Neuroprotective and mechanistic study of GJ-4 on okadaic acid-induced memory impairment in mice / 药学学报

GJ-4 is crocin enrichments extracted from Gardenia jasminoides J. Ellis, and our previous studies have shown that GJ-4 significantly improved learning and memory impairment induced by Aβ in mice. Herein, a memory deficit model was developed by injecting okadaic acid (OA) into the lateral ventricle of mice, and the neuroprotection and underlying mechanism of GJ-4 on neuronal injury caused by Tau hyperphosphorylation were investigated. The Animal Care & Welfare Committee, Institute of Materia Medica, CAMS & PUMC has approved all procedures (No.00000318). GJ-4 at different doses was intragastric administration to mice for 16 days. Step-down test and Morris water maze test showed that GJ-4 could significantly improve OA-induced memory impairment in mice, and reduced the loss of Nissl bodies in the hippocampus of mice. GJ-4 could also decrease the phosphorylation level of Tau protein at Ser396, Thr231 and Ser404 via increasing protein phosphatase 2A (PP2A) activity and inhibiting glycogen synthase kinase-3β (GSK-3β) activity. Besides, further researches indicated that GJ-4 could inhibit the level of oxidative stress in the brain of OA mice, reduce neuronal apoptosis and inhibit the neuroinflammation mediated by activation of astrocytes in the hippocampus of mice, and eventually achieve its effects in improving learning and memory impairment in mice. According to these findings, we anticipated that GJ-4 might be a potential therapeutic drug for Alzheimer's disease.

Flavonoids from stem and leaf of Scutellaria baicalonsis Georgi inhibit PHF abnormality and regulatory mechanism of protein phosphatase in rats' brain induced by okadaic acid / 中国病理生理杂志

AIM:To investigate the effect of flavonoids from stem and leaf of Scutellaria baicalonsis Georgi(SSF)on paired helical filament(PHF)abnormality and the regulatory mechanism of protein phosphatase(PP)in rats' brain induced by okadaic acid(OA).METHODS:Male Sprague-Dawley(SD)rats were microinjected with OA(200 ng/kg)by the lateral ventricle to establish a memory impairment model.Morris water maze was used to screen the memory impairment model.The successful model rats were continuous intragastric infusion(ig)SSF for 36 days.The relative pro-tein expression of PHF,PP1,PP2A-Cα,PP2A-Cβ,PP2CA and PP2CB in the rat cerebral cortex and hippocampus were detected by Western blot.GinKgo biloba leaf flavonoids(GLF)were used as positive control drug.RESULTS:Compared with the sham-operated rats ,the relative protein expression of PHF in the cerebral cortex and hippocampus and PP 1 in cor-tex of model rats were significantly increased(P<0.01),and the protein expression of PP2A-Cα,PP2A-Cβin the cere-bral cortex and hippocampus and PP2CB in the hippocampus were decreased(P<0.05),while the relative protein expres-sion of PP2CA and PP2CB in the cortex were significantly increased(P<0.01).SSF reversed the abnormality in the pro-tein expression of PHF,PP2A-Cαand PP2A-Cβin rat cortex and hippocampus and PP1 in rat cortex induced by OA(P<0.01),which had no significant effect on the relative protein expression of PP 2CA and PP2CB.GLF also showed similar results to SSF.CONCLUSION:SSF significantly reduces the abnormal formation of PHF in rats ' brain induced by OA ,which may be related to the regulation of PP 1,PP2A-Cαand PP2A-Cβexpression,but not with PP2CA and PP2CB ex-pression.

Role of bone marrow mesenchymal stem cells in repair of neural cell injury induced by okadaic acid / 解放军医学杂志

Objective To study whether the human bone marrow mesenchymal stem cells (HBMSCs) can repair damaged neural cells induced by okadaic acid (OA).Methods Neuroblastoma cell line SH-SY5Y cells were used to incubate with 20nmol/L okadaic acid for 24h,establishing Alzheimer's Disease cell model;Three groups were set up:normal group,okadaic acid-damaged (OA-damaged) group,hBMSCs-treatment group.The cells were injured for 24h with 20nmol/L OA in OA-damaged group,and treated with conditioned medium obtaining hBMSCs for 24h after 24h OA injury in the treatment group.Then CCK-8 was used for detecting cell vitality,immune fluorescence dyed microtubules and micro filaments for determining the dendritic cell length and fluorescence intensity,in addition,Western blotting for analyzing the protein level of phosphorylated tau and total tau proteins.Results Okadaic acid damaged SH-SY5Y cells,contributed to shrinkage,collapse,cavitation of the SH-SY5Y cell body,dendritic shortening and fracture,and irregular arrangement of microtubule microfilaments;while BMSCs conditioned medium made SHSYSY cell body become round and longer,dendrites restored,and microtubules and microfilaments arranged regularly,fluorescence intensity enhanced.Meanwhile,it also down-regulated the level of OA-induced tau phosphorylation.Conclusion hBMSCs have repair effects on the neural cell damage induced by okadaic acid.

