Neuroprotective Effects of Bushen Decoction Against Glutamate-Induced Neurotoxicity in PC12 Cells

Aim The enhanced effect of Bushen (Kidney-tonifying) decoction (BS) oncultured PC12 cell proliferation and its antagonistic action on neurotoxicity induced by glutamatewere investigated by serum pharmacological method of the Chinese material medica (CMM) in vitro.Methods The effect of BS on cultured PC12 cell activity and its antagonistic action on neurotoxicityinduced by glutamate was observed by MTT method. Flow cytometry and fluorescence microscopetechniques were employed to observe the antagonistic effect of BS on early period apoptosis of PC12cells induced by glutamate. Results The serum with BS was able to enhance activity of PC12 cells andexert antagonistic effect on glutamate-induced neurotoxicity. Meanwhile, these beneficial effectsproduced by BS were found to be the strongest in 20% concentration of in serum BS. Moreover, it caninhibit apoptosis of PC12 cells induced by glutamate , which occurs in the early period. ConclusionBS may exert a potential neuroprotective effect.

Protective effects of organic extracts of Alpinia oxyphylla against hydrogen peroxide-induced cvtotoxicitv in PC12 cells

Alpinia oxyphylla,a traditional herb,is widely used for its neuroprotective,antioxidant and memory-improving effects.However,the neuroprotective mechanisms of action of its active ingredients are unclear.In this study,we investigated the neuroprotective effects of various organic extracts of Alpinia oxyphylla on PC12 cells exposed to hydrogen peroxide-induced oxidative injury in vitro.Alpinia oxyphylla was extracted three times with 95%ethanol(representing extracts 1–3).The third 95%ethanol extract was dried and resuspended in water,and then extracted successively with petroleum ether,ethyl acetate and n-butanol(representing extracts 4–6).The cell counting kit-8 assay and microscopy were used to evaluate cell viability and observe the morphology of PC12 cells.The protective effect of the three ethanol extracts(at tested concentrations of 50,100 and 200μg/mL)against cytotoxicity to PC12 cells increased in a concentration-dependent manner.The ethyl acetate,petroleum ether and n-butanol extracts(each tested at 100,150 and 200μg/mL)had neuroprotective effects as well.The optimum effective concentration ranged from 50–200μg/mL,and the protective effect of the ethyl acetate extract was comparatively robust.These results demonstrate that organic extracts of Alpinia oxyphylla protect PC12 cells against apoptosis induced by hydrogen peroxide.Our findings should help identify the bioactive neuroprotective components in Alpinia oxyphylla.

Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations（between 1 × 10–10 M and 1 × 10–5 M） of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions（1 × 10–6 M β-asarone and eugenol）. The survival rates of PC12 cells significantly increased, while expression levels of the m RNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl m RNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

Effects of hypoxia, soman and their combination on PC12 cells

To investigate the effects of hypoxia, soman and their combined ones on PC12 cells. Methods: After the PC12 cells were exposed to an atmosphere containing different concentrations of oxygen and cultured in a medium containing different concentrations of soman, the amount of lactic dehydrogenase (LDH) released by the cells and their survival rate were determined to observe the dose-dependent and time-dependent cytotoxic effects. Student’s t test and two-way ANOVA were employed to determine the statistical differences and interaction between hypoxia and soman exposure. Results: 1) Both hypoxia and soman exposures exerted dose-dependent cytotoxic effects on PC12 cells and the interaction between the two injurious factors was significant; 2)The combined effects of the two factors were equal to the sum of those exerted by each one separately; and the combined application of the two factors resulted in a more severe cytotoxicity than that caused by either agent used singly; 3) The amount of LDH released from PC12 cells could serve as a more sensitive indicator of cytotoxicity than the survival rate of the cells. Conclusion: This study demonstrates the cytotoxic effects of the combined exposure to hypoxia and soman acted in a summative manner, which suggests that the two factors might induce intracellular release of LDH in PC12 cells through different mechanisms.

Neuroprotective Effects of Ginkgo Biloba Extract (GbE) on Oxygen-Glucose Deprivation (OGD) in PC12 Cells

Ischemic cerebrovascular disease is a global health problem. According to the World Health Organization, ischemic stroke is actually the most common cause of death in the world. Ginkgo biloba extract (GbE) is a traditional Chinese medicine for angina pectoris. Ginkgo biloba plays a role in expanding blood vessels, increasing coronary and cerebral blood flow, preventing platelet aggregation, inhibiting thrombosis, and improving the microcirculation. In the present study, we investigated the mechanisms involved in the neuroprotective effects of GbE in a model of hypoxic-ischemic brain disease. We used NGF(100 ng/ml for 6 days)and OGD(5% CO2and 95% N2, 1 mmol/l NaS2O4insugar-free DMEM for 16 h) to stimulate PC12 cells and convert them into neurons in order to establish an ischemia model. The results showed that PC12 cells transformed into cells that looked like neurons and that MAP2 was up-regulated in NGF-treated PC12 cells. Cell apoptosis was found to be up-regulated after NGF stimulation and OGD. The apoptosis rate after 16 hours of OGD was 19.44%. GbE (50ng/ml) reduces apoptosis rate to 11.35%. These results may help to show that NGF treatment can be combined with OGD to establish anin vitromodel of acute ischemic brain damage. In the present study, we find that GbE effectively increases the survival rate of PC12 cells and relieves OGD damage. These results suggest that GbE has the neuroprotective effects of ischemic brain damage.

