathway proteins was detected by Western blot. RESULTS: The results showed that hgher purity neuronal cells can be obtained by primary culture. Compared with model group, Buyang Huanwu Decoction and the like all can reduce the release of TNF-α and IL-1β in hippocampal neurons, significantly reduced the number of apoptosis. At the same time, the three traditional Chinese medicine compounds all can inhibit the expression of TLR4 and NF-κB protein in TLR4/NF-κB signaling pathway, and protect the inflammatory response of oxygenated sugar deprivation and reoxygenated hippocampal neurons. The addition of TLR4-specific inhibitor TAK-242 blocked the conduction of this signaling pathway and reduced the protective effect of Buyang Huanwu Decoction and the like. CONCLUSION: Buyang Huanwu Decoction and the like have protective effects on OGD/R-induced inflammatory response in hippocampal neurons, its mechanism may be related to TLR4/NF-κB signaling pathway, and the anti-inflammatory protective effect of Naoxintong Capsule is relatively obvious.

Nao-Fu-Cong ameliorates diabetic cognitive dysfunction by inhibition of JNK/CHOP/Bcl2-mediated apoptosis in vivo and in vitro / 中国天然药物

Chinese herbal compound Nao-Fu-Cong (NFC) has been mainly used to treat cognitive disorders in Traditional Chinese Medicine (TCM). The present study aimed to investigate whether its neuroprotective effects might be related to the inhibition of JNK/CHOP/Bcl2-mediated apoptosis pathway or not. We randomly assigned STZ (60 mg·kg)-induced diabetic rats into control group, diabetic model group and NFC groups (low-dose, medium-dose and high-dose). The primary culture of hippocampal neurons were transferred into different culture media on the third day. The cells were then divided into control group, high-glucose group, NFC (low-dose, medium-dose and high-dose) groups, CHOP si-RNA intervention group, JNK pathway inhibitor SP600125 group and oxidative stress inhibitor N-acetylcysteine (NAC) group. NFC significantly improved the cognitive function of diabetic rats, and had neuroprotective effect on hippocampal neurons cultured in high glucose. Further research results showed that NFC could reduce the apoptosis of hippocampal neurons in rats with diabetic cognitive dysfunction. NFC had inhibitory effects on CHOP/JNK apoptosis pathway induced by high glucose, and also decreased the levels of ROS and increased the mitochondrial membrane potential. These suggested that the neuroprotective effect of NFC might be related to the inhibition of CHOP and JNK apoptotic signaling pathways, and the cross pathway between oxidative stress and mitochondrial damage pathway.

Vitamin E reduces radiation injury of hippocampal neurons in mice by inhibiting ferroptosis / 南方医科大学学报

OBJECTIVE@#To explore the protective effect of vitamin E (VE) against radiation injury of hippocampal neurons in mice and explore the possible mechanism.@*METHODS@#Cultured HT-22 and U251 cells with or without exposure to 8 Gy irradiation were treated with VE (200 μmol/L for 24 h), ferroptosis inhibitor (ferrostatin-1, 5 μmol/L for 24 h), apoptosis inhibitor (ZVAD-FMK, 2 μmol/L), or necroptosis inhibitor (100 μmol/L). MTT assay was used to evaluate the cell viability after the treatments, and reduced glutathione (GSH), malondialdehyde (MDA), lipid reactive oxygen species (lipid ROS), and intracellular iron ion levels were detected for assessment of ferroptosis. The mice exposed to 16 Gy irradiation with or without vitamin E (500 U/kg) treatment for 6 weeks were assessed for behavioral changes and cognitive functions using Morris water maze test.@*RESULTS@#Treatment with VE significantly promoted the cell survival following irradiation in HT-22 cells ( < 0.05) but not in U251 cells ( > 0.05). Ferrostatin-1, but not ZVAD or the necroptosis inhibitor, promoted the survival of HT-22 cells following the irradiation. Exposure to irradiation significantly increased ferroptosis-related oxidative stress level in HT-22 cells, manifested by decreased GSH level and increased MDA, lipid ROS and intracellular iron ion levels ( < 0.05); treatment with VE and ferrostatin-1 both obviously reversed radiation-induced ferroptosis-related oxidative stress in the cells ( < 0.05). In Morris water maze test, the mice with radiation exposure showed obviously increased exploration time and distance ( < 0.05), which were significantly decreased after treatment with VE ( < 0.05).@*CONCLUSIONS@#Vitamin E reduces radiation injury by inhibiting ferroptosis in the hippocampal neurons in mice.

