目的探讨左归降糖解郁方调控小胶质细胞免疫受体分子样家族成员f(immune receptor molecule-like family member f,CD300f)改善糖尿病并发抑郁症(diabetes-related depression,DD)海马突触微环境损伤的保护作用及机制。方法原代分离、...目的探讨左归降糖解郁方调控小胶质细胞免疫受体分子样家族成员f(immune receptor molecule-like family member f,CD300f)改善糖尿病并发抑郁症(diabetes-related depression,DD)海马突触微环境损伤的保护作用及机制。方法原代分离、培养、纯化和鉴定小胶质细胞和海马神经元,复制模拟DD环境下的海马突触微环境损伤体外细胞模型,设置正常组、模型组、CD300f阻断剂CLM-1组、左归降糖解郁方含药血清组和左归降糖解郁方含药血清+CLM-1组。采用细胞成像分析观察小胶质细胞和海马神经元形态结构;采用ELISA法检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)、5-羟色胺(5-hydroxyteyptamine,5-HT)、多巴胺(dopamine,DA)含量;采用CCK-8实验及活死细胞染色评估海马神经元活性;采用尼氏染色观察海马神经元突触损伤;采用免疫荧光检测小胶质细胞中CD300f、Toll样受体4(Toll-like receptor 4,TLR4)和海马神经元中突触素(synaptophysin,SYN)、突触后密度蛋白95(postsynaptic density protein 95,PSD-95)蛋白表达;采用Western blotting检测SYN、PSD-95蛋白表达。结果左归降糖解郁方能有效改善小胶质细胞和海马神经元形态结构损伤,抑制细胞上清中神经炎症因子TNF-α、IL-1β和IDO水平(P<0.05、0.01),促进神经递质5-HT、DA水平(P<0.01),进而缓解海马神经元细胞活性及突触损伤。进一步的机制研究发现,左归降糖解郁方能显著增加海马小胶质细胞中CD300f表达(P<0.01),降低TLR4表达(P<0.05),并上调海马神经元中突触前膜SYN和突触后膜PSD-95表达(P<0.01),最终抑制海马突触微环境损伤。结论左归降糖解郁方能有效改善体外DD状态下的海马突触微环境损伤,其机制可能与上调小胶质细胞CD300f有关。展开更多
探讨柴金解郁安神片调控前扣带皮层(ACC)-腹侧海马(vHPC)谷氨酸能神经环路异常改善抑郁症大鼠腹侧海马神经元突触重塑的分子机制。首先运用化学遗传将谷氨酸能腺相关病毒(AAV)定位注射至大鼠ACC脑区,并通过慢性温和不可预知性应激(CUMS...探讨柴金解郁安神片调控前扣带皮层(ACC)-腹侧海马(vHPC)谷氨酸能神经环路异常改善抑郁症大鼠腹侧海马神经元突触重塑的分子机制。首先运用化学遗传将谷氨酸能腺相关病毒(AAV)定位注射至大鼠ACC脑区,并通过慢性温和不可预知性应激(CUMS)联合孤笼饲养复制大鼠抑郁模型,实验设正常组、模型组、AAV空载组、AAV病毒组、AAV病毒+糖皮质激素受体(GR)阻断剂组、AAV病毒+趋化因子受体1(CX3CR1)阻断剂组、AAV病毒+柴金解郁安神片组,采用水迷宫(Morris water maze)、旷场(open-field)和强迫游泳(forced-swimming)实验联合动物行为分析系统评估大鼠抑郁样行为;苏木素-伊红(HE)染色检测大鼠ACC及vHPC脑区神经元形态结构变化;免疫荧光及核磷酸蛋白(c-Fos)检测大鼠ACC-vHPC谷氨酸能神经环路激活情况;高尔基染色和透射电镜检测大鼠vHPC神经元树突、树突棘及突触亚微结构变化;免疫荧光、Western blot分别检测大鼠vHPC谷氨酸能神经元细胞内突触重塑相关蛋白谷氨酸受体2A(GRIN2A)、谷氨酸受体2B(GRIN2B)、Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、丝裂原激活蛋白激酶激活蛋白激酶2(MK2)、丝切蛋白(cofilin)表达水平。结果表明,谷氨酸能AAV病毒激活后模型组大鼠抑郁样行为表型、ACC及vHPC神经元形态结构、突触超微结构损伤更加加重,而GR、CX3CR1阻断剂均能不同程度逆转其异常改变,提示ACC脑区内胶质细胞GR/CX3CR1双信号介导的ACC-vHPC谷氨酸能神经环路异常激活可能与抑郁的发生发展密切相关。有趣的是,柴金解郁安神片也能显著抑制AAV病毒诱导的ACC-vHPC神经环路激活及Glu含量异常升高,同时有效逆转模型组大鼠进一步加重的抑郁样行为和vHPC谷氨酸能神经元突触重塑,并揭示其改善腹侧海马神经元突触损伤的分子机制可能与调控突触重塑相关信号NR/CaMKⅡ、MK2/cofilin有关。综上,该文证实了柴金解郁安神片能有效调控ACC-vHPC谷氨酸能神经环路异常进而改善抑郁症大鼠腹侧海马谷氨酸能神经元突触重塑,其分子机制可能与调节突触相关NR/CaMKⅡ、MK2/cofilin信号通路有关,这可能是其发挥抗抑郁作用的重要机制。展开更多
