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1.
Chinese Journal of Information on Traditional Chinese Medicine ; (12): 116-122, 2024.
Article in Chinese | WPRIM | ID: wpr-1026854

ABSTRACT

Objective To observe the protective effect of modified Baishile Decoction on hippocampal neuronal cells cultured in vitro;To explore its mechanism of treating post-stroke depression.Methods Hippocampal neuronal cells from mammary rats were isolated and cultured in vitro,cell injury was induced by oxygen glucose deprivation combined with lipopolysaccharide.The cells were divided into normal group,model group,blank serum group(10%)and modified Baishile Decoction containing serum group(10%).Invertedmicroscope was used to observe cell morphological changes,CCK-8 method was used to detect cell survival rate,Hoechst33342 staining was used to observe apoptosis,ELISA was used to detect Glu,5-HT,TNF-α,IL-1β,and IL-6 contents in cell supernatant,the expressions of purinergic P2X7 receptor(P2X7R)and NOD-like receptor protein 3(NLRP3)were detected by immunofluorescence staining.Results Compared with the normal group,the hippocampal neurons in the model group showed significant changes in cell morphology,the cell survival rate significantly decreased(P<0.01),the cell apoptosis increased(P<0.01);Glu,TNF-α,IL-1β,IL-6 contents in cell supernatant significantly increased(P<0.05,P<0.01),5-HT content significantly decreased(P<0.01),P2X7R and NLRP3 expressions in hippocampal neuronal cells significantly increased(P<0.01).Compared with the model group,the morphology of hippocampal neurons in modified Baishile Decoction containing serum group was significantly improved,the cell survival rate significantly increased(P<0.01),the cell apoptosis reduced(P<0.01);Glu,TNF-α and IL-1β content in cell supernatant significantly reduced(P<0.05,P<0.01),5-HT content significantly increased(P<0.01),and P2X7R and NLRP3 expressions in hippocampal neuronal cells significantly decreased(P<0.01).Conclusion Modified Baishile Decoction may exert a protective effect on oxidative glucose deprivation combined with lipopolysaccharide induced hippocampal neuronal inflammation damage by inhibiting the P2X7R/NLRP3 signaling pathway,regulating neurotransmitter secretion,and inhibiting inflammatory factor release,thus treating post-stroke depression.

2.
European J Med Plants ; 2023 Feb; 34(2): 1-12
Article | IMSEAR | ID: sea-219534

ABSTRACT

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.

3.
Journal of Apoplexy and Nervous Diseases ; (12): 113-117, 2023.
Article in Chinese | WPRIM | ID: wpr-1032096

ABSTRACT

@#Objective To explore the impact of dexmedetomidine (Dex) on the apoptosis of rat hippocampal neurons induced by β-amyloid (Aβ) 1-42 via mitogen-activated protein kinase (MAPK)/signal transduction and transcriptional activators 3 (STAT3) pathway. Metuods The patients were randomly divided into sham operation group,Aβ1-42、Dex(Dex-L)、Dex(Dex-H)、Dex-H+Anisomycin、Dex-H+colivelin group.The experimental observation was carried out after different treatment of administration.Results In the Aβ1-42 group,the neuronal structure of the hippocampus of rats in the Aβ1-42 group was lost,the cell body morphology was abnormal,and the Aβ deposition was severe,the escape latency,the levels of inflammatory factors (IL-1β,IL-6,TNF-α),the number of apoptotic cells,the expressions of p-p38 MAPK/p38 MAPK,p-STAT3/STAT3,and cleaved caspase-3 were unusually higher than those in the sham operation group,the times of crossing the original platform and the expression of Bcl-2 were unusually lower (P<0.05);the above indicators could be reversed after intervention with different doses of Dex (P<0.05);MAPK pathway activators and STAT3 activators could alleviate the protective effect of Dex on AD rats (P<0.05). Conclusion Dex can reduce the levels of inflammatory factors and Aβ deposition to reduce cell damage and apoptosis,and protect rat hippocampal neurons,which may be related to the inhibition of MAPK/STAT3 pathway.

