MAK-5 treatment enhances the nerve growth factor-mediated neurite outgrowth in PC12 cells

The effects of an ayurvedic compound (MAK-5) alone or together with nerve growth factor (NGF) on the neurite outgrowth of PC12 cells was studied. PC12 cells treated with NGF alone showed a clear neurite outgrowth with a decrease of the proliferation at the dose higher than 5 ng/ml. MAK-5 alone does not induce significant neurite outgrowth in the PC12 cells and does not decrease the proliferation. The PC12 cells treated with NGF supplemented with MAK-5 showed a well-evident morphological differentiation also at low doses of NGF (less than 5 ng/ml), however, the proliferation does not decrease. We suggest that MAK-5 could contain some differentiating agents that are able to potentiate NGF inducing neuronal differentiation in PC12 cells without decreasing the cell proliferation.     

人参炔醇对神经细胞的营养和保护作用

 目的探讨人参炔醇对神经细胞的营养及保护作用。方法通过PC12细胞、新生鼠脊髓背根神经节研究人参炔醇的神经营养和保护作用。结果人参炔醇可时间、剂量依赖性地促进PC12细胞轴索生长,促进细胞分化标志物MAP-2B表达; 8 μmol·L^-1的人参炔醇可与5 μg·L^-1的神经生长因子协同促进大鼠脊髓背根神经轴索的延长;对叠氮钠致PC12细胞损伤有剂量依赖性保护作用。结论人参炔醇对PC12细胞具有类似神经生长因子的神经营养和神经保护作用。     

阿魏酸对bFGF诱导PC12细胞分化的影响

目的:探讨阿魏酸在低浓度碱性成纤维生长因子(bFGF)诱导大鼠肾上腺髓质嗜铬瘤细胞(PC12细胞)分化的作用及其初步机制。方法:在0 ng/mL或1 ng/mL bFGF条件下培养PC12细胞,以PC12细胞的突起长度及诱导率确定药效。结果:阿魏酸在无bFGF的情况下不能诱导PC12分化,但在低浓度bFGF(1 ng/mL)情况下,与bFGF(1 ng/mL)对照组比较可明显诱导PC12细胞分化(P<0.01),这种作用能被有丝分裂原激活的蛋白激酶(MAPK)特异性抑制剂PD98059所抑制。结论:阿魏酸可增强bFGF诱导PC12细胞分化,其机制可能与MAPK信号通路有关。     

Sterol and triterpenoid constituents of Verbena littoralis with NGF-potentiating activity

Two new sterols, stigmast-5-ene 3beta,4beta,7alpha,22alpha-tetraol (1) and stigmast-5-ene 3beta,7alpha,22alpha-triol (2), were isolated from the aerial parts of a Paraguayan medicinal plant, Verbena littoralis, together with the known compounds ursolic acid (3) and oleanolic acid (4). The structures of 1 and 2 were elucidated by spectroscopic analyses. Compounds 2-4 showed an enhancing activity of nerve growth factor (NGF)-mediated neurite outgrowth in PC12D cells.     

Bioactive ent-clerodane diterpenoids from the aerial parts of Baccharis gaudichaudiana

Three new ent-clerodane diterpenes (1-3) were isolated from the aerial parts of Baccharis gaudichaudiana, and their structures were elucidated as 15,16-epoxy-15alpha-methoxy-ent-clerod-3-en-18-oic acid (1), 13-epi-15,16-epoxy-15alpha-methoxy-ent-clerod-3-en-18-oic acid (2), and 7-oxo-16-hydroxy-ent-clerod-3-en-15-oic acid methyl ester-18,19-olide (3), on the basis of spectroscopic data analysis. A known compound, 7-oxo-ent-clerod-3-en-15,16:18,19-diolide, was also identified. Compounds 1 and 2 showed enhancing activity of nerve growth factor (NGF)-induced neurite outgrowth in PC 12D cells.     