目的研究左归降糖解郁方调控模拟糖尿病并发抑郁症脑环境下的体外海马神经干细胞增殖与分化作用及分子机制。方法纯化和培养SD大鼠海马神经干细胞并通过标记巢蛋白(Nestin)进行荧光鉴定。取第3代海马神经干细胞进行实验,分为正常组、模...目的研究左归降糖解郁方调控模拟糖尿病并发抑郁症脑环境下的体外海马神经干细胞增殖与分化作用及分子机制。方法纯化和培养SD大鼠海马神经干细胞并通过标记巢蛋白(Nestin)进行荧光鉴定。取第3代海马神经干细胞进行实验,分为正常组、模型组、空白血清组、左归降糖解郁方含药血清组、左归降糖解郁方含药血清+海马无翅基因3a(recombinant wingless type MMTV integration site family member 3a,Wnt3a)信号抑制剂XAV-939组。在干细胞培养液中添加葡萄糖和皮质酮模拟糖尿病并发抑郁症脑环境进行造模,空白血清组和左归降糖解郁方含药血清组于造模同期分别给予不同血清。干预18 h后,采用WST-1检测干细胞活力,采用Brdu免疫荧光结合高内涵细胞成像技术评价干细胞的增殖能力,分别采用双皮质素(doublecortin,DCX)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经元核抗原(neuron specific nuclear protein,NeuN)免疫荧光评估干细胞的分化能力,采用Western blotting检测各组细胞Wnt3a、低密度脂蛋白受体相关蛋白5(low density lipoprotein receptor-related protein 5,LRP5)、LRP6、β-连环蛋白(β-catenin)表达,采用qRTPCR法检测神经原素2(neurogenin 2,Ngn2)、细胞周期蛋白(cyclinD1)基因表达。结果左归降糖解郁方能显著增加受损海马神经干细胞的活力(P<0.01),增加Brdu、DCX、GFAP、NeuN的表达(P<0.01),增强其增殖和分化的能力。机制研究发现,左归降糖解郁方能显著增加神经干细胞中的Wnt3a、LRP5、LRP6、总β-catenin、核β-catenin蛋白的表达(P<0.05、0.01),激活Wnt3a信号并增加β-catenin的蓄积和入核,从而促进下游Ngn2、cyclinD1靶基因的转录(P<0.05、0.01)。XAV-939可通过增加β-catenin的降解(P<0.01),减少其进入细胞核,减少下游Ngn2、cyclinD1的m RNA转录(P<0.05、0.01),从而降低干细胞的增殖与分化能力。结论左归降糖解郁方能明显改善葡萄糖与皮质酮对体外海马神经干细胞增殖与分化的抑制作用,其改善作用与激活Wnt3a/β-catenin信号有关。展开更多
Objective To investigate the efficacy and mechanism of action of Compound Chaijin Jieyu Tablets(复方柴金解郁片,CCJJYT)in rats with insomnia complicated with depression.Methods Seventy-two Sprague-Dawley rats were rand...Objective To investigate the efficacy and mechanism of action of Compound Chaijin Jieyu Tablets(复方柴金解郁片,CCJJYT)in rats with insomnia complicated with depression.Methods Seventy-two Sprague-Dawley rats were randomly assigned into eight groups:the control,chronic unpredictable mild stress(CUMS),sleep deprivation(SD),CUMS+SD,positive drug(venlafaxine hydrochloride+diazepam),CCJJYT high-dose(CCJJYT˗2×),medium-dose(CCJJYT˗1×),and low-dose(CCJJYT˗0.5×)groups,with nine rats in each group.Depression-like behavior was evaluated by body weight,food intake,and behavioral tests such as the sucrose preference test(SPT),open field test(OFT),forced swimming test(FST),and pentobarbital-induced sleep test(PST).Hematoxylin-eosin(HE)staining and Golgi-Cox staining were used to observe changes in pathological tissue and synaptic morphology,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of orexin-A and acetylcholine.The expression levels of orexin receptor 1(OXR1),melatonin receptor 1(MT1A),melatonin receptor 2(MT1B),acetylcholinesterase(AChE),and choline acetyltransferase(ChAT)were detected by immunohistochemistry and Western blot.Results In the present study,rats in the model group showed significant behavioral changes as well as a reduction