金银花对H_2O_2诱导的PC12细胞氧化损伤的保护作用及机制研究

目的:探讨金银花水提物对过氧化氢(H_2O_2)诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞氧化损伤的保护作用。方法:采用H_2O_2损伤PC12细胞建立氧化损伤模型,以不同浓度金银花水提物(5、25和50μg/ml)进行研究,采用MTT法检测细胞活力,DCFH-DA检测ROS的水平,TUNEL法检测细胞凋亡,免疫印迹实验检测细胞中Sirt3和凋亡相关蛋白蛋白表达。结果:不同浓度金银花水提物(5、25和50μg/ml)能够抑制H_2O_2诱导的细胞死亡并呈剂量依赖性。H_2O_2组细胞活力和凋亡率较对照组显著增加,给予金银花水提物作用48 h后,明显降低细胞内活性氧(ROS)和丙二醛(MDA)含量,增加超氧化物歧化酶(SOD)活力,抑制H_2O_2诱导的细胞凋亡并上调Sirt3、IDH2、Bcl-2蛋白表达,下调Bax、caspase3蛋白表达。结论:金银花水提物能够抑制H_2O_2诱导的PC12细胞的氧化损伤,其保护作用可能与抑制氧化应激引起的细胞凋亡并调控ROS/Sirt3途径有关。     

Neuroprotective effects of Buyang Huanwu decoction against hydrogen peroxide induced oxidative injury in Schwann cells

Ethnopharmacological relevance

Buyang Huanwu decoction (BYHWD) is a traditional Chinese medicine and can be used to promote peripheral nerve regeneration. However the regenerative mechanism of BYHWD remains unclear. The objective of this study was to investigate the protective mechanisms of BYHWD in Schwann cells damaged by hydrogen peroxide (H₂O₂).

Materials and methods

Schwann cells which were derived from neonatal sciatic nerves of rats were used in subsequent experiments. Schwann cells were injured by various concentrations of H₂O₂ (0.25, 0.5 and 1 mM final concentration). BYHWD (600 μg/ml final concentration) was added to the medium either simultaneously or 1 h later after the addition of H₂O₂. Subsequently, methyl thiazolyl tetrazolium (MTT) assay was performed. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were also examined after 12 h. The expression of Caspase 3 and the concentration of intercellular Ca²⁺ ([Ca²⁺]i) were also determined.

Results

Among three concentrations of H₂O₂, 0.5 mM H₂O₂ induced Schwann cells swelled and neuritis disappeared after 12 h. In the presence of BYHWD, MTT assay showed that more cells were viable in comparison with the H₂O₂ injury group. Moreover, the addition of BYHWD has also increased the SOD activity with decreased in MDA level. Furthermore, the concentration of [Ca²⁺]i and expression of Caspase 3 were decreased with the addition of BYHWD in culture.

Conclusions

Our results revealed that BYHWD protected Schwann cells from oxidative injury. The mechanism of BYHWD promoting neural regeneration possibly associated with its anti-oxidative activity.     

褐藻多糖硫酸酯通过溶酶体组织蛋白酶D调节过氧化氢诱导的PC12细胞凋亡

目的:研究褐藻多糖硫酸酯(fucoidan)对过氧化氢(H2O2)诱导的大鼠嗜铬细胞瘤细胞株(pheochromocytomacells,PC12)凋亡的影响,探讨fucoidan对阿尔茨海默病(Alzheimer's disease,AD)的作用机制.方法:50μg·L-1神经生长因子(nerve growth factor,NGF)诱导分化PC12细胞(differentiated PC12,dPC12)7 d,0.5 mmol·L-1H2O2刺激30 min造成dPC12细胞凋亡.磺酰罗丹明B(sulforhodrimine B sodium salt,SRB)法检测不同质量浓度fucoidan(0.1,1,10,50,100 mg·L-1)对细胞存活的影响,筛选适宜药物浓度;荧光底物法检测凋亡细胞内caspase-3的活性及细胞浆中溶酶体组织蛋白酶D的活性;酶-底物法检测不同质量浓度fucoidan(0.01,0.1,1,10,100 mg·L-1)对人肝脏溶酶体组织蛋白酶D活性的影响.结果:fucoidan能够拈抗H2O2所致细胞损伤,以10 mg·L-1作用最为明显;并能降低异常升高的凋亡执行酶caspase-3的活性.fucoidan对H2O2损伤后dPC12细胞胞浆内和人肝脏提取的溶酶体组织蛋白酶D活性均具有抑制作用.结论:fucoidan可以抑制H2O2诱导的dPc12细胞凋亡,其作用机制可能与抑制溶酶体组织蛋白酶D的活性有关.     