Protective effects of compatibility of effective constituents in Salvia miltiorrhiza and Ligusticum chuanxiong on hippocampal neurons injured by oxygen glucose deprivation / 中草药

Objective: To observe the protective effect of Salvia miltiorrhizae and Ligusticum chuanxiong effective constituents: danshensu, protocatechuic aldehyde, ligustrazine, and ferulic acid combination on primary cultured hippocampal neurons injured by oxygen glucose deprivation, and find out an optimized combination. Methods Primary cultured rats hippocampal neurons waschosen as research objects by adopting immunohistochemistry of the neuron-specific enolase IgG to authenticate, then the OGD model of the hippocampal neurons injured by oxygen glucose deprivation was established. The non-cytotoxic dose range of danshensu, protocatechuic aldehyde, ligustrazine, ferulic acid, and nimodipine was studied by MTT method. The compatibility of components was arranged by L9 (34) orthogonal design. Primary cultured rats hippocampal neurons was divided into 12 groups: control group, model group, Nimodipine positive control group, and orthogonal design 1-9 group. The activity of LDH was measured by colorimetry, the activity of SOD was tested by WST-1 and the levels of MDA were examined by TBA. The levels of TNF-α, IL-1β, and IL-6 in cell culture supernate were examined by ELISA, the apoptosis of hippocampal neurons was detected by Fluorochrome Hoechst33258 staining and the cell early apoptosis rate was detected with flow cytometry. The results of orthogonal test was analyzed by using range analysis method. Results The orthogonal compatibility of reagents played significant roles against the hypoxia damage of hippocampal neurons, improved the cellular morphology obviously, reduced the activity of LDH, increased the activity of SOD and decreased the content of MDA significantly, inhibited the release of TNF-α, and reduced the content of IL-1β and IL-6, reduced the apoptosis of cells apparently. The effect of active ingredients of S. miltiorrhiza and L. Chuanxiong on LDH activity was danshensu > ligustrazine > protocatechuic aldehyde > ferulic acid, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on SOD activity was ferulic acid > ligustrazine > danshensu > protocatechuic aldehyde. The best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (40 μg/mL). The order of influence on MDA content was danshensu > protocatechuic aldehyde > ferulic acid > ligustrazine, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on TNF-α content were: ligustrazine > protocatechuic aldehyde > danshensu > ferulic acid, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (60 μg/mL), ligustrazine (40 μg/mL), and ferulic acid (10 μg/mL). The order of influence on the content of IL-1β was ligustrazine > ferulic acid > danshensu > protocatechuic aldehyde, and the best combination was danshensu (30 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (20 μg/mL). The effect on the content of IL-6 was protocatechuic aldehyde > ligustrazine > ferulic acid > danshensu, and the best combination was danshensu (120 μg/mL), protocatechuic aldehyde (120 μg/mL), ligustrazine (80 μg/mL), and ferulic acid (10 μg/mL). The effect on the early apoptosis rate was ferulic acid > protocatechuic aldehyde > ligustrazine > danshensu, and the best combination was danshensu (60 μg/mL), protocatechuic aldehyde (30 μg/mL), ligustrazine (20 μg/mL), and ferulic acid (40 μg/mL). Conclusion The protective mechanism of the effective constituents of S. miltiorrhizae and L. chuanxiong were relied on reducing the oxidative damage, reducing inflammation damage, and antagonizing cell apoptosis. According to the experimental results, we need to change the prescription ratio and guide clinical medication for different clinical courses.

