目的观察左归降糖解郁方对模拟糖尿病并发抑郁症(DD)环境下海马神经元的影响,探讨其可能的作用机制。方法海马神经元原代细胞取自受孕18 d SD大鼠胎鼠,葡萄糖联合皮质酮干预构建DD海马神经元细胞模型。将培养的海马神经元随机分为正常...目的观察左归降糖解郁方对模拟糖尿病并发抑郁症(DD)环境下海马神经元的影响,探讨其可能的作用机制。方法海马神经元原代细胞取自受孕18 d SD大鼠胎鼠,葡萄糖联合皮质酮干预构建DD海马神经元细胞模型。将培养的海马神经元随机分为正常组、空白血清组、模型组、阳性药(二甲双胍+氟西汀)药物血清组、中药(左归降糖解郁方)药物血清组和阻断剂组。正常组和模型组给予等量培养液,阻断剂组给予促肾上腺皮质激素释放激素1型受体(CRHR1)阻断剂安塔拉明200μmol/L,其余组给予体积分数为10%的药物血清或空白血清,造模干预18 h后,尼氏染色检测海马神经元损伤情况,高内涵细胞成像分析(HCA)技术检测CRHR1、突触可塑性相关蛋白切丝蛋白(Cofilin)和肌动蛋白(Actin)蛋白的表达,实时荧光定量PCR检测CRHR1、Cofilin和Actin mRNA的表达。结果与正常组及空白血清组比较,模型组海马神经元神经网络、树突棘断裂或消失,尼氏小体数量显著减少,CRHR1表达明显上调(P<0.01),Cofilin和Actin表达明显下调(P<0.05);与模型组比较,阳性药药物血清组、中药药物血清组和阻断剂组可一定程度上逆转上述变化(P<0.05,P<0.01)。结论左归降糖解郁方对模拟DD环境下海马神经元具有保护作用,其机制与调控CRHR1、Cofilin、Actin的表达有关。展开更多
目的采用慢性不可预见性中度应激联合快速和慢性改良多平台水环境睡眠剥夺的方法复制抑郁症失眠大鼠模型,对两种造模方法比较,选择最佳造模方式。方法84只大鼠按体重随机分为空白组、环境对照组、慢性应激组、72 h睡眠剥夺组、慢性应激+...目的采用慢性不可预见性中度应激联合快速和慢性改良多平台水环境睡眠剥夺的方法复制抑郁症失眠大鼠模型,对两种造模方法比较,选择最佳造模方式。方法84只大鼠按体重随机分为空白组、环境对照组、慢性应激组、72 h睡眠剥夺组、慢性应激+72 h睡眠剥夺组、21 d睡眠剥夺组、慢性应激+21 d睡眠剥夺组,共7组,每组12只。应激造模14 d和复合造模结束后进行行为学测试大鼠抑郁失眠样行为,ELISA法检测大鼠血清中促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、皮质酮(CORT)的含量以及下丘脑中谷氨酸(Glu)、γ-氨基丁酸(GABA)含量,HPLC-ECD测定大鼠下丘脑中NE、5-HT、DA含量。HE染色观察各组大鼠下丘脑的病理变化。结果与空白组相比,CUMS+72 h SD组和CUMS+21 d SD组大鼠均表现出体重增长率和摄食量显著下降,旷场实验中自主活动次数明显减少,粪便粒数增多,不动时间显著增长,糖水消耗率显著减少,睡眠潜伏期延长,睡眠时长明显缩短,其中后者表现更为显著;CUMS+21 d SD组大鼠血清中CRH、ACTH、CORT含量也明显升高,下丘脑中Glu含量显著增加、GABA含量显著减少,单胺递质含量降低,下丘脑神经元细胞排列紊乱、间隙变大、空泡样性状显著。结论采用慢性不可预见性中度应激联合每天18 h,连续21 d的改良多平台水环境睡眠剥夺的方法可稳定复制抑郁症失眠大鼠模型。展开更多
Objective To explore the protective effects and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方,ZGJTJYF)on hippocampal neurons in rats of diabetes complicated with depression(DD)via the TRP/KYN metabolic pa...Objective To explore the protective effects and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方,ZGJTJYF)on hippocampal neurons in rats of diabetes complicated with depression(DD)via the TRP/KYN metabolic pathway.Methods(i)In vivo experiments:60 specified pathogen free(SPF)grade male Sprague-Dawley(SD)rats were randomly divided into six groups with 10 rats in each groups:control,DD model,positive(1.8 mg/kg fluoxetine+0.18 g/kg metformin),high-dose ZGJTJYF(ZGJTJYFH,40.500 g/kg ZGJTJYF),middle-dose ZGJTJYF(ZGJTJYF-M,20.250 g/kg ZGJTJYF),and lowdose ZGJTJYF(ZGJTJYF-L,10.125 g/kg ZGJTJYF)groups.Except for the control group,other groups were established DD model by high-fat emulsion intake with single tail vein streptozotocin(STZ)and four weeks of chronic unpredictable mild stress(CUMS).All drug administration groups were treated by gavage during CUMS modeling,and the control and model groups were given equal amount of distilled water.After four weeks,the serum levels of blood glucose and glycosylated hemoglobin were measured to determine the hypoglycemic effect of ZGJTJYF.Moreover,the open field test and Morris water maze test were performed to evaluate the antidepressant effect of ZGJTJYF.Changes in 5-hydroxytryptamine(5-HT)level were detected via high-performance liquid chromatography with electrochemical detection(HPLC-ECD);the levels of tryptophan(TRP),kynurenine(KYN),and indoleamine 2,3-dioxygenase(IDO)in the hippocampus were detected using enzyme-linked immunosorbent assay(ELISA);the protein expression levels of synaptophysin(SYN)and postsynaptic density material-95(PSD-95)were detected via immunohistochemistry(IHC);and the protein expression levels of N-methyl-D-aspartate receptor(NR)2 A and NR2 B were detected using Western blot.