Protective effects of LiCl on synaptic atrophy induced by okadaic acid in differentiated SK-N-SH neuroblastoma cells / 中国药理学通报

Aim To explore the protective effects of lithium chloride ( LiCl ) on neurous injuries and phos-phorylation of tau protein at serine262 induced by okada-ic acid( OA) . Methods The neuroblastoma SK-N-SH cells were differentiated by all-trans-retinoic acid ( AT-RA) . The differentiated SK-N-SH cells were treated with OA to establish the Alzheimer′s disease cellular model. SK-N-SH cells′ viability and proliferation were measured by SRB test. Giemsa staining was used to observe cell morphology. The neurite length of SK-N-SH cells was measured by Image-Proplus software. Syn-aptophysin and phosphorylated tau protein at serine262 expression levels were tested by Western blot. Results The SK-N-SH cells which were treated with 10 μmol ·L-1 ATRA for 7 days displayed mature neuronal fea-tures. The synaptic length of SK-N-SH cells became longer. And the levels of serine262 phospho-tau was sig-nificantly elevated. 20~100 nmol·L-1 OA effectively inhibited the viability of differentiated SK-N-SH cells in a concentration-dependent manner and in a time-de-pendent manner. The OA treatment induced obvious synaptic atrophy in differentiated SK-N-SH cells. And the phosphorylation level of tau protein serine262 also greatly increased. The pretreatment with 10 mmol · L-1 LiCl significantly ameliorated the synaptic atrophy, the decrease of synaptophysin expression and the in-crease of tau phosphorylation at serine262 induced by OA in differentiated SK-N-SH cells. Conclusion LiCl could effectively inhibit OA-induced synaptic atro-phy in differentiated SK-N-SH cells, and it could also result in the increase of synaptophysin expression and the decrease of the phosphorylation of tau protein at serine262 .

The protective effects of Ginsenosides Rg1 in AD-like pathological model induced by okadaic acid / 中国药理学通报

Aim To the investigate the protective effect of ginsenoside Rg1 in Alzheimer's disease ( AD)-like neurotoxicity model induced by okadaic acid ( OKA) in the cellular level , and explore the mecha-nism preliminarily. Methods The PC12 cells model, simulate neurons, induced by OKA was given Rg1 (1, 5,10 μmol·L-1), and melatonin (Melat) 10 μmol· L-1 was given as a positive control. MTT and LDH were carried out to assess the cell viability and mortality. To detect the accumulation of ROS, the DCFH-DA fluores-cent probe was conducted. And to assess the change of the activity of a variety of antioxidant enzymes, various kits were used, including ABTS、CAT、SOD、GSH-Px and GSSG/GSH. Results Compared with the control group, the survival rate of PC12 cell in OKA group re-duces significantly, the mortality rate was increased sig-nificantly , the number of early apoptotic cells was in-creased significantly (P<0. 01). Oxidative stress-relat-ed indicators show that ROS accumulation within the cells of OKA group increases significantly ( P<0. 01 ) , and the total antioxidant capacity ( ABTS ) decreases significantly ( P < 0. 01 ) , the activity of peroxidase (Catalase, CAT) (P <0. 01), glutathione peroxidase (glutathione peroxidase, GSH-Px) and superoxide dis-mutase ( superoxide dismutase, SOD) decreased signifi-cantly ( P <0. 05 ) , the rate of GSSG/GSH increased significantly ( P <0. 01 ) . Compared with the model group, the different doses of Rg1 could improve the sur-vival rate and decrease the mortality rate of PC12 cell significantly in the group of OKA, and could decrease the level of the accumulation of ROS, improve the activ-ity of antioxidant enzymes. Conclusion Ginsenoside Rg1 can decrease PC12 cell apoptosis by exerting an-tioxidant effects, and protect the nerve cells in AD-like pathology model induced by OKA.