Intervention Effect of Kidney-tonifying and Essence-filling Therapy on Hippocampal Autophagy in Kidney-deficiency Alzheimer Mice / 中国实验方剂学杂志

Objective:To study the effect of Liuwei Dihuangwan on the improvement of autophagy level of hippocampal neurons in mice with kidney deficiency Alzheimer' s disease (AD) and its partial mechanism, in order to explore part of therapeutic mechanisms of kidney-tonifying and essence-filling therapy for AD. Method:Healthy male C57-B6 mice were divided into control group, AD group, kidney deficiency AD group and Liuwei Dihuangwan group(1.08 g·kg-1). The control group and the AD group were subcutaneously injected with normal saline (15 mL·kg-1) daily, and the kidney deficiency AD group and the Liuwei Dihuangwan group were subcutaneously injected with hydrocortisone injection (15 mL·kg-1) daily for 20 consecutive days. On the 21st day, the other three groups were injected with 6 μg amyloid beta protein 25-35(Aβ25-35) in the lateral ventricle, while the control group was injected with sterile saline into the lateral ventricle. The levels of serum cortisol and testosterone in each group were detected by enzyme-linked immunosorbent assay (ELISA), the morphological changes in hippocampal neurons were observed by transmission electron microscopy, the expression of microtubule-associated protein 1 light chain 3 (LC3) was detected by immunofluorescence, and the expression of selective autophagic junction protein (p62) was detected by Western blot. Result:Compared with normal group, serum cortisol and testosterone levels in AD group and kidney deficiency AD group were significantly reduced (PPPPPPPPPConclusion:Kidney-tonifying and essence-filling therapy can protect hippocampal neurons, increase LC3 expression in hippocampal neurons, decrease p62 expression level and increase autophagy level of hippocampal neurons. It has a certain therapeutic effect on kidney-deficiency Alzheimer' s disease.

Protective effect and mechanism of Xiaoyao San on lipopolysaccharide-induced hippocampal neurons injury / 中国中药杂志

To investigate the relationship between anti-depressant effect and hippocampal nerve growth of Xiaoyao San,the inflammatory model of hippocampal neuron was induced by lipopolysaccharide( LPS). The effect of Xiaoyao San serum( final concentration of4%,8%) on the cell proliferation activity was detected by immunofluorescence,the levels of BDNF and β-NGF in the supernatant of hippocampal neurons were detected by ELISA,and the expressions of BDNF,NGF,Trk B,Trk A and CREB mRNA in cell lysate of hippocampal neuron were detected by PCR. Western blot was used to detect the expressions of Trk B,CREB,p-CREB and SYP protein in cell lysate of hippocampal neuron,and to reveal the neuroprotective effect and mechanism of Xiaoyao San. The results showed that8% Xiaoyao San serum could significantly increase in Brdu/Neu N ratio( P<0. 01). 4%,8% Xiaoyao San serum could significantly improve the levels of BDNF and β-NGF in supernatant( P<0. 05 or P<0. 01),up-regulate the expression of BDNF,NGF,Trk B,Trk A,CREB mRNA and Trk B,p-CREB,SYP protein in cell lysate( P< 0. 05 or P< 0. 01). 8% Xiaoyao San serum could significantly increase CREB protein in cell lysate( P<0. 05),and elevate in p-CREB/CREB ratio( P<0. 01). All the above results indicate that Xiaoyao San has a certain protective effect on LPS induced hippocampal neuron injury,which suggests that the protective effect of Xiaoyao San is related to the promotion of hippocampal nerve growth,which is one of its antidepressant mechanisms.