(ii)In vitro experiments:five SPF grade SD pregnant rats(E16–18)were used to obtain primary hippocampal neurons(Ne),six SD new-born rats were used to collected primary astrocytes(As)and microglia(MG),and to establish a Ne-As-MG co-culture system.All co-culture systems were divided into six groups:control(PBS),model[150 mmol/L glucose+200μmol/L corticosterone(G&P)+PBS],blank(G&P+blank serum),positive(G&P+positive drug-containing serum),ZGJTJYF(G&P+ZGJTJYF serum),and 1-methyl-D-tryptophan(1-MT,IDO inhibitor)(G&P+1-MT)groups.After 18 h of intervention by corresponding treatment,immunofluorescence was used to analyze the protein expression levels of SYN,PSD-95,NR2 A,and NR2 B;ELISA was performed to measure the levels of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,and TRP/KYN metabolic pathway-related factors[TRP,KYN,kynurenine acid(KYNA),quinolinic acid(QUIN)].Results(i)In vivo experimental results showed that ZGJTJYF-M and ZGJTJYF-L significantly improved the elevated blood glucose state of DD rats(P<0.01 and P<0.05,respectively);ZGJTJYF-H,ZGJTJYF-M,and ZGJTJYF-L increased their autonomous activity,learning,and memory ability(P<0.01,P<0.01,and P<0.05,respectively).Moreover,the levels of 5-HT and TRP were significantly increased(P<0.01),and the levels of KYN and IDO were significantly decreased in the hippocampus(P<0.01)of rats after ZGJTJYF-M treatment.The protein expression levels of SYN and PSD-95 were significantly upregulated in hippocampal neurons(P<0.01),while the abnormal activation of NR2A and NR2B was markedly inhibited in hippocampus(P<0.05)of rats after ZGJTJYF-M treatment.(ii)In vitro experimental results showed that ZGJTJYF-containing serum significantly increased the protein expression levels of SYN and PSD-95 in hippocampal neurons(P<0.01),decreased the levels of IL-1β(P<0.01),IL-6(P<0.05),TNF-α(P<0.01),IDO(P<0.05),KYN(P<0.05),and QUIN(P<0.01),and increased the levels of TRP and KYNA(P<0.01)in the simulated DD state.ZGJTJYF also had an significantly inhibitory effect on the abnormal activation of NR2A and NR2B in neurons(P<0.05)in a stimulated DD state.Conclusion ZGJTJYF can effectively improve 5-HT deficiency in the hippocampus of rats by inhibiting IDO expression and regulating the TRP/KYN metabolic pathway,and it has a favorable protective effect on hippocampal neuron injury caused by DD.Therefore,ZGJTJYF is an effective potential therapeutic drug for the prevention and treatment of DD.展开更多