in hippocampal dendritic branch length and synaptic number,along with increasing the content of orexin A and acetylcholine(P<0.05),and altered expression levels of OX1R,MT1A,MT1B,ChAT,and AChE in the hippocampus and prefrontal cortex after modeling(P<0.05).CCJJYT can improve depressive insomnia behavior and synaptic plasticity of rats(P<0.05),which is similar to that of the positive drug group.It can also decrease the content of orexin A and acetylcholine,and reduce the expression levels of OXR1 and ChAT in hippocampus and prefrontal cortex(P<0.05),and increase the expression levels of MT1A,MT1B,and AChE proteins(P<0.05).Conclusion CCJJYT has good antidepressant and insomnia effects,probably through the regulation of orexin-A,melatonin,and acetylcholine content in hippocampus and prefrontal cortex of rats,improving synaptic plasticity and thus exerting antidepressant and insomnia effects.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
盐诱导激酶(salt-inducible kinases,SIKs)属于腺苷酸活化激酶(AMP-activated protein kinase,AMPK)家族,主要调控环磷酸苷效应元件结合蛋白(cAMP-response element binding protein,CREB)转录共激活因子(CREB-regulated transcription ...盐诱导激酶(salt-inducible kinases,SIKs)属于腺苷酸活化激酶(AMP-activated protein kinase,AMPK)家族,主要调控环磷酸苷效应元件结合蛋白(cAMP-response element binding protein,CREB)转录共激活因子(CREB-regulated transcription coactivators,CRTCs)的胞核-胞质分布,对CRTC-CREB复合物的合成起到调节作用,从而间接影响CREB目的基因的转录与表达,影响多种生理过程,例如能量代谢、细胞周期进程和细胞凋亡。过去对SIKs的相关研究主要集中在外周系统疾病,包括肿瘤、高血压和糖异生等。近年来越来越多的研究开始探索SIKs和中枢神经系统疾病的关联性,如抑郁症、睡眠障碍、癫痫和阿尔茨海默病等都存在SIKs水平异常的情况,这提示SIKs信号失调参与这些疾病的病理过程,SIKs具有成为此类疾病新治疗靶点的潜力。本文就SIKs在抑郁症、睡眠障碍及其他神经系统疾病中的作用和调控机制进行综述。展开更多
文摘目的探讨左归降糖解郁方调控小胶质细胞免疫受体分子样家族成员f(immune receptor molecule-like family member f,CD300f)改善糖尿病并发抑郁症(diabetes-related depression,DD)海马突触微环境损伤的保护作用及机制。方法原代分离、培养、纯化和鉴定小胶质细胞和海马神经元,复制模拟DD环境下的海马突触微环境损伤体外细胞模型,设置正常组、模型组、CD300f阻断剂CLM-1组、左归降糖解郁方含药血清组和左归降糖解郁方含药血清+CLM-1组。采用细胞成像分析观察小胶质细胞和海马神经元形态结构;采用ELISA法检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)、5-羟色胺(5-hydroxyteyptamine,5-HT)、多巴胺(dopamine,DA)含量;采用CCK-8实验及活死细胞染色评估海马神经元活性;采用尼氏染色观察海马神经元突触损伤;采用免疫荧光检测小胶质细胞中CD300f、Toll样受体4(Toll-like receptor 4,TLR4)和海马神经元中突触素(synaptophysin,SYN)、突触后密度蛋白95(postsynaptic density protein 95,PSD-95)蛋白表达;采用Western blotting检测SYN、PSD-95蛋白表达。结果左归降糖解郁方能有效改善小胶质细胞和海马神经元形态结构损伤,抑制细胞上清中神经炎症因子TNF-α、IL-1β和IDO水平(P<0.05、0.01),促进神经递质5-HT、DA水平(P<0.01),进而缓解海马神经元细胞活性及突触损伤。进一步的机制研究发现,左归降糖解郁方能显著增加海马小胶质细胞中CD300f表达(P<0.01),降低TLR4表达(P<0.05),并上调海马神经元中突触前膜SYN和突触后膜PSD-95表达(P<0.01),最终抑制海马突触微环境损伤。结论左归降糖解郁方能有效改善体外DD状态下的海马突触微环境损伤,其机制可能与上调小胶质细胞CD300f有关。