Antagonistic effect of nimodipine on DBP-induced learning and memory impairment in KM mice / 中国药理学通报

Aim To explore the antagonistic effect of nimodipine (Nim) on dibutyl phthalate (DBP)-in-duced learning and memory impairment in KM mice. Methods Thirty-six male KM mice were treated with saline (control), 50 mg • kg-1 DBP, 2 mg • kg-1 Nim, and DBP + Nim lasted for 28 days. The latency of KM mice in each group was measured. Levels of calmodulin (CaM), calmodulin/calmodulin-dependent protein kinase II ( CaMKII ) , protein kinase C (PKC) , cytochrome C (Cyt C) and caspase-3 in hippocampus of KM mice in each group were detected. And expressions of ERK1/2 and p-ERKl/2 were evaluated. In addition, the pathological changes of hipp-ocampal CAI region were also analyzed by HE, Nissl staining, and TUNEL assay. Results Compared with 50 mg • kg-1 DBP group, the learning and memory im-pairment of KM mice in DBP + Nim group was alleviated, the pathological damage and apoptosis in CA1 region of hippocampus were reduced, the levels of PKC, Cyt C, caspase-3 and p-ERKl/2 decreased, while the levels of CaM and CaMKII increased accordingly (P < 0.05). Conclusions DBP affects Ca2 +-related proteins and up-regulates p-ERKl/2 expression, inducing hippocampal neuronal damage and apoptosis, whereas Nim can improve DBP-induced learning and memory impairment in KM mice, which may be related to the ability of Nim to reduce the levels of p-ERKl/2 and caspase-3 in brain tissues of mice after DBP exposure by blocking DBP-induced Ca2+ concentration.

Cyanidin-3-glucoside inhibits amyloid β₂₅₋₃₅-induced neuronal cell death in cultured rat hippocampal neurons

Increasing evidence implicates changes in [Ca²⁺]i and oxidative stress as causative factors in amyloid beta (Aβ)-induced neuronal cell death. Cyanidin-3-glucoside (C3G), a component of anthocyanin, has been reported to protect against glutamate-induced neuronal cell death by inhibiting Ca²⁺ and Zn²⁺ signaling. The present study aimed to determine whether C3G exerts a protective effect against Aβ₂₅₋₃₅-induced neuronal cell death in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using MTT assay for cell survival, and caspase-3 assay and digital imaging methods for Ca²⁺, Zn²⁺, MMP and ROS. Treatment with Aβ25–35 (20 µM) for 48 h induced neuronal cell death in cultured rat pure hippocampal neurons. Treatment with C3G for 48 h significantly increased cell survival. Pretreatment with C3G for 30 min significantly inhibited Aβ₂₅₋₃₅-induced [Zn²⁺]i increases as well as [Ca²⁺]i increases in the cultured rat hippocampal neurons. C3G also significantly inhibited Aβ₂₅₋₃₅-induced mitochondrial depolarization. C3G also blocked the Aβ₂₅₋₃₅-induced formation of ROS. In addition, C3G significantly inhibited the Aβ₂₅₋₃₅-induced activation of caspase-3. These results suggest that cyanidin-3-glucoside protects against amyloid β-induced neuronal cell death by reducing multiple apoptotic signals.

Neuroprotective effect and mechanism of liraglutide on hippocampal neurons in diabetic rats / 中华内分泌代谢杂志

Objective To investigate the neuroprotective effect and mechanism of liraglutide on diabetic rats. Methods 24 healthy male SPF Goto-Kakizaki (GK) rats with random blood glucose greater than 11.1 mmol/L were selected as the experimental group, and randomly divided into diabetes mellitus group ( n＝12) and liraglutide group (n＝12). Ten healthy male SPF Wistar rats with the same age and weight as GK rats were selected as normal control group. After adaptively feeded for 2 weeks, the liraglutide group was given liraglutide (400 μg·kg－1·d－1, subcutaneous injection), while the control group and diabetes mellitus group were given the same volume of saline, and continued to be administered for 8 weeks. After 10 weeks, data and biochemical indicators were recorded. Effects of liraglutide on learning and memory in diabetes mellitus rats were detected by Morris water maze test. HE staining observed the hippocampal neurons morphology. Western blotting method detected the expression of p- IκB kinase (IKK) β, p-NF-κB, NF-κB, Klotho, and PRX2 in hippocampus. Results Morris water maze test showed that liraglutide can improve the spatial learning and memory ability of diabetes mellitus rats. HE staining showed that liraglutide significantly reduced the pathological damage of hippocampal neurons of diabetes mellitus rats. Western blotting showed that liraglutide inhibited NF-κB signaling pathway in hippocampus of diabetes mellitus rats. The expression of Klotho protein in hippocampus of diabetes mellitus group was significantly lower than that of control group, while the expression of PRX2 protein was higher than control group (t＝8.298,－7.398,all P<0.01). The expression of Klotho and PRX2 protein in hippocampus of liraglutide group were higher than diabetes mellitus group (t＝－13.059, 14.113, all P<0.01). The expression of Klotho protein of liraglutide group was similar to that of control group ( t ＝ －1. 137, P>0. 05 ). The expression of PRX2 protein was significantly higher than control group (t＝－28.055, P<0.01). Conclusions Liraglutide may enhance the expression of antioxidant stress protein including Klotho and PRX2, by inhibiting NF-κB signaling pathway in hippocampus of diabetes mellitus rats, reduced oxidative stress and improved the injury of hippocampal neuronal in diabetes mellitus rats, which seems to play a neuroprotective effect, to prevent and delay the occurrence of diabetic encephalopathy.