地骨皮甲素对鱼藤酮诱导的PC12细胞损伤的保护作用

目的 考察地骨皮甲素（kukoamine A,KuA）对鱼藤酮诱导的PC12细胞损伤的保护作用并对其作用机制进行初步研究,为发现具有抗帕金森病的活性先导化合物提供依据。方法 通过体外建立鱼藤酮诱导的帕金森模型,运用MTT、LDH、Hoechst33342染色等方法对KuA体外抗鱼藤酮诱导的PC12细胞损伤进行初步确认。利用比色法和荧光染色分别考察KuA对超氧化物歧化酶（SOD）活性、丙二醛（MDA）及活性氧（ROS）含量、线粒体膜电位（MMP）的影响。采用Western blotting探讨KuA体外抗鱼藤酮诱导PC12细胞损伤的作用机制。结果 0.5 μmol/L鱼藤酮可降低PC12细胞存活率,不同浓度KuA可减轻鱼藤酮诱导的PC12细胞损伤。与模型组相比,KuA可降低细胞内ROS及MDA含量,并提高SOD活性。此外,KuA可提高基质金属蛋白酶（MMP）水平,下调Bax/Bcl-2值、抑制细胞色素C释放以及下调Caspase-3、Caspase-9、α-synuclein的蛋白表达。结论 KuA对鱼藤酮诱导的PC12细胞帕金森病模型具有保护作用,其可能的机制与抑制ROS生成、保护MMP、调控线粒体凋亡途径相关蛋白表达和降低α-synuclein蛋白表达有关。     

Neuroprotective effects of Total Saikosaponins of Bupleurum yinchowense on corticosterone-induced apoptosis in PC12 cells

Ethnopharmacological relevance

The root of Bupleurum yinchowense Shan et Y. Li, a well-known medicinal plant in China, was originally documented in the “Shennong's Herbal”, which is the oldest Chinese materia medica monographs. It has the action of soothing liver and relieving constraint for improving symptoms of emotional instability such as depression, anxiety and phobia. The in vivo experiment of our previous study has showed an efficacy of Total Saikosaponins (TSS) from Bupleurum yinchowense in acute stress and chronic unpredictable mild stress models. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of TSS on corticosterone-induced apoptosis in PC12 cells. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of TSS.

Materials and methods

The PC12 cells were treated with 250 μM corticosterone in the absence or presence of different concentrations of TSS for 24 h, then the cell viability, lactate dehydrogenase (LDH) release, Hoechst 33342 and propidium iodide (PI) double staining and the DNA fragmentation of the apoptotic PC12 cells were determined. The mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca²⁺ ([Ca²⁺]i) concentration and western blot analysis of caspase-3, glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible proteins 153 (GADD-153), X-box DNA-binding protein-1 (XBP-1), Bax, Bcl-2 were investigated.

Results

Pretreatment of PC12 cells with TSS (3.125, 6.25, 12.5, 25 μg/ml) partly reversed corticosterone-induced neurotoxicity in a dose dependent manner. TSS (25 =g/ml) reversed the increase of dead cells in the Hoechst 33342 stain, the accumulation in LDH leakage and the number of TUNEL positive cells induced by corticosterone to PC12 cells. Moreover, the cytoprotection of TSS was proved to be associated with the homeostasis of intracellular Ca²⁺, the stabilization of ER stress via the down-regulation of GRP78, GADD-153, XBP-1, and the restoration of mitochondrial function, which included mPTP, MMP and caspase-3 activity. Furthermore, TSS (25 μg/ml) markedly ameliorated up-regulation of Bax and down-regulation of Bcl-2 in corticosterone-induced PC12 cells.

Conclusion

The result depicted that antidepressant-like effect of TSS in vivo may be associated with the cytoprotection of neuron, and the neuroprotective mechanisms were correlated with inhibiting the ER stress and the mitochondrial apoptotic pathways.     