文摘探讨柴金解郁安神片调控前扣带皮层(ACC)-腹侧海马(vHPC)谷氨酸能神经环路异常改善抑郁症大鼠腹侧海马神经元突触重塑的分子机制。首先运用化学遗传将谷氨酸能腺相关病毒(AAV)定位注射至大鼠ACC脑区,并通过慢性温和不可预知性应激(CUMS)联合孤笼饲养复制大鼠抑郁模型,实验设正常组、模型组、AAV空载组、AAV病毒组、AAV病毒+糖皮质激素受体(GR)阻断剂组、AAV病毒+趋化因子受体1(CX3CR1)阻断剂组、AAV病毒+柴金解郁安神片组,采用水迷宫(Morris water maze)、旷场(open-field)和强迫游泳(forced-swimming)实验联合动物行为分析系统评估大鼠抑郁样行为;苏木素-伊红(HE)染色检测大鼠ACC及vHPC脑区神经元形态结构变化;免疫荧光及核磷酸蛋白(c-Fos)检测大鼠ACC-vHPC谷氨酸能神经环路激活情况;高尔基染色和透射电镜检测大鼠vHPC神经元树突、树突棘及突触亚微结构变化;免疫荧光、Western blot分别检测大鼠vHPC谷氨酸能神经元细胞内突触重塑相关蛋白谷氨酸受体2A(GRIN2A)、谷氨酸受体2B(GRIN2B)、Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)、丝裂原激活蛋白激酶激活蛋白激酶2(MK2)、丝切蛋白(cofilin)表达水平。结果表明,谷氨酸能AAV病毒激活后模型组大鼠抑郁样行为表型、ACC及vHPC神经元形态结构、突触超微结构损伤更加加重,而GR、CX3CR1阻断剂均能不同程度逆转其异常改变,提示ACC脑区内胶质细胞GR/CX3CR1双信号介导的ACC-vHPC谷氨酸能神经环路异常激活可能与抑郁的发生发展密切相关。有趣的是,柴金解郁安神片也能显著抑制AAV病毒诱导的ACC-vHPC神经环路激活及Glu含量异常升高,同时有效逆转模型组大鼠进一步加重的抑郁样行为和vHPC谷氨酸能神经元突触重塑,并揭示其改善腹侧海马神经元突触损伤的分子机制可能与调控突触重塑相关信号NR/CaMKⅡ、MK2/cofilin有关。综上,该文证实了柴金解郁安神片能有效调控ACC-vHPC谷氨酸能神经环路异常进而改善抑郁症大鼠腹侧海马谷氨酸能神经元突触重塑,其分子机制可能与调节突触相关NR/CaMKⅡ、MK2/cofilin信号通路有关,这可能是其发挥抗抑郁作用的重要机制。
文摘目的研究左归降糖解郁方调控模拟糖尿病并发抑郁症脑环境下的体外海马神经干细胞增殖与分化作用及分子机制。方法纯化和培养SD大鼠海马神经干细胞并通过标记巢蛋白(Nestin)进行荧光鉴定。取第3代海马神经干细胞进行实验,分为正常组、模型组、空白血清组、左归降糖解郁方含药血清组、左归降糖解郁方含药血清+海马无翅基因3a(recombinant wingless type MMTV integration site family member 3a,Wnt3a)信号抑制剂XAV-939组。在干细胞培养液中添加葡萄糖和皮质酮模拟糖尿病并发抑郁症脑环境进行造模,空白血清组和左归降糖解郁方含药血清组于造模同期分别给予不同血清。干预18 h后,采用WST-1检测干细胞活力,采用Brdu免疫荧光结合高内涵细胞成像技术评价干细胞的增殖能力,分别采用双皮质素(doublecortin,DCX)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经元核抗原(neuron specific nuclear protein,NeuN)免疫荧光评估干细胞的分化能力,采用Western blotting检测各组细胞Wnt3a、低密度脂蛋白受体相关蛋白5(low density lipoprotein receptor-related protein 5,LRP5)、LRP6、β-连环蛋白(β-catenin)表达,采用qRTPCR法检测神经原素2(neurogenin 2,Ngn2)、细胞周期蛋白(cyclinD1)基因表达。结果左归降糖解郁方能显著增加受损海马神经干细胞的活力(P<0.01),增加Brdu、DCX、GFAP、NeuN的表达(P<0.01),增强其增殖和分化的能力。机制研究发现,左归降糖解郁方能显著增加神经干细胞中的Wnt3a、LRP5、LRP6、总β-catenin、核β-catenin蛋白的表达(P<0.05、0.01),激活Wnt3a信号并增加β-catenin的蓄积和入核,从而促进下游Ngn2、cyclinD1靶基因的转录(P<0.05、0.01)。XAV-939可通过增加β-catenin的降解(P<0.01),减少其进入细胞核,减少下游Ngn2、cyclinD1的m RNA转录(P<0.05、0.01),从而降低干细胞的增殖与分化能力。结论左归降糖解郁方能明显改善葡萄糖与皮质酮对体外海马神经干细胞增殖与分化的抑制作用,其改善作用与激活Wnt3a/β-catenin信号有关。
基金National Major New Drug Development Project(2017 ZX09309026)National Natural Science Foundation of China(82104846)+1 种基金Natural Science Foundation of Hunan Province(2022JJ40323 and 2022JJ80092)Natural Science Foundation of Changsha(Kq2202266).