Protective effect and mechanism of ginsenoside Rg1 on H2O2induced hippocampal neurons aging due to down-regulate NOX2 mediated NLRP1 inflammasome activation in vitro / 中国药理学与毒理学杂志

OBJECTIVE To explore the protective effects and mechanisms of Ginsenoside Rg1 (Rg1) on H2O2-induced hippocampal neurons aging in vitro. METHODS The primary culture hippo-campal neurons(7 d)were randomly placed into six groups:normal control group,H2O2(200 μM)treat-ment group,and H2O2+Rg1(1,5 and 10μM)groups.The neurons were with Rg1(1,5 and 10 μmol·L-1) for 6h. H2O2(200 μmol·L-1) was added to the medium and incubate for 18 h. The Dihydroethidium (DHE) staining was performed for ROS production assessment. The LDH release and Hoechst 33258 were performed to examine the neuronal damage and apoptosis. The immunoblot was used to deter-mine the expression of β-Gal,NOX2,p22phox,p47phox,NLRP-1,ASC and Caspase-1 in hippocampal neurons.The ELISA was performed to detect the levels of IL-1β and IL-18 released in the supernatant in hippocampal neurons.RESULTS Rg1(5 and 10 μmol·L-1)significantly reduced the ROS production, attenuated H2O2-induced neuronal damage and apoptosis (P<0.05, P<0.01). The immunoblot results showed that Rg1(5 and 10 μmol·L-1)treatment significantly decreased the expression of β-Gal,NOX2, p22phox,p47phox,NLRP-1,ASC and Caspase-1 in hippocampal neurons(P<0.05,P<0.01).Additionally, Rg1(5 and 10 μmol·L-1)treatment significantly decreased IL-1β and IL-18 release in the supernatant. CONCLUSION The protective effect of Rg1 in H2O2-induced hippocampal neurons aging may be due to inhibit NOX2-NLRP1 activation.

Protective effect and mechanism of glycyrrhizic acid on the hippocampal neuron injury in epileptic rat models / 中国比较医学杂志