4.
Acta Universitatis Medicinalis Anhui ; (6): 589-596, 2023.
Article in Chinese | WPRIM | ID: wpr-1038506

ABSTRACT

Objective@#To explore and optimize the primary culture method of neonatal mouse hippocampal neurons in vitro.To construct a G-protein-coupled receptor kinase 2 ( GRK2) knockout HT22 cell line.@*Methods @#Hippocampal tissue of C57BL6 /J mice on day 1-2 was taken,digested with trypsin and pipetted to form a cell suspension,and supplement was added to Neurobasal-A medium to maintain cell growth. CRSIPR / Cas9 gene editing technique was used to construct HT22-GRK2 -/ - cell line,and the knockout efficiency of GRK2 was detected by immunofluorescence staining and Western blot. @*Results @#Primary hippocampal neurons of newborn mice were put into six-well plates with 3 × 107 /well using a serum-free culture method,which could get a high purity and good activity ; HT22-GRK2 -/ - cell line was constructed successfully.@*Conclusion@#The primary culture method of mouse hippocampal neurons was successfully established and optimized,and HT22-GRK2 -/ - cell line was successfully constructed by CRSIPR / Cas9 gene editing technique.

5.
China Journal of Chinese Materia Medica ; (24): 3874-3881, 2023.
Article in Chinese | WPRIM | ID: wpr-981520

ABSTRACT

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.


Subject(s)
Mice , Animals , Depression/genetics , Interleukin-18 , Cyclooxygenase 2/genetics , Hippocampus , Glucose , Neoplasms
6.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 73-82, 2023.
Article in Chinese | WPRIM | ID: wpr-969601

ABSTRACT

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.

7.
Chinese Pharmacological Bulletin ; (12): 1189-1194, 2023.
Article in Chinese | WPRIM | ID: wpr-1013795

ABSTRACT

Aim To explore the protective effect of Zishen Huoxue Prescription on OGD/R-induced primary hippocampal neuron damage in rats and the possible mechanism. Methods After the isolated primary hippocampal neurons were identified by immunofluorescence, OGD/R induced neuronal damage, and the changes of autophagic flux at different re-oxygenation time were observed by confocal laser scanning microscopy. After OGD/R-induced primary hippocampal neurons were intervened with serum containing Zishen Huoxue Prescription, cell viability was detected by CCK-8, cell apoptosis was detected by flow cytometry, autophagosomes were detected by transmission electron microscopy, and autophagy-related protein expressions were detected by Western blot. Results 10% Zishen Huoxue Prescription-containing serum could significantly improve cell viability and reduce the proportion of cell apoptosis, increase the number of autophagosomes in neurons, and up-regulate the expression of autophagy-related protein PINK1, Parkin, and pATG16L1. Conclusions Zishen Huoxue Prescription can effectively resist OGD/R-induced apoptosis of primary hippocampal neurons in rats, and its effect may be related to the regulation of PINK1-Parkin pathway to promote mitophagy.