Effects of intrahippocampal administration of the phosphatase inhibitor okadaic acid: dual effects on memory formation / Efeitos da administração intra-hipocampal do inibidor de fosfatases ácido okadaico: efeito duplo sobre a formação de memória

Protein phosphorylation mediated by serine-threonine kinases in the hippocampus is crucial to the synaptic modifications believed to underlie memory formation. The role of phosphatases has been the focus of comparatively little study. Objectives: Here we evaluate the contribution of the serine-threonine protein phosphatases 1 and 2A (PP1, PP2A) on memory consolidation. Methods: We used immediate post-training bilateral hippocampal infusions of okadaic acid (OA, 0.01 and 10 pmol/side), a potent inhibitor of PP1 and PP2A, and measured short- [3 h] and long-term memory [24 h] (STM, LTM) of step-down inhibitory avoidance. Results: At the lower dose, OA inhibited both STM and LTM whereas at the higher dose it instead enhanced LTM. Pre-test infusion of these two doses of OA had no effect on retrieval. Conclusions: These two doses of OA are known to selectively inhibit PP1 and PP2A respectively. These findings point to the importance of these enzymes in memory formation and also suggest a deleterious influence of endogenous hippocampal PP2A on LTM formation.

A fosforilação de proteínas mediada por serina-treonina quinases no hipocampo é crucial para as modificações sinápticas que se acredita sejam necessárias para a formação de memórias. O papel das fosfatases tem sido comparativamente pouco estudado. Objetivos: Aqui avaliamos a contribuição das fosfatases serina-treonina 1 e 2 (PP1, PP2A) sobre a consolidação da memória. Métodos: Usamos infusões imediatamente após o treino de ácido okadaico (OA, 0.01 e 10 pmol/lado), um potente inibidor de PP1 e medimos memória de curta [3 h] e longa duração [24 h] (STM, LTM) de esquiva inibitória de evitar descer de uma plataforma. Resultados: Na dose menor, OA inibiu tanto STM como LTM. Na dose maior, produziu, em vez disso, uma melhora da LTM. A infusão pré-teste de qualquer uma das duas doses de OA não teve efeito sobre a evocação. Conclusões: Estas duas doses de OA são conhecidas por inibir seletivamente PP1 a PP2 respectivamente. Estes resultados apontam à importância das duas enzimas na formação de memória e sugerem, adicionalmente, uma influência deletérea da PP2A endógena sobre a formação de LTM.

Involvement of serine/threonine protein phosphatases 1/2A intolerance established by hypoxic preconditioning inhuman umbilical vein endothelial cells / 中国药理学与毒理学杂志

AIM To investigate the role of serine/threonine protein phosphatases 1 and 2A (PP1/2A) in regulation of cell signal transduction involved in the tolerance of human umbilical vein endothelial cells (HUVEC) to hypoxia. METHODS HUVEC tolerance was established by hypoxic preconditioning. The tolerance of HUVEC was evaluated by the cell survival rate, lactic dehydrogenase (LDH) releasing and total antagonistic-oxidative capability (T-AOC). Subcellular localization of nuclear factor E2-related factor 2 (Nrf2) was determined by immunocytochemistry combined with Western blot. The expression of stress protein of heme oxygenase-1 (HO-1) was measured by Western blot. RESULTS Hypoxia 90 min decreased the survival rate and T-AOC of HUVEC significantly, increased the release of LDH in cultured HUVEC. Compared with the hypoxic group, hypoxic preconditioning (4, 8 and 24 h after hypoxia 10 min) up-regulated the tolerance against hypoxia in HUVEC, the survival rate of HUVEC and T-AOC increased and the release of LDH down-regulated when insulted with hypoxia (90 min) in HUVEC. Hypoxic preconditioning established the translocation of Nrf2 from cytoplasm to nucleus and up-regulated the expression of downstream protein HO-1. Pretreatment with okadaic acid (40 nmol·L-1), a powerful inhibitor of PP1/2A, for 10 min in hypoxic preconditioning HUVEC partly inhibited the translocation of Nrf2 from cytoplasm to nucleus and the expression of HO-1, abolished the tolerance of HUVEC established by hypoxic preconditioning. CONCLUSION PP1/2A at least partly take part in Regulation of translocation of Nrf2 and expression of HO-1, with is associated with the tolerance of HUVEC established by hypoxic preconditioning.