Objective To investigate the effectiveness of compound Chai Jin Jie Yu Tablets(CJJYT)in ameliorating cognitive impairment associated with depression and its potential mechanism of action.Methods In vitro experiments,t...Objective To investigate the effectiveness of compound Chai Jin Jie Yu Tablets(CJJYT)in ameliorating cognitive impairment associated with depression and its potential mechanism of action.Methods In vitro experiments,the hippocampus was isolated from the whole brain of the fetal rat and cultured into hippocampal neuron cells.50μM corticosterone(CORT)was added to each group 18 h before the experiment for modeling depression,with the exception of the control group.After modeling,the blank serum group was added with 10%blank serum,the CJJYT group and the venlafaxine group were added with the corresponding 10%drug-containing serum,and the control group and the model group were added with equal volumes of culture medium.The intracellular Ca^2+mean fluorescence intensity,miniature excitatory postsynaptic current(mEPSC)current amplitude,and frequency of different hippocampal neurons were evaluated as indicators of synaptic function in the hippocampal neurons.In addition,the expression of synaptic plasticity related proteins,synaptophysin-α(SYN-α),N-methyl-D-aspartate receptor 2A(NR2A),N-methyl-Daspartate receptor 2B(NR2B),post synaptic density 95 protein(PSD-95),calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ)and synaptic associated protein(SynGAP)were detected in the hippocampal neurons by immunofluorescence staining and high content analysis(HCA)system.Then,reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression levels of SYN-α,NR2A,NR2B,PSD-95,CaMKⅡand SynGAP.For in vivo experiments,except for those in the blank control group,all rats were treated within a single cage for chronic unpredictable stress-induced depression modeling and subjected to corresponding drug interventions.Behavioral tests were used to detect depressive behavior and determine learning,memory and other cognitive abilities,whereas enzyme-linked immunosorbent assay(ELISA)was used to detect the CORT levels.Golgi-Cox staining was used to observe changes in the synaptic morphology of parahippocampal gyrus CA1 area(CA1)and dentategyrus(DG).Results In vitro,CJJYT treatment reduced the intracellular Ca^2+mean flurorescence intensity in the hippocampal neurons(P<0.05),causing a reduction in the frequency and current amplitude of mEPSC(P<0.05),and thus inhibited the excessive activation of post-synaptic receptors.CJJYT treatment reduced the protein and mRNA expression of SYN-α,NR2A,NR2B and PSD-95 in the hippocampal neurons(P<0.05),increased the mRNA and protein expression of CaMKⅡand SynGAP(P<0.05),and thereby improved the synaptic plasticity of the hippocampal neurons.In vivo,CJJYT intervention improved sucrose preference,voluntary activity,learning and memory ability of Morris water maze test,and suppressed appetite(P<0.05),and increased the despair feeling of forced swimming test(P<0.05).The CORT level was reduced(P<0.05),leading to the repair of synaptic damage in the hippocampal neurons.Conclusions CJJYT can improve the synaptic function of hippocampal neurons and has obvious protective effects on neurons.It can repair the structural damage in the hippocampal neurons,improving the cognitive ability of the depressed model rats.The mechanism of CJJYT improving cognition in depressed rats may be related to the transmission and function of SYN-α/NR and its downstream neurotransmitters.展开更多