甘草苷对原代海马神经细胞的保护和营养作用

目的:研究甘草苷(liquiritin,LQ)对β-淀粉样蛋白(Aβ_25～35)所致大鼠原代神经细胞损伤的保护作用和营养作用。方法:采用大鼠原代海马神经元Aβ_25～35损伤模型,四甲基偶氮唑盐(MTT)法测定细胞活力、流式细胞术检测神经细胞凋亡、荧光探针法检测细胞内钙离子浓度,考察LQ神经保护作用;通过考察LQ诱导海马神经元轴突延长的作用和流式细胞术检测其对海马神经干细胞分化为胆碱能神经元作用观察其神经营养作用。结果:10μmol.L^-1Aβ_25～35显著降低细胞的存活率;明显升高细胞内钙离子水平;诱导细胞凋亡发生,凋亡率达28%。预先给预0.1,1,10μmol.L^-1浓度的LQ处理细胞6 h,可显著抑制Aβ_25～35导致的细胞死亡,能够明显降低Aβ_25～35导致的细胞内钙离子浓度升高,流式细胞检测凋亡率分别降低到10%,15%,22%,提示LQ能够拮抗Aβ_25～35导致的细胞凋亡。LQ能够显著增加神经生长因子(NGF)介导的海马神经元轴突延长,特异性的促分裂原活化蛋白激酶(MAPK)抑制剂能够部分阻断该作用。另外,LQ可以诱导神经干细胞体外定向分化为胆碱能神经元。结论:甘草苷对Aβ_25～35导致的大鼠原代神经细胞损伤具有神经保护作用,同时甘草苷对原代海马神经元有营养作用。     

竹叶提取物抗肿瘤和神经保护作用研究

目的研究竹叶提取物抑制肿瘤细胞增殖的活性及其分子生物学机制。方法选取血管内皮生长因子(VEGF)诱导型肿瘤细胞人肝癌细胞Hep G2和不诱导型肿瘤细胞人神经母细胞瘤细胞SH-SY5Y为模型,用不同质量浓度竹叶提取物处理Hep G2和SH-SY5Y细胞。采用MTT法检测竹叶提取物对2种细胞增殖的抑制作用;采用RT-PCR法检测VEGF和血管内皮生长因子受体2(VEGFR2)基因在Hep G2和SH-SY5Y细胞中的表达差异以及竹叶提取物处理后Hep G2细胞中VEGF基因表达的变化;用荧光倒置显微镜观察竹叶提取物处理后SH-SY5Y细胞神经突触的生长情况;采用RT-PCR法检测竹叶提取物处理后SH-SY5Y细胞中神经生长因子(NGF)表达的变化。结果竹叶提取物对Hep G2细胞增殖的抑制活性高于对SH-SY5Y细胞的增殖抑制作用。进一步研究发现Hep G2细胞中VEGF和VEGFR2基因的表达显著高于SH-SY5Y细胞,且竹叶提取物可显著抑制Hep G2细胞的VEGF表达。适当剂量的竹叶提取物可以刺激SH-SY5Y细胞神经突触的生长,并可上调其NGF的表达。结论竹叶提取物可能通过抑制VEGF的表达影响VEGF诱导型肿瘤细胞的增殖,同时,可能通过上调NGF的表达刺激神经细胞神经突触的生长,竹叶提取物可能具有潜在的抗肿瘤和神经保护的双重功效。     

Spirocyclic nortriterpenoids with NGF-potentiating activity from the fruits of Leonurus heterophyllus

Four new spirocyclic nortriterpenoids, leonurusoleanolide A (1), leonurusoleanolide B (2), leonurusoleanolide C (3), and leonurusoleanolide D (4), were isolated from the MeOH extract of the fruits of Leonurus heterophyllus. Compounds 1 and 2, and compounds 3 and 4, were found to exist as equilibrium mixtures of trans and cis isomers. Mixtures of 1 and 2, and 3 and 4, significantly enhanced the neurite outgrowth of nerve growth factor-treated PC12 cells at concentrations ranging from 1 to 30 μM. Compound 8 was also found to have a neurite outgrowth-promoting effect at concentrations of 1 and 10 μM. The structure-activity relationship of these compounds is discussed.     