Objective To explore the protective effect and mechanism of glycyrrhizic acid on hippocampal neuron injury in epileptic rat models. Methods The epileptic rat models were established by lithium and pilocarpine kindling. The successful models were randomly divided into epilepsy group and glycyrrhizic acid group. In addition, the rats without any treatment were used as the normal control group, with 12 rats in each group. Hippocampal neuron injury and apoptosis were detected by Nissl and TUNEL staining. Mitochondrial membrane potential in the hippocampal neurons of rats was detected by JC-1 method. The activity of aspartic acid protein hydrolase 3 (caspase 3) and caspase 9 was detected by colorimetric assay. The expressions of cleaved-caspase 3, 9, B lymphocyte tumor-2 (Bcl-2), Bcl-2 related X protein (Bax), cytochrome C (CytC) and apoptotic protease-activating factor (Apaf-1) protein in the hippocampal tissues of rats were detected by western blot assay. Results Compared with the normal group, the number of neurons was reduced (P＜0. 01), the number of TUNEL-positive cells was increased (P＜ 0. 01), mitochondrial membrane potential was decreased (P＜ 0. 01), caspase 3 and 9 activity was increased (P＜ 0. 01), the expressions of Bcl-2 and mitochondrial CytC were down-regulated (P＜ 0. 01), the expressions of Bax, cytoplasm CytC and Apaf-1 were up-regulated (P＜ 0. 01) in the epilepsy group. Compared with the epilepsy group, the number of neurons was increased (P ＜ 0. 01), the number of TUNEL-positive cells was reduced (P＜ 0. 01), mitochondrial membrane potential was increased (P＜ 0. 01), caspase 3 and 9 activity was decreased (P ＜ 0. 01 ), the expressions of Bcl-2 and mitochondrial CytC were up-regulated (P ＜0. 01), the expressions of Bax, cytoplasm CytC and Apaf-1 were down-regulated (P ＜ 0. 01) in the glycyrrhizic acid group. Conclusions These results suggest that glycyrrhizic acid can inhibit the hippocampal neuron injury in epileptic rats by blocking the mitochondrial pathway.

Role of SIRT1∕NF-κB signaling pathway in oxygen-glucose deprivation and restoration injury to hip-pocampal neurons of mice / 中华麻醉学杂志

Objective To evaluate the role of sirtuin 1 ( SIRT1)∕nuclear factor kappa B ( NF-κB) signaling pathway in oxygen-glucose deprivation and restoration ( OGD∕R) injury to hippocampal neurons of mice. Methods The HT22 hippocampal neurons were seeded in a culture plate ( 96-well plate, 100 μl∕well; 6-well plate, 2 ml∕well) at the density of 5×104 cells∕ml or in a culture dish (6 cm in diameter), and then divided into 4 groups ( n=24 each) using a random number table method: control group ( group C) , OGD∕R group ( group OGD) , SIRT1 inhibitor EX-527 preconditioning group ( group EX) and SIRT1 agonist SRT1720 preconditioning group ( group SRT) . Neurons were cultured in normal culture atmosphere at 37 ℃ in group C. In OGD, EX and SRT groups, the culture medium was replaced with oxygen-poor flu-id, and neurons were exposed to 5％ CO2-95％ N2 for 12 h in an incubator at 37℃, oxygen-poor fluid was replaced with the culture medium, and neurons were cultured for 24 h in normal culture atmosphere at 37℃. SIRT1 inhibitor EX-5271 μmol∕L and SIRT1 agonist SRT172010 μmol∕L were added at 12 h beforeOGD in EX and SRT groups, respectively. The cell viability was measured by CCK8 assay, the activity of LDH was detected by chemical colorimetry, cell apoptosis rate was determined by flow cytometry, the ex-pression of SIRT1, NF-κB, IκBα, Bcl-2 and Bax was detected by Western blot, and the Bcl-2∕Bax ratio was calculated. Results Compared with group C, the cell viability was significantly decreased, LDH ac-tivity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regula-ted, the expression of NF-κB and Bax was up-regulated, and the Bcl-2∕Bax ratio was decreased in OGD, EX and SRT groups ( P<0. 05) . Compared with group OGD, the cell viability was significantly increased, the LDH activity and cell apoptosis rate were decreased, the expression of SIRT1, IκBαand Bcl-2 was up-regulated, the expression of NF-κB and Bax was down-regulated, and the Bcl-2∕Bax ratio was increased in group SRT, and the cell viability was significantly decreased, LDH activity and cell apoptosis rate were in-creased, the expression of SIRT1, IκBαand Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2∕Bax ratio was decreased in group EX (P<0. 05). Conclusion SIRT1∕NF-κB signaling pathway inhibition is involved in OGD∕R injury to hippocampal neurons of mice.

Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy.</p><p><b>METHODS</b>The primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca2+], endoplasmic reticulum calcium [Ca2+]ER, and mitochondrial calcium [Ca2+]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate (SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels.</p><p><b>RESULTS</b>The results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum.</p><p><b>CONCLUSION</b>The calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.</p>