文摘目的观察左归降糖解郁方对模拟糖尿病并发抑郁症(DD)环境下海马神经元的影响,探讨其可能的作用机制。方法海马神经元原代细胞取自受孕18 d SD大鼠胎鼠,葡萄糖联合皮质酮干预构建DD海马神经元细胞模型。将培养的海马神经元随机分为正常组、空白血清组、模型组、阳性药(二甲双胍+氟西汀)药物血清组、中药(左归降糖解郁方)药物血清组和阻断剂组。正常组和模型组给予等量培养液,阻断剂组给予促肾上腺皮质激素释放激素1型受体(CRHR1)阻断剂安塔拉明200μmol/L,其余组给予体积分数为10%的药物血清或空白血清,造模干预18 h后,尼氏染色检测海马神经元损伤情况,高内涵细胞成像分析(HCA)技术检测CRHR1、突触可塑性相关蛋白切丝蛋白(Cofilin)和肌动蛋白(Actin)蛋白的表达,实时荧光定量PCR检测CRHR1、Cofilin和Actin mRNA的表达。结果与正常组及空白血清组比较,模型组海马神经元神经网络、树突棘断裂或消失,尼氏小体数量显著减少,CRHR1表达明显上调(P<0.01),Cofilin和Actin表达明显下调(P<0.05);与模型组比较,阳性药药物血清组、中药药物血清组和阻断剂组可一定程度上逆转上述变化(P<0.05,P<0.01)。结论左归降糖解郁方对模拟DD环境下海马神经元具有保护作用,其机制与调控CRHR1、Cofilin、Actin的表达有关。
文摘目的采用慢性不可预见性中度应激联合快速和慢性改良多平台水环境睡眠剥夺的方法复制抑郁症失眠大鼠模型,对两种造模方法比较,选择最佳造模方式。方法84只大鼠按体重随机分为空白组、环境对照组、慢性应激组、72 h睡眠剥夺组、慢性应激+72 h睡眠剥夺组、21 d睡眠剥夺组、慢性应激+21 d睡眠剥夺组,共7组,每组12只。应激造模14 d和复合造模结束后进行行为学测试大鼠抑郁失眠样行为,ELISA法检测大鼠血清中促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、皮质酮(CORT)的含量以及下丘脑中谷氨酸(Glu)、γ-氨基丁酸(GABA)含量,HPLC-ECD测定大鼠下丘脑中NE、5-HT、DA含量。HE染色观察各组大鼠下丘脑的病理变化。结果与空白组相比,CUMS+72 h SD组和CUMS+21 d SD组大鼠均表现出体重增长率和摄食量显著下降,旷场实验中自主活动次数明显减少,粪便粒数增多,不动时间显著增长,糖水消耗率显著减少,睡眠潜伏期延长,睡眠时长明显缩短,其中后者表现更为显著;CUMS+21 d SD组大鼠血清中CRH、ACTH、CORT含量也明显升高,下丘脑中Glu含量显著增加、GABA含量显著减少,单胺递质含量降低,下丘脑神经元细胞排列紊乱、间隙变大、空泡样性状显著。结论采用慢性不可预见性中度应激联合每天18 h,连续21 d的改良多平台水环境睡眠剥夺的方法可稳定复制抑郁症失眠大鼠模型。
基金National Natural Science Foundation of China(81874464and 82104793)the Scientific Research Project of Education Department of Hunan Province(19K066)。
文摘Objective To explore the protective effects and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方,ZGJTJYF)on hippocampal neurons in rats of diabetes complicated with depression(DD)via the TRP/KYN metabolic pathway.Methods(i)In vivo experiments:60 specified pathogen free(SPF)grade male Sprague-Dawley(SD)rats were randomly divided into six groups with 10 rats in each groups:control,DD model,positive(1.8 mg/kg fluoxetine+0.18 g/kg metformin),high-dose ZGJTJYF(ZGJTJYFH,40.500 g/kg ZGJTJYF),middle-dose ZGJTJYF(ZGJTJYF-M,20.250 g/kg ZGJTJYF),and lowdose ZGJTJYF(ZGJTJYF-L,10.125 g/kg ZGJTJYF)groups.Except for the control group,other groups were established DD model by high-fat emulsion intake with single tail vein streptozotocin(STZ)and four weeks of chronic unpredictable mild stress(CUMS).All drug administration groups were treated by gavage during CUMS modeling,and the control and model groups were given equal amount of distilled water.After four weeks,the serum levels of blood glucose and glycosylated hemoglobin were measured to determine the hypoglycemic effect of ZGJTJYF.Moreover,the open field test and Morris water maze test were performed to evaluate the antidepressant effect of