银杏内酯诱导嗜铬细胞瘤细胞表达低氧诱导因子-1α

 目的研究银杏内酯(ginkgolides,Gin)对神经生长因子(nerve growth factor,NGF)诱导分化的嗜铬细胞瘤(PC12)细胞的影响以及与其相关的信号通路。方法通过MTT比色法观察不同浓度Gin对PC12细胞活性的影响,RT-PCR和Western Blot法分析Gin对PC12细胞低氧诱导因子-1(αhypoxia inducible factor-1α,HIF-1α)表达以及MAPK信号通路的影响。结果一定浓度范围的Gin可促进PC12细胞的活性,在37.5 mg·L^-1作用24 h效果最明显。37.5 mg·L^-1 Gin单独处理PC12细胞24 h可诱导HIF-1αmRNA表达增强和蛋白水平上调。Gin还引起p-ERK水平的明显提高。Gin增加PC12细胞HIF-1α蛋白的稳定性存在一定的时间和剂量依赖关系,PD98059可部分抑制Gin的增强作用,金雀异黄素可完全阻断之;与此相对应的是,Gin以时间和剂量依赖方式诱导PC12细胞p-ERK水平的增高,PD98059和金雀异黄素均能完全阻断Gin的诱导作用。结论Gin可诱导PC12细胞表达HIF-1α,主要与MEK-ERK信号通路激活有关,可能是其促进分化的PC12生长的原因。     

Neurotrophic activity of DA-9801, a mixture extract of Dioscorea japonica Thunb. and Dioscorea nipponica Makino, in vitro

Ethnopharmacological relevance

Dioscorea japonica Thunb. has been traditionally used to treat polyuria and diabetes in Korea.

Aim of the study

We previously report the effects of Dioscorea japonica Thunb. extract on glucose control, NGF induction, and neuroprotection in a rodent diabetic model. Since the most potent fraction, DA-9801, was identified from a mixture of Dioscorea japonica Thunb. (DJ) and Dioscorea nipponica Makino (DN) following bioactivity-guided fractionation, here, we investigated the potential mechanism of the extract activity against diabetic peripheral neuropathy (DPN).

Materials and methods

A 1:3 mixture of DJ and DN was extracted with ethanol (DA-9801) and further fractionated into an ethylacetate-soluble fraction (DA-9801E). Effects of these extracts on neurite outgrowth were measured in PC-12 cells and DRG neurons. Effects on cell viability and TrkA phosphorylation were evaluated in PC-12 cells. NGF induction effect was determined in primary Schwann cells as well as IMS32 cells (immortalized Schwann cells).

Results

No cytotoxicity was observed in PC-12 cells at the concentration below 500 μg/ml of either DA-9801 or DA-9801E. DA-9801 and DA-9801E at 100 μg/ml and 10 μg/ml, respectively, showed a significant effect on neurite outgrowth in PC-12 cells and DRG neurons in the presence of or absence a low concentration of NGF (2 ng/ml). The Trk-A phosphorylation effect of DA9801 was confirmed in PC-12 cells. An NGF induction effect of these extracts was not detected in either IMS-32 cells, or primary Schwann cells.

Conclusions

The NGF agonistic activity of DA-9801 and DA-9801E was demonstrated, which may contribute to their neuroprotective effect against DPN. Studies of the detailed mechanism of these extracts as well as identification of the active components are warranted for the development of an anti-DPN drug from DJ and DN.     

Verbenachalcone, a novel dimeric dihydrochalcone with potentiating activity on nerve growth factor-action from Verbena littoralis.

A novel dimeric dihydrochalcone, verbenachalcone (1), was isolated from the aerial parts of Verbena littoralis. Its structure was elucidated, on the basis of spectral data interpretation, as 4,2',4',2' ",4' "-pentahydroxy-3' '-methoxy-3-O-4' '-tetrahydrobichalcone. This compound caused a significant enhancement of nerve growth factor-mediated neurite outgrowth from PC12D cells.     

Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis

Three new acetylated flavonoid glycosides, 5,6,4'-trihydroxyflavone 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (1), apigenin 7-O-alpha-L-3-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (2), and apigenin 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3), were isolated from Scoparia dulcis together with the known compound eugenyl beta-D-glucopyranoside (4). Their structures were elucidated by spectroscopic analyses. Compounds 2 and 3 showed an enhancing activity of nerve growth factor-mediated neurite outgrowth in PC12D cells.     