Effects of okadaic acid on voltage-gated potassium and calcium channels in cultured rat trigeminal neurons / 中国药理学与毒理学杂志

AIM To investigate the effects of serine/threonine protein phosphatases in regulation of cell signal transduction on voltage-gated potassium and calcium channels in cultured rat trigeminal ganglion (TRG) neurons. METHODS Whole-cell patch clamp technique was used to record the potassium and calcium currents from adult rat TRG neurons before and after perfusion of okadaic acid, a potent inhibitor of the serine/threonine protein phosphatases 1 and 2A. RESULTS Okadaic acid 1 μmol·L-1 inhibited transient outwards potassium currents (IA) by 28.6%, increased delay rectified potassium currents (IK) and calcium currents (ICa) by 22.7% and 20.0%, respectively. okadaic acid 1 μmol·L-1 produced significant hyperpolarizing shifts in the conductance-voltage (G-V) curves and inactivation curves of IA , also produced significant hyperpolarizing shifts in the G-V curves of IK, while it had no effect on the activation and inactivation kinetics of ICa. CONCLUSION Serine/threonine protein phosphatases 1 and 2A may be involved in the modulation of voltage-gated potassium and calcium channels on rat TRG neurons. In addition, voltage-gated potassium and calcium channels show different dependence on the dephosphorylation reactions of PP1 and PP2A phosphatases.

Anti-apoptotic Effects of Cornus Officinals Iridoid Glycoside on Cellular Model of Alzheimer Disease Induced by Protein Phosphatase Inhibitor Okadaic Acid / 中国药房

OBJECTIVE:To investigate the effects and the mechanisms of cornel iridoid glycoside(CIG)on antiapoptosis in cellular model of alzheimer disease(AD)induced by the protein phosphatase inhibitor okadaic acid(OA).METHODS:The human neuroblastoma cell line SK-N-SH cells were preincubated with CIG for 24 h,and then incubated with OA 10 nmol for 6 h.The changes of apoptotic cells were observed by TUNEL method.Western blotting was used to determine the expression of apoptosis-regulating factors Bcl-2,Bcl-2 related X protein(Bax)and Caspase-3.RESULTS:The normal SK-N-SH cells spread well and no apoptotic cells appeared.In OA-treated model group,the number of apoptotic cells increased significantly,the expressions of Bax and Caspase-3 increased significantly,but Bcl-2 expression decreased significantly.In CIG(100 and 200 ?g?mL-1)-treated group,the number of apoptotic cells decreased significantly as compared with OA-treated model group,and the expressions of Bax and Caspase-3 decreased significantly whereas Bcl-2 expression increased significantly.CONCLUSION:CIG can inhibit the apoptosis of nerve cells through affecting apoptosis-regulating factors,suggesting its potential as a therapy for AD.

Effects of inhibitor of serine/threonine protein phosphatases 1 and 2A on voltage-dependent sodium channels in rat trigeminal ganglion neurons / 中国药理学与毒理学杂志

AIMTo investigate the role of serine/threonine protein phosphatases in regulation of cell signal transduction on voltage-dependent sodium channels in rat trigeminal ganglion (TRG) neurons. METHODSWhole-cell patch clamp techniques were used to record the total sodium current (INa-T) and the tetrodotoxin-resistant sodium current (INa-TTX-R) before and after okadaic acid, a potent inhibitor of the serine/threonine protein phosphatases 1 and 2A, perfusion on adult rat TRG neurons. RESULTS1μmol*L-1 okadaic acid inhibited INa-T by (20±13)% (n=9, P<0.05) and INa-TTX-R by (4±3)% (n=6, P<0.05), respectively. The inhibition on INa-T was significantly greater than that on INa-TTX-R (P<0.05). Furthermore, 1μmol*L-1 okadaic acid produced significant 3-4 mV hyperpolarizing shifts in the conductance-voltage curves of INa-T, while it had no effect on that of INa-TTX-R. CONCLUSIONThe serine/threonine protein phosphatases take part in the regulation of total and TTX-R sodium channels on rat TRG neurons. In addition, small-diameter TRG neurons express various voltage-gated sodium channel with different sensitivity to okadaic acid.