ZGJTJYF.Changes in 5-hydroxytryptamine(5-HT)level were detected via high-performance liquid chromatography with electrochemical detection(HPLC-ECD);the levels of tryptophan(TRP),kynurenine(KYN),and indoleamine 2,3-dioxygenase(IDO)in the hippocampus were detected using enzyme-linked immunosorbent assay(ELISA);the protein expression levels of synaptophysin(SYN)and postsynaptic density material-95(PSD-95)were detected via immunohistochemistry(IHC);and the protein expression levels of N-methyl-D-aspartate receptor(NR)2 A and NR2 B were detected using Western blot.(ii)In vitro experiments:five SPF grade SD pregnant rats(E16–18)were used to obtain primary hippocampal neurons(Ne),six SD new-born rats were used to collected primary astrocytes(As)and microglia(MG),and to establish a Ne-As-MG co-culture system.All co-culture systems were divided into six groups:control(PBS),model[150 mmol/L glucose+200μmol/L corticosterone(G&P)+PBS],blank(G&P+blank serum),positive(G&P+positive drug-containing serum),ZGJTJYF(G&P+ZGJTJYF serum),and 1-methyl-D-tryptophan(1-MT,IDO inhibitor)(G&P+1-MT)groups.After 18 h of intervention by corresponding treatment,immunofluorescence was used to analyze the protein expression levels of SYN,PSD-95,NR2 A,and NR2 B;ELISA was performed to measure the levels of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,and TRP/KYN metabolic pathway-related factors[TRP,KYN,kynurenine acid(KYNA),quinolinic acid(QUIN)].Results(i)In vivo experimental results showed that ZGJTJYF-M and ZGJTJYF-L significantly improved the elevated blood glucose state of DD rats(P<0.01 and P<0.05,respectively);ZGJTJYF-H,ZGJTJYF-M,and ZGJTJYF-L increased their autonomous activity,learning,and memory ability(P<0.01,P<0.01,and P<0.05,respectively).Moreover,the levels of 5-HT and TRP were significantly increased(P<0.01),and the levels of KYN and IDO were significantly decreased in the hippocampus(P<0.01)of rats after ZGJTJYF-M treatment.The protein expression levels of SYN and PSD-95 were significantly upregulated in hippocampal neurons(P<0.01),while the abnormal activation of NR2A and NR2B was markedly inhibited in hippocampus(P<0.05)of rats after ZGJTJYF-M treatment.(ii)In vitro experimental results showed that ZGJTJYF-containing serum significantly increased the protein expression levels of SYN and PSD-95 in hippocampal neurons(P<0.01),decreased the levels of IL-1β(P<0.01),IL-6(P<0.05),TNF-α(P<0.01),IDO(P<0.05),KYN(P<0.05),and QUIN(P<0.01),and increased the levels of TRP and KYNA(P<0.01)in the simulated DD state.ZGJTJYF also had an significantly inhibitory effect on the abnormal activation of NR2A and NR2B in neurons(P<0.05)in a stimulated DD state.Conclusion ZGJTJYF can effectively improve 5-HT deficiency in the hippocampus of rats by inhibiting IDO expression and regulating the TRP/KYN metabolic pathway,and it has a favorable protective effect on hippocampal neuron injury caused by DD.Therefore,ZGJTJYF is an effective potential therapeutic drug for the prevention and treatment of DD.