8.
Journal of Apoplexy and Nervous Diseases ; (12): 503-509, 2022.
Article in Chinese | WPRIM | ID: wpr-1038957

ABSTRACT

@#Objective To observe the influences of circular RNA HECT domain E3 ubiquitin ligase 1 (Circ_HECTD1) on the proliferation and apoptosis of HT22 cells and its regulatory mechanism on the microRNA-135a-5p (miR-135a-5p)/tumor protein 53-induced nuclear protein 1 (TP53INP1) axis by in vitro oxygen-glucose deprivation/reoxygenation (OGD/R)-induced hippocampal neuron damage.Methods HT22 cells were routinely cultured,and the cells were separated into Con group,OGD/R group,si-NC group,and si-Circ_HECTD1 group,miR-NC group,miR-135a-5p group,si-Circ_HECTD1+anti-miR-NC group,and si-Circ_HECTD1+anti-miR-135a-5p group.qRT-PCR method was used to detect the expression of Circ_HECTD1,miR-135a-5p and TP53INP1 mRNA;MTT was used to detect cell viability;flow cytometry was used to detect apoptosis;dual-luciferase reporter gene experiment was applied to verify the targeting relationship between Circ_HECTD1 and miR-135a-5p,miR-135a-5p and TP53INP1;Western blot was applied to measure the protein expressions of Bax,Bcl-2 and TP53INP1.Results After OGD/R induction,the expressions of Circ_HECTD1 and TP53INP1 in HT22 cells were up-regulated,the expression of miR-135a-5p was down-regulated,the cell survival rate and the expression of Bcl-2 protein were remarkably decreased,the apoptosis rate and the expression of Bax protein were remarkably increased (all P<0.05).Silencing the expression of Circ_HECTD1 could remarkably up-regulate the expression of miR-135a-5p in OGD/R-induced HT22 cells,down-regulate the expression of TP53INP1,increase cell survival rate and Bax protein expression,decrease cell apoptosis rate and Bcl-2 protein expression (all P<0.05).There was a targeting relationship between Circ_HECTD1 and miR-135a-5p,between miR-135a-5p and TP53INP1.Overexpression of miR-135a-5p could remarkably down-regulate the expression of TP53INP1,increase cell survival rate and Bcl-2 protein expression,decrease cell apoptosis rate and Bax protein expression (all P<0.05).Inhibition of miR-135a-5p expression could partially reverse the protective effect of silencing Circ_HECTD1 on HT22 cell damage.Conclusion Silencing Circ_HECTD1 can regulate the miR-135a-5p/TP53INP1 axis and promote cell survival,inhibit cell apoptosis,and protect against OGD/R-induced hippocampal neuron damage.

9.
Chinese Pharmacological Bulletin ; (12): 874-879, 2022.
Article in Chinese | WPRIM | ID: wpr-1014085

ABSTRACT

Aim To explore the effeet of soy isofla- vones (SI) on p-amyloid 1 -42 ( Ap, _42 ) -induced hippocampal neuroinflammation and neuronal apoptosis and the underlying mechanism.Methods The prima¬ry hippocampal neurons cultured in vitro were divided into control group (control), Ap,_42 treatment group f model) , SI low-dose group ( Sl-L, 10 mg • L 1 ) , and SI medium-dose group (SI-M, 20 mg • L_l ) and SI high-dose group (SI-H, 40 mg • L 1 ).The model group was treated with 30 (xmol • L"1 Ap, _42 for 48 h; the SI-L, SI-M and SI-H groups were treated with SI for 2 hours, and Ap,_42 was treated for 48 h; the con¬trol group was routinely cultured for 48 h.MTT method was used to detect the survival rate of hippocampal neurons; TUNEL staining was used to detect the apop¬tosis rate of hippocampal neurons; Western blot was used to detect COX-2, TNF-a, NF-kB p65 , P-NF-kB p65, Bcl-2 and caspase-3 protein expression levels.Results Compared with the control group, the surviv¬ al rate of hippocampal neurons was significantly re- duced (P <0.01) , and the apoptotie rate significantly increased (P<0.01).COX-2, TNF-a, p-NF-KB p65 , caspase-3 protein expressions markedly increased (P <0.05 or P <0.01 ) , and the expression of Bcl-2 protein significantly decreased in the model group ( P <0.01 ).Compared with the model group, the surviv¬al rate of hippocampal neurons, Bcl-2 protein in-creased, and the apoptotic rate, the expression of COX-2, TNF-a, p-NF-KB p65 , caspase-3 protein de¬creased (P < 0.05 or P < 0.01 ) in SI each dose group.Conclusion SI can reduce the hippocampal neuroinflammation and neuronal apoptosis induced by APi _42 by inhibiting the activation of NF-kB p65 signa¬ling pathway.