Protective effects of paeoniflorin against corticosterone-induced neurotoxicity in PC12 cells

Neuroprotection has been proposed as one of the acting mechanisms of antidepressants. Paeoniflorin, a monoterpene glycoside, has been reported to display antidepressant-like effects in animal models of behavioural despair. The present study aimed to examine the protective effect of paeoniflorin treatment on corticosterone-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Paeoniflorin was shown to elevate cell viability, decrease levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) in corticosterone-treated PC12 cells. Paeoniflorin also reversed the reduced nerve growth factor (NGF) mRNA level caused by corticosterone in PC12 cells. The results suggest that paeoniflorin exerts a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, at least in part, via the inhibition of oxidative stress and the up-regulation of NGF expression. This neuroprotective effect may be one of the action pathways that accounts for the in vivo antidepressant activity of paeoniflorin.     

利用干细胞筛选中药神经保护作用活性成分的新方法

目的：利用干细胞构建可用于药物筛选的细胞模型,初步建立寻找中药活性成分的新方法。方法：首先,克隆神经生长因子（NGF）启动子的序列,连接到含有荧光素酶报告基因/E2红色荧光蛋白的质粒上,并将合成得到的质粒转入HEK-293细胞中,构建基于启动子的初步筛选稳定的细胞模型。使用阳性药进行验证细胞模型的准确性,并用该模型筛选来自中药的小分子化合物。然后使用筛选得到的化合物诱导大鼠的骨髓间充质干细胞,检测相关神经营养因子的转录水平,验证待测小分子化合物可能对神经生长的生物活性。结果：来自中药益智的三种化合物,白杨素、圆柚酮和杨芽黄素均可以激活NGF启动子。白杨素、圆柚酮诱导MSC不同时间后,细胞表面神经标志物:神经生长因子（NGF）、半乳糖神经酰胺（GalC）、巢蛋白（Nestin）、神经元特异烯醇化酶（NSE）、波形蛋白（Vimentin）对应RNA的转录水平均有不同程度的提高。而杨芽黄素没有明确促进神经细胞分化的活性。结论：白杨素、圆柚酮可以促进干细胞向神经细胞分化,是中药益智发挥神经保护作用物质基础的组成部分。     

Differentiation induced by Achyrocline satureioides (Lam) infusion in PC12 cells

Epidemiological studies have shown that flavonoid‐rich plants induce beneficial health effects that are likely beyond their potent antioxidant capacity. Thus, the mechanisms by which Achyrocline satureioides (AS), a popular South American medicinal plant, protects cells and neurons in culture, are still unclear. In this sense, a recently described trophic capacity for flavonoids, similar to that evoked by growth factors, could be one of the mechanisms involved in AS cellular protection. Since this trophic activity causes differentiation of PC12 cells, the cell differentiation capacity of AS and some of its flavonoids were evaluated. PC12 cells were treated with AS infusion (10 or 20 µg/mL of total polyphenols), quercetin (Q) (12.5 or 25 µm ), luteolin (L) (25 µm ), Q + L (12.5 µm each one) or nerve growth factor (NGF) for 3 days. Four morphological parameters (percentage of cells with neurites longer than one cell body diameter, percentage of cells with neurites, average number of neurites per cell and percentage of fusiform cells) were explored. The AS infusion showed differentiation capacity on all parameters with similar potency when compared with NGF. Besides, AS was more potent than some of its constituent flavonoids: Q, L or their combination. Copyright © 2009 John Wiley & Sons, Ltd.     

苦参素通过调控神经生长因子表达抑制背根神经节生长和疼痛递质释放研究

目的:研究苦参素对神经生长和疼痛递质的表达影响和作用机制。方法:从新生大乳鼠提取脊柱两侧的背根神经节,修剪并在体外进行培养,通过测量其生长径评价神经节生长能力。应用Real-time PCR检测神经节中神经生长因子(NGF)、疼痛递质P物质(SP)和降钙素基因相关肽(CGRP)的mRNA表达; 应用ELISA检测培养体系上清中NGF和疼痛递质SP、CGRP的分泌量。结果:将提取的原代背根神经节应用S100标记进行鉴定。苦参素和空白对照组比较,能明显抑制神经节生长径,且苦参素可抑制培养体系中背根神经节NGF和疼痛递质SP、CGRP的表达(P<0.05)。在培养体系中应用外源性NGF后,能够逆转苦参素抑制背根神经节的生长。结论:苦参素通过抑制NGF表达延缓背根神经节生长,抑制疼痛递质释放。     

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-neuroinflammatory and neuroprotective activities

Ethnopharmacological relevance

Dioscorea nipponica (Dioscoreaceae) have been used as traditional medicines for diabetes, inflammatory and neurodegenerative diseases in Korea. The aim of the study was to isolate the bioactive components from the rhizomes of Dioscorea nipponica and to evaluate their anti-neuroinfalmmatory and neuroprotective activities.