Effect of Hypericum perforatum on marble-burying by mice

The effect of Hypericum perforatum extract (LI 160) at a dose that exerts an antidepressive-like effect was studied in mice in the marble-burying test. Acute Hypericum perforatum (150, 300 and 500 mg/kg, p.o.) reduced immobility time in the forced swimming test. The number of marbles buried, but not locomotor activity, was reduced by acute treatment with Hypericum perforatum (150 and 300 mg/kg, p.o.). However, this effect was not seen after chronic treatment (21 days) with Hypericum perforatum (300 mg/kg, p.o.). Thus, Hypericum perforatum extract, at antidepressant dose, exerts an acute anxiolytic drug effect on the marble-burying test, which could indicate a potential anti-obsessive effect, although the development of tolerance could be an important drawback.     

Inhibitory effects of flavonoids from Hypericum perforatum on nitric oxide synthase

The inhibitory effects of six flavonoids from Hypericum perforatum were assessed spectrophotometrically using nitric oxide synthase (NOS) in blood and cerebral homogenate of rats. Of the assayed compounds, quercetin and hyperoside showed concentration-dependent enzyme inhibitory actions. The IC50 values of quercetin for inhibiting NOS in rat cerebral homogenate and blood were 63.06 and 57.54 microM, and those of hyperoside 56.23 and 158.49 microM, respectively. The competitive patterns were discerned with the inhibition of the two flavonoids on NOS in serum and cerebral homogenate (except a mixed type inhibition was observed with quercetin in inhibiting cerebral NOS). Furthermore, similar inhibitions were found for quercetin upon NOS in cerebral homogenate and blood. However, a stronger inhibitory effect of hyperoside on the enzyme was discerned in cerebrum than in blood. These results suggested that the galactose moiety in hyperoside may be associated with the selectivity of the NOS inhibition.     

葡萄籽原花青素对H2O2损伤PC12细胞的保护作用

目的：检测葡萄籽原花青素对H2O2引起PC12细胞氧化损伤的保护作用及其作用机制。方法：用盐酸-香草醛法检测葡萄籽原花青素中低聚花青素的含量，并用DPPH（1，1-diphenyl-2-picrylhydrazyl）法检测其对自由基的清除能力，以PC12细胞为实验对象，进一步检测葡萄籽原花青素对氧化损伤的PC12细胞的保护效应。结果：低聚原花青素含量为45．16％的葡萄籽原花青素在浓度为64μg/ml时对DPPH自由基的清除率为76．69％，对H2O2损伤的PC12细胞在浓度为100μg／ml时保护率为83．1％，同时可使细胞内超氧化物歧化酶（SOD），谷胱甘肽（GSH）活性增加，细胞的脂质过氧化水平降低。结论：葡萄籽原花青素是通过清除自由基和提高细胞内源抗氧化酶来保护PC12细胞免受氧化损伤。     

葡萄籽原花青素对H_2O_2损伤PC12细胞的保护作用

目的:检测葡萄籽原花青素对H2O2引起PC12细胞氧化损伤的保护作用及其作用机制。方法:用盐酸-香草醛法检测葡萄籽原花青素中低聚花青素的含量,并用DPPH(1,1-d iphenyl-2-p icrylhydrazyl)法检测其对自由基的清除能力,以PC12细胞为实验对象,进一步检测葡萄籽原花青素对氧化损伤的PC12细胞的保护效应。结果:低聚原花青素含量为45.16%的葡萄籽原花青素在浓度为64μg/m l时对DPPH自由基的清除率为76.69%,对H2O2损伤的PC12细胞在浓度为100μg/m l时保护率为83.1%,同时可使细胞内超氧化物歧化酶(SOD),谷胱甘肽(GSH)活性增加,细胞的脂质过氧化水平降低。结论:葡萄籽原花青素是通过清除自由基和提高细胞内源抗氧化酶来保护PC12细胞免受氧化损伤。     

HPLC法测定贯叶连翘中贯叶金丝桃素含量

目的：测定贯叶连翘中贯叶金丝桃素的含量。方法：采用HPLC法,流动相：乙腈－水（140：1,内含1％冰醋酸）,流速：1mL/min,检测波长276nm,结果：该法的平均回收率为98．47％,RSD＝1．33％。结论：方法可用于贯叶金丝桃药材及其制剂 质量控制。     