文摘Objective To investigate the efficacy and mechanism of action of Compound Chaijin Jieyu Tablets(复方柴金解郁片,CCJJYT)in rats with insomnia complicated with depression.Methods Seventy-two Sprague-Dawley rats were randomly assigned into eight groups:the control,chronic unpredictable mild stress(CUMS),sleep deprivation(SD),CUMS+SD,positive drug(venlafaxine hydrochloride+diazepam),CCJJYT high-dose(CCJJYT˗2×),medium-dose(CCJJYT˗1×),and low-dose(CCJJYT˗0.5×)groups,with nine rats in each group.Depression-like behavior was evaluated by body weight,food intake,and behavioral tests such as the sucrose preference test(SPT),open field test(OFT),forced swimming test(FST),and pentobarbital-induced sleep test(PST).Hematoxylin-eosin(HE)staining and Golgi-Cox staining were used to observe changes in pathological tissue and synaptic morphology,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of orexin-A and acetylcholine.The expression levels of orexin receptor 1(OXR1),melatonin receptor 1(MT1A),melatonin receptor 2(MT1B),acetylcholinesterase(AChE),and choline acetyltransferase(ChAT)were detected by immunohistochemistry and Western blot.Results In the present study,rats in the model group showed significant behavioral changes as well as a reduction in hippocampal dendritic branch length and synaptic number,along with increasing the content of orexin A and acetylcholine(P<0.05),and altered expression levels of OX1R,MT1A,MT1B,ChAT,and AChE in the hippocampus and prefrontal cortex after modeling(P<0.05).CCJJYT can improve depressive insomnia behavior and synaptic plasticity of rats(P<0.05),which is similar to that of the positive drug group.It can also decrease the content of orexin A and acetylcholine,and reduce the expression levels of OXR1 and ChAT in hippocampus and prefrontal cortex(P<0.05),and increase the expression levels of MT1A,MT1B,and AChE proteins(P<0.05).Conclusion CCJJYT has good antidepressant and insomnia effects,probably through the regulation of orexin-A,melatonin,and acetylcholine content in hippocampus and prefrontal cortex of rats,improving synaptic plasticity and thus exerting antidepressant and insomnia effects.
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.
文摘盐诱导激酶(salt-inducible kinases,SIKs)属于腺苷酸活化激酶(AMP-activated protein kinase,AMPK)家族,主要调控环磷酸苷效应元件结合蛋白(cAMP-response element binding protein,CREB)转录共激活因子(CREB-regulated transcription coactivators,CRTCs)的胞核-胞质分布,对CRTC-CREB复合物的合成起到调节作用,从而间接影响CREB目的基因的转录与表达,影响多种生理过程,例如能量代谢、细胞周期进程和细胞凋亡。过去对SIKs的相关研究主要集中在外周系统疾病,包括肿瘤、高血压和糖异生等。近年来越来越多的研究开始探索SIKs和中枢神经系统疾病的关联性,如抑郁症、睡眠障碍、癫痫和阿尔茨海默病等都存在SIKs水平异常的情况,这提示SIKs信号失调参与这些疾病的病理过程,SIKs具有成为此类疾病新治疗靶点的潜力。本文就SIKs在抑郁症、睡眠障碍及其他神经系统疾病中的作用和调控机制进行综述。