10.
Chinese Pharmacological Bulletin ; (12): 956-960, 2022.
Article in Chinese | WPRIM | ID: wpr-1014097

ABSTRACT

Aim To establish a stable and efficient method for the primary culture of hippocampal neurons from serum-free fetal rats.so as to obtain hippoeampal neurons with higher purity and better vitality.Methods The hippocampal tissues of SI) rat fe¬tus rats on the ( 18 ± 1) day of pregnancy were cultured by Neu- robasal or DMEM/F12 medium and divided into serum-free cul-ture group, fetal bovine serum culture group and 5-fluoro-2'de- oxyuridine culture group.After cells of three groups were cul¬tured for 12 hours, all medium was changed to Neurobasal medi¬um.When the neurons in FUI)R culture group were cultured to 3 days.FLDR was added to inhibit the growth of glial cells.'Hie growing status of hippo cam pal neurons at different culture time points was observed,neuronal activity was detected,cell apopto¬sis was assessed, and the purity of hippocampal neurons was i- dentified.Results Compared with 10% serum culture group, the neurons cultured in serum-free culture group showed higher viability,lower apoptosis rate and higher purity; FUI)R reduced cell viability and increased cell apoptosis.Conclusions 'Hie neurons cultured by serum-free culture have good activity and high purity, instead of adding glial cell inhibitors, which is a fea¬sible and optimized primary culture method for hippocampal neu¬rons.

11.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 63-71, 2021.
Article in Chinese | WPRIM | ID: wpr-905065

ABSTRACT

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.

12.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 72-80, 2021.
Article in Chinese | WPRIM | ID: wpr-905066

ABSTRACT

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.

13.
China Pharmacy ; (12): 1319-1324, 2021.
Article in Chinese | WPRIM | ID: wpr-877252

ABSTRACT

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.

14.
Chinese Pharmacological Bulletin ; (12): 828-832, 2021.
Article in Chinese | WPRIM | ID: wpr-1014443

ABSTRACT

Aim To investigate the distribution of postsynaptic density protein 95 (PSD95) and AMPA receptor GluA2 subunit during the maturation of rat hippocampal neurons in vitro. Methods Cultured rat hippocampal neurons at 4(days in vitro, DIV), 7DIV, 14DIV and 20DIV were used in an immunofluorescence assay to test co-localization of PSD95 and GluA2 by laser confocal microscope at a magnification of 60 ×. Results PSD95 puncta were gradually distributed densely in dendrites and dendritic spines during neuron maturation progresses. GluA2 subunits were uniformly distributed in the cell body, axons, dendrites and dendritic spines. The Pearson' s correlation of PSD95 and GluA2 was 0. 830 ±0. 033 and 0. 734 ±0. 019 respectively at the dendrites of 4DIV and 7DIV neurons, which showed strong co-localization. While at the dendrites of 14DIV and 20DIV neurons, a moderate correlation was demonstrated by the Pearson correlation 0. 547 ± 0. 021 and 0. 574 ± 0. 024. Conclusions GluA2 has no specific intracellular distribution during neuron maturation, while the functional localization of PSD95 depends on the appearance of dendritic spines. The co-localization of GluA2 with PSD95 in dendritic spines suggests that there are stable synapses containing AMPA receptors.

15.
Chinese Pharmaceutical Journal ; (24): 728-736, 2020.
Article in Chinese | WPRIM | ID: wpr-857720

ABSTRACT

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.

16.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 704-713, 2020.
Article in English | WPRIM | ID: wpr-827786

ABSTRACT

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.

17.
Journal of Southern Medical University ; (12): 1097-1102, 2020.
Article in Chinese | WPRIM | ID: wpr-828912

ABSTRACT

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.