通窍活血汤含药血清对谷氨酸损伤的PC12细胞的保护作用

目的:从细胞水平研究通窍活血汤含药血清(TQHXD)对谷氨酸(Glu)损伤的PC12细胞的保护作用,为该方剂的临床应用提供依据。方法:SD大鼠灌胃给予通窍活血汤提取液,每日2次,每次4 g.kg-1,连续5 d,制备通窍活血汤含药血清。采用PC12细胞和终浓度为12.5 mmol.L-1的谷氨酸共培养,造成神经细胞损伤模型。光镜观察5%,10%,20%的TQHXD对损伤PC12细胞形态改变的影响;台盼兰染色法、AO/EB染色法及LDH法观察TQHXD对损伤的细胞膜通透性的变化影响;MTT法检测TQHXD对PC12细胞增殖及活性的影响及对培养上清液中NO浓度的影响。结果:倒置显微镜下可见,正常组细胞胞体较损伤组略大,呈强折光性,细胞数量多,突起长。模型组细胞数量显著减少,折光性明显减弱,细胞突起减少、缩短。TQHXD和PC12细胞共培养24 h后,活细胞数明显增加,细胞折光性增加,损伤细胞形态接近于正常细胞;台盼兰染色后,TQHXD组活细胞比率明显上升,AO/EB染色后被EB染色的数量较少。MTT法检测结果显示,与模型组比较,各浓度TQHXD组的吸光度A均升高且有显著性差异。各浓度的TQHXD组细胞培养上清中LDH,NO含量相对模型组明显减少,两者有显著性差异。结论:TQHXD对Glu损伤的PC12细胞有明显的保护作用。     

黄芪多糖对过氧化氢诱导的内皮细胞损伤的保护作用

目的:观察黄芪多糖(Astragalus polysaccharide,APS)对过氧化氢(H2O2)诱导的人脐静脉内皮细胞HUVECs损伤的保护作用及其可能的作用机制。方法:不同浓度的黄芪多糖孵育内皮细胞1h后加入400μmol/L的H2O2继续培养24h,用MTT法检测细胞活性;DAPI/PI双染法观察细胞形态并统计细胞凋亡率;硝酸还原酶法测定NO释放量;Western blot法测定Cu/Zn-SOD蛋白表达。结果:H2O2模型组细胞活性较之空白对照组下降42.87%,细胞核皱缩形态不规则化,细胞凋亡率升高了7倍、NO释放量降低42.96%,Cu/Zn-SOD蛋白表达下降45.31%;与模型组相比黄芪多糖各剂量组可不同程度改善这些变化,其中黄芪多糖1μg/ml组作用最为明显,其细胞活性和NO释放量分别增加24.96%、32.13%,细胞凋亡率降低36.02%,Cu/Zn-SOD蛋白表达升高37.14%。结论:黄芪多糖可能通过影响内皮细胞NO释放量和Cu/Zn-SOD蛋白表达,改善细胞NO与氧自由基的平衡发挥保护作用。     

贯叶连翘提取物抗抑郁作用研究

目的：研究贯叶连翘提取物对小鼠的抑制郁作用。方法：选取了强迫小鼠游泳实验，小鼠尾悬吊应激实验，拮抗利血平所致的抑制症状等实验指标。结果：贯叶连翘提取物150mg/kg,300mg/kg显著缩短迫游泳小鼠及尾悬吊小鼠的不动时间；显著拮抗利血平所致的体温下降作用和小鼠眼睑下垂作用。结论：贯叶连翘提取物具有一定的抗抑郁作用。     

芥子碱对连二亚硫酸钠引起的PC12细胞凋亡损伤的保护作用

目的：探讨芥子碱对连二亚硫酸钠诱导PC12细胞缺氧损伤的作用并探讨其机制。方法：用连二亚硫酸钠消除培养基中的氧，建立细胞缺氧损伤模型，MTT法检测细胞的存活率；应用流式细胞仪检测细胞凋亡率及线粒体跨膜电位的改变：测定乳酸脱氢酶（LDH）的释放量，培养基和细胞内丙二醛（MDA）含量。结果：与对照组和单纯缺氧模型组相比，芥子碱能显著提高损伤后细胞的存活率，降低细胞内LDH的漏出，使细胞内的MDA减少，降低细胞的凋亡率并提高线粒体的膜稳定性。结论：芥子碱具有显著的神经保护作用，其机制可能与提高细胞的抗氧化能力，减少细胞凋亡有关。     

基于TCMGIS的贯叶连翘生态适宜性研究

目的：分析贯叶连翘的全国适宜产地。方法：以贯叶连翘的主产地湖北板桥镇为基点,运用《中药材产地适宜性分析地理信息系统》（TCMGIS）进行分析。结果：全国共有湖北、重庆、安徽、湖南、四川、陕西、贵州、云南等8个省区的163个县市为贯叶连翘适宜产区,适宜产区面积总和为45773．53km^2。结论：为贯叶连翘引种栽培、合理布局及资源可持续利用提供了依据。     

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。     

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.     