Material and methods

The phytochemical investigation of 50% EtOH extract of Dioscorea nipponica using successive column chromatography over silica gel, Sephadex LH-20, and preparative high performance liquid chromatography (HPLC) resulted in the isolation and identification of 17 phenolic derivatives, including four new phenolic compounds (1–4). The structural elucidation of these compounds was based on spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques, mass spectrometry, and optical rotation. All isolated compounds were evaluated for their effects on nerve growth factor (NGF) secretion in a C6 rat glioma cell line and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV2 cells. The neurite outgrowth of compound 16 was further evaluated by using mouse neuroblastoma N2a cell lines.

Results

Three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new phenanthrene glycoside, diosniposide B (4), together with 13 known compounds were isolated from the rhizomes of Dioscorea nipponica. Of the tested compounds (1–17), phenanthrene, 3,7-dihydroxy-2,4,6-trimethoxy-phenanthrene (16) was the most potent NGF inducer, with 162.35±16.18% stimulation, and strongly reduced NO levels with an IC₅₀ value of 19.56 μM in BV2 microglial cells. Also, it significantly increased neurite outgrowth in N2a cells.

Conclusions

This study supports the ethnopharmacological use of Dioscorea nipponica rhizomes as traditional medicine.     

Rosmarinus officinalis polyphenols activate cholinergic activities in PC12 cells through phosphorylation of ERK1/2

Aim of the study

This paper aimed to elucidate the traditional use of Rosmarinus officinalis through the investigation of cholinergic activities and neuronal differentiation in rat pheochromocytoma PC12 cells. These effects were examined in relation to the plant's habitat, the extraction procedure, and the major active compounds of R. officinalis.

Materials and methods

Cell viability, cell differentiation, acetylcholinesterase (AChE) activity, total choline, acetylcholine (ACh) and extracellular signal-regulated kinases (ERK1/2) were determined in PC12 cells treated with extracts and HPLC-identified polyphenols of R. officinalis originated from Tunisian semi-arid and subhumid area in comparison with nerve growth factor (NGF).

Results

R. officinalis extracts potentiated cell differentiation and significantly enhanced AChE activity in PC12 cells. The highest AChE activity was induced by semi-arid hydro-ethanolic extract (137% of control). Among HPLC-identified and screened polyphenols, carnosic acid (CA) and rosmarinic acid (RA) significantly induced cell differentiation, increased ACh level, and enhanced AChE activity in PC12 cells. U0126, inhibitor of ERK1/2, significantly reduced CA and RA effects on cell differentiation and AChE activity.

Conclusions

R. officinalis’ CA and RA exhibited neurotrophic effects in PC12 cells through cell differentiation induction and cholinergic activities enhancement. These effects could be regulated by mitogen-activated protein kinase (MAPK), ERK1/2 signaling pathway.     

巴戟天寡糖对皮质酮损伤的PC12细胞的保护作用

目的 :探讨巴戟天寡糖 (MW 97)抗抑郁作用的可能机制。方法与结果 :MW-97或NGF与皮质酮 2×10 ^-4 mol·L^-1共孵PC12细胞 48h ,可防止皮质酮所致的PC12神经细胞损伤。进一步运用RT PCR方法研究表明 ,MW 97(10 0mg·kg^-1)及去甲丙米嗪 (10mg·kg^-1)慢性ip 21d ,可使大鼠前脑皮层NGF、脑源性神经营养因子(BDNF)及海马BDNFmRNA表达升高。结论 :MW 97抗抑郁作用机理可能与神经细胞营养因子表达升高 ,从而对皮质酮诱导的神经元损伤产生保护作用有关。