Subject(s)
Animals , Mice , Ferroptosis , Hippocampus , Neurons , Radiation Injuries , Vitamin E
18.
Journal of Apoplexy and Nervous Diseases ; (12): 317-321, 2020.
Article in Chinese | WPRIM | ID: wpr-1039777

ABSTRACT

@#Objective To investigate the protective effect of dexmedetomidine (DEX) on apoptosis of hippocampal neurons in rats with status epilepticus (SE) by regulating MAPK/ERK-CREB pathway.Methods SE rat model was established by intraperitoneal injection of lithium pilocarpine,and was randomly divided into 4 groups,with 10 rats in each group.1 μmol/L of DEX was injected intraperitoneally in the SE+DEX group and 1 μmol/L of phenobarbital was injected intraperitoneally in the positive control group,after 24 hours of drug intervention,Nissl and TUNEL methods were used to detect the damage and apoptosis of hippocampal neurons,and Western blot was used to detect the expression of MAPK,p-ERK,p-CREB and caspase-3,Bcl-2,Bax in hippocampus of rats.Results Compared with the control group,the Racine scores of SE group,positive control group and SE+DEX group were significantly higher (P<0.05),the number of hippocampal neurons and the expression of Bcl-2 protein expressions were decreased significantly (P<0.05),and the number of sepia TUNEL positive cells,MAPK,p-ERK,p-CREB,caspase-3,Bax,Bax/Bcl-2 protein levels increased significantly (P<0.05).Compared with the SE group,the Racine scores of the positive control group and the SE+DEX group were significantly lower (P<0.05),the number of hippocampal neurons and the expression of Bcl-2 protein were significantly increased (P<0.05),and the number of sepia TUNEL positive cells,MAPK,p-ERK,p-CREB,caspase-3,Bax,Bax/Bcl-2 protein expressions decreased significantly (P<0.05).Conclusions DEX may inhibit apoptosis of hippocampal neurons by inhibiting MAPK/ERK-CREB pathway,and then improve epilepsy in rats.

19.
Journal of Apoplexy and Nervous Diseases ; (12): 4-10, 2020.
Article in Chinese | WPRIM | ID: wpr-1039814

ABSTRACT

@#Objective To observe whether there is abnormal expression of PTEN in hippocampal neurons of depressed rats and explore the correlation between its expression and autophagy. Methods 50 healthy adult male SD rats were randomly divided into control group and model group:25 rats in each group. A depressive rat model was established by chronic unforeseeable mild stress(CUMS). Behavioral tests such as open field,water maze,shuttle box,tail suspension and sugar water preference were used to detect the depression of rats. Hippocampal tissue was taken 0.5,1,3,7,and 14 days after molding. Immunohistofluorescence,Weston blot and real-time fluorescence quantitative PCR were used to detect the expression changes of hippocampal autophagy related proteins LC3,Beclin1 and tumor suppressive gene PTEN. Results In the model group,the rats showed lack of curiosity,impaired spatial learning and memory,behavioral hopelessness and anhedonia(P<0.01). LC3 and Beclin1,two autophagy-related proteins,were co-expressed in the model group,in which LC3 was co-expressed with PTEN protein. The expression of LC3,Beclin1 and PTEN in the hippocampus of the depression model group was higher than that of the control group(P<0.05),and increased with time. The expression of two kinds of autophagy-related genes and PTEN gene in the depression model group was higher than that in the control group(P<0.01). Conclusion Abnormal PTEN expression exists in hippocampal neurons of depressed rats,which is related to autophagy.

20.
Braz. arch. biol. technol ; 63: e20190072, 2020. graf
Article in English | LILACS | ID: biblio-1132180

ABSTRACT

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.


Subject(s)
Animals , Male , Mice , Oxidation-Reduction/drug effects , Benzofurans/pharmacology , Reactive Oxygen Species , Neuroprotective Agents/pharmacology , Hydrogen Peroxide , Benzofurans/chemical synthesis , Cell Death , Primary Cell Culture , Hippocampus/cytology , Neurons/metabolism
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