Effects of ginsenoside on cellular model of Alzheimer disease induced by protein phosphatase inhibitor okadaic acid / 中国药理学通报

Aim To investigate the effects of ginsenoside(GS) on phosphorylation of tau protein,microtubule,cellular apoptosis and its related factors in cellular model of Alzheimer disease(AD) induced by the protein phosphatase 1 and 2A inhibitor okadaic acid(OA).Methods The human neuroblastoma cell line SK-N-SH cells were cultured with GS for 24 h,the culture medium was changed,and then incubated with OA 10 nmol?L-1 for 6 h.The changes of cell morphology were observed by inverted microscope.The laser confocal microscopy was used to observe the microtubule changes.Western blot was applied to determine the expression of phosphorylation of tau protein,and apoptosis-regulating factors Bcl-2,Bax and Caspase-3.The changes of apoptotic cells were observed by TUNEL method.Results The normal SK-N-SH cells spread well.OA-treated cells showed that the cell axons and the microtubules were broken and decreased under the inverted microscope and laser confocal microscope.Preincubation of GS demonstrated the significantly protective effects against the morphologic damage induced by OA.In OA-treated group,the phosphorylation of tau protein at Ser-199/202 and Ser-404 sites was higher than that in normal group,and the non-phosphorylation of tau protein at the same sites was lower;Incubation of GS at the dose of 50 mg?L-1 and 100 mg?L-1 with the cells decreased the phosphorylation of tau protein Ser-199/202 and Ser-404 sites.GS group at the dose of 50 mg?L-1 and 100 mg?L-1 decreased the expression of at non-phosphorylation of tau protein at the Ser202 site.The apoptotic cells were not found in normal group.The number of apoptotic cells were obviously increased.the expression of Bax and caspase-3 significantly enhanced,and Bcl-2 expression decreased in the OA-treated model group.GS significantly decreased the apoptotic cell number of nerve cells,inhibited the expression of Bax and caspase-3.Conclusion GS can protect the nerve cells from pathological change induced by OA.Maybe because it can inhibit the hyperphosphorylation of tau protein and protect the nerve cells from apoptosis,thus GS may have potential to treat Alzheimer disease.

Up-regulation of osteopontin gene in BALB/c 3T3 cells induced by tumor promoters of TPA,okadaic acid and cadmium chloride / 第三军医大学学报

Objective To explore the changes of osteopontin (OPN) gene expression induced by tumor promoter of 12-O-tetradecanoylphorbol13-acetate(TPA),okadaic acid(OA) or cadmium chloride(CdCl_(2)).Methods The two-stage transformation test of BALB/c 3T3 cells was established with MNNG as initiator,TPA,OA or CdCl_(2) as promoter respectively.Mouse toxicology gene chip was used to detect the gene expression of BALB/c 3T3 cells transformed with TPA,OA or CdCl_(2) respectively,and the expression of OPN gene was validated by real-time RT-PCR assay.Results TPA,OA or CdCl_(2) could increase the transcriptional expression of OPN gene in BALB/c 3T3 cells.The expression of OPN gene was up-regulated in the eight transformed cell colonies induced by TPA,OA or CdCl_(2) respectively.Moreover,the up-regulation of OPN gene expression was confirmed by real-time RTPCR assay.Conclusion The up-regulation of OPN gene expression is closely related to cell transformation of BALB/c 3T3 cells induced by TPA,OA or CdCl_(2).

Advances in research of okadaic acid / 中国海洋药物

The origin,distribution,chemical structure and biological properties of okadaic acid are described in this paper.Research prospects of okadaic acid and several research fields with priority are proposed according to the present research progress in China.

Effect of hyperphosphorylated tau protein on the formation of brain amyloid / 临床神经病学杂志

Objective To explore the effect of hyperphosphorylated tau protein on the formation of brain amyloid.Methods Okadaic acid was injected into lateral ventricle of rats, once a day for eight weeks. The place navigation and spatial probe ability of rats were assessed by Morris water maze. Immunohistochemistry techniques were used to detect the expressions of neurofibrillary tangles and amyloid.Results In the Okadaic acid group, the spatial learning and memory abilities of rats were significantly impaired. The mean incubation period of Morris water maze was longer than control group. The accumulation of neurofibrillary tangles and plenty of ?-amyloid positive cells were detected in hippocampus CA1, CA3 and CA4 regions, dentate gyrus and cortex. Deposition of ?-amyloid was observed in hippocampus CA1 and CA3 regions, dentate gyrus and cortex.Conclusion The hyperphosphorylated tau protein may significantly increase deposition of amyloid in brain.