基金funding support from the National Major New Drug Development Project(No.2017ZX09309026)Provincial Department of Graduate Research Innovation Project of Hunan(No.CX20190565)。
文摘Objective To investigate the effectiveness of compound Chai Jin Jie Yu Tablets(CJJYT)in ameliorating cognitive impairment associated with depression and its potential mechanism of action.Methods In vitro experiments,the hippocampus was isolated from the whole brain of the fetal rat and cultured into hippocampal neuron cells.50μM corticosterone(CORT)was added to each group 18 h before the experiment for modeling depression,with the exception of the control group.After modeling,the blank serum group was added with 10%blank serum,the CJJYT group and the venlafaxine group were added with the corresponding 10%drug-containing serum,and the control group and the model group were added with equal volumes of culture medium.The intracellular Ca^2+mean fluorescence intensity,miniature excitatory postsynaptic current(mEPSC)current amplitude,and frequency of different hippocampal neurons were evaluated as indicators of synaptic function in the hippocampal neurons.In addition,the expression of synaptic plasticity related proteins,synaptophysin-α(SYN-α),N-methyl-D-aspartate receptor 2A(NR2A),N-methyl-Daspartate receptor 2B(NR2B),post synaptic density 95 protein(PSD-95),calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ)and synaptic associated protein(SynGAP)were detected in the hippocampal neurons by immunofluorescence staining and high content analysis(HCA)system.Then,reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression levels of SYN-α,NR2A,NR2B,PSD-95,CaMKⅡand SynGAP.For in vivo experiments,except for those in the blank control group,all rats were treated within a single cage for chronic unpredictable stress-induced depression modeling and subjected to corresponding drug interventions.Behavioral tests were used to detect depressive behavior and determine learning,memory and other cognitive abilities,whereas enzyme-linked immunosorbent assay(ELISA)was used to detect the CORT levels.Golgi-Cox staining was used to observe changes in the synaptic morphology of parahippocampal gyrus CA1 area(CA1)and dentategyrus(DG).Results In vitro,CJJYT treatment reduced the intracellular Ca^2+mean flurorescence intensity in the hippocampal neurons(P<0.05),causing a reduction in the frequency and current amplitude of mEPSC(P<0.05),and thus inhibited the excessive activation of post-synaptic receptors.CJJYT treatment reduced the protein and mRNA expression of SYN-α,NR2A,NR2B and PSD-95 in the hippocampal neurons(P<0.05),increased the mRNA and protein expression of CaMKⅡand SynGAP(P<0.05),and thereby improved the synaptic plasticity of the hippocampal neurons.In vivo,CJJYT intervention improved sucrose preference,voluntary activity,learning and memory ability of Morris water maze test,and suppressed appetite(P<0.05),and increased the despair feeling of forced swimming test(P<0.05).The CORT level was reduced(P<0.05),leading to the repair of synaptic damage in the hippocampal neurons.Conclusions CJJYT can improve the synaptic function of hippocampal neurons and has obvious protective effects on neurons.It can repair the structural damage in the hippocampal neurons,improving the cognitive ability of the depressed model rats.The mechanism of CJJYT improving cognition in depressed rats may be related to the transmission and function of SYN-α/NR and its downstream neurotransmitters.