Herbal formula SYJN protect PC12 cells from neurotoxicity induced by corticosterone

Aim of the study

SYJN is a Chinese herbal formula, containing four herbs: Bupleurum chinense DC., Curcuma aromatica Salisb., Perilla frutescens (Linn.) Britt. and Acorus tatarinowii Schott. Previous studies on the formula in our laboratory revealed an antidepressant-like effect on animal models of behavioral despair. However,the mechanisms underlying such antidepressant-like effect are yet to be understood. The aim of this work was to verify the previously established antidepressant-like effects on cell level using corticosterone-induced neurotoxicity in rat pheochromocytoma (PC12) cells to see if SYJN possesses any neuroprotective properties.

Materials and methods

PC12 cells were treated with 200 μM corticosterone in the absence or the presence of various concentrations of SYJN for 48 h. Then, cell viability, apoptosis, intracellular Ca²⁺ ([Ca²⁺]i) concentration and caspase-3 activity were determined.

Results

Following the exposure of PC12 cells to 200 μM corticosterone for 48 h, there were reductions in cell survival rate but increases in lactate dehydrogenase (LDH) release. In parallel, corticosterone caused significant elevations in DNA fragmentation, [Ca²⁺]i concentration and caspase-3 activity. However, when the PC12 cells were incubated with SYJN at different concentrations (10, 50 and 100 mg/L) in the presence of 200 μM corticosterone for 48 h, the above effects were evidently alleviated in a dose-dependent manner.

Conclusion

SYJN could generate a neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells, suggesting a possible action pathway of SYJN in vivo by decreasing the [Ca²⁺]i concentration and caspase-3 activity.     

金丝桃苷对叠氮钠诱导PC12细胞凋亡的保护作用(英文)

目的:研究金丝桃苷对叠氮钠诱导的PC12细胞凋亡的保护作用。方法:将PC12细胞与不同浓度金丝桃苷共同孵育4h后,加入20mmol·L-1的叠氮钠继续孵育3～24h,观察金丝桃苷的保护作用。采用MTT、Hoechst33342分别检测细胞存活率和细胞形态变化。以Ac-DEVD-AMC为底物检测caspase-3活性,CM-H2DCFDA法用于测定ROS水平。Western blotting用于分析Bcl-2和Bax表达水平。结果:金丝桃苷在0.01,0.1及1μmol·L-1浓度时,可显著提高叠氮钠作用后PC12细胞的存活率,抑制细胞凋亡,减少细胞内活性氧自由基生成和caspase-3活性,提高抗凋亡蛋白Bcl-2表达,降低促凋亡因子Bax表达。同时结果显示caspase-3抑制剂呈时间依赖性地减少细胞内活性氧自由基含量。结论:金丝桃苷对叠氮钠诱导的PC12细胞凋亡具有保护作用,活性氧自由基生成与凋亡进程中caspase-3活性密切相关。     

甘木通总黄酮保护H_2O_2诱导的PC12细胞损伤

目的探讨甘木通(Clematis filamentosa)总黄酮(TFC)对H_2O_2引起的大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)损伤的保护作用。方法用H_2O_2损伤PC12细胞建立神经元氧化应激损伤模型,噻唑蓝(MTT)法检测细胞存活率,观察甘木通总黄酮对神经元损伤的作用;形态学以及Hoechst 33258染色观察TFC对细胞形态的影响;生化法测定超氧化物歧化酶(SOD)活性以及丙二醛(MDA)的量。结果与200μmol/L H_2O_2诱导的模型组对比,中(10μg/m L)、高(100μg/m L)剂量甘木通总黄酮预处理细胞形态好于模型组,细胞活性明显增强(P0.05),细胞内的SOD活性增强(P0.05),MDA水平减低(P0.05)。结论甘木通总黄酮对H_2O_2引起的神经损伤有保护作用。