Effect of insulin on 1-methyl-4-phenylpyridinium ion-induced apoptosis of PC12 cells

BACKGROUND： Insulin receptor （IR） expression in the substantia nigra of patients with Parkinson disease （PD） is not only significantly lower than that in the substantia nigra of normal persons of the same age, but also significantly lower than that in other regions in brain of himself/herself. It suggests that the abnormal effect of insulin receptor-mediated insulin, as a neurotrophic factor, is very possibly related to the loss of dopaminergic neurons in the substantia nigra and striatum in patients with Parkinson disease. OBJECTIVE ： TO observe the interventional effect of insulin on 1-methyl-4-phenylpyridinium ion （MPP^＋）-induced apoptosis of PC12. DESIGN： Controlled observation SETTINGS： Department of Neurology, Beijing China-Japan Friendship Hospital; Department of Neurology Huashan Hospital Affiliated to Fudan University. MATERIALS： PC12 cells were provided by the Cell Bank, Shanghai Institute of Cell Biology, Chinese Academy of Science. MPP^＋, MTT, HOECHST 33258 （Invitrogen Life Technologies）, reverse transcription-polymerase chain reaction （RT-PCR） reagent （Takara Shuzo Co., Ltd.）, flow cytometer （Bacton Dickionson, San Jose, CA）, enzyme labelling instrument （Bio-Tek, Winooski, VT） and PCR circulation instrument （Takara Shuzo Co., Ltd） were used in this study. METHODS ： This study was carried out in the Department of Neurology, Huashan Hospital Affiliated to Fudan University during June 2003 to August 2004. （1） Cell culture and experimental grouping： PC12 cells were cultured according to the method from Peng et al, then were randomized into 3 groups; blank control group, MPP^＋ group and insulin group. （2） Detection of relative survival rate of cells： The relative survival rate of cells at different MPP^＋ final concentrations （0, 50, 100, 200, 300, 1 000 μmol/L） and at different culture time （0, 4, 8, 12, 18, 24 hours） in the 300 Fmol/L MPP^＋ group and different concentrations of insulin （0, 15, 50, 100 nmol/L） in the insulin group was detected with MTT method according to the method from Hansen et al. （3） Observation of cell apoptosis： After stained by HOECHST 33258, the apoptotic cells were observed under the fluorescence miscroscope with the method from Chen et al. （4） Dection of apoptotic rate of cells： Apoptotic rate of cells was detected with flow cytometry according to the method from Zhang et al. （5） The expression of tyrosine hydroxylase （TH） mRNA in PC12 cells was detected with RT-PCR methods according to the modified method from Peng et al. MAIN OUTCOME MEASURES ： Comparison of relative survival rate, apoptosis rate, the expression of IR mRNA and TH mRNA and cell apoptosis. RESULTS： （1） After 12-hour incubation of 100, 200, 300 and 1 000 μmol/L MPP^＋, the relative survival rate of PC12 cells was （72.88±2.91）%, （60.64±0.81）%, （54.56±0.76）% and （16.89±2.83）%, respectively, which was significantly lower than that of blank control group （100%, P 〈 0.05）; After 12, 18 and 24-hour incubation, the relative survival rate of PC12 cells was （54.56±0.76）%, （42.43±0.16）% and （23.56±0.17）% respectively, which was significantly lower than that of blank control group （100%, P〈 0.05）; When 15, 50 and 100 nmol/L insulin was pre-added to cells, the relative survival rate was （70.10±0.16）%, （78.01 ±2.43）% and （83.55±1.43）%, respectively, which was significantly higher than MPP^＋ alone [（54.56±0.76）%, P 〈 0.05]. （2） Appototic bodies were rarely seen in the blank control group, but densely gathered in the MPP^＋ group and were significantly decreased in the insulin group. （3） Apoptosis rate of PC12 cells in the MPP^＋ group was significantly higher than that in the blank control group [（36.56±0.89）% vs. （2.34±0.23）%, P〈 0.05], and that in the 15, 50, 100 nmol/L insulin group [（30.01±0.04）%, （24.23±0.37）%, （20.01 ±1.01）%, respectivelyl was significantly lower than that in MPP^＋ group （P 〈 0.05）. （4） The TH mRNA expression in PC12 cells in MPP^＋ group was significantly lower than that in blank control group; The expression of TH mRNA in insulin group was gradually increased in an insulin dose-dependent manner. There were no significant changes in the expression of IR mRNA under different experimental conditions. CONCLUSION： Insulin can resist MPP^＋-induced apoptosis of PC12 cells, lessen the damage of PC12 cells, but does not change the gene expression of target cell insulin receptor.

Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.

The neuroprotective effect of walnut-derived peptides against glutamate-induced damage in PC12 cells: mechanism and bioavailability

In our previous study, defatted walnut meal hydrolysate(DWMH) could attenuate D-galactose-induced acute memory deficits in vivo, and six potent active peptides including WSREEQ, WSREEQE, WSREEQEREE, ADIYTE, ADIYTEEAG and ADIYTEEAGR were identified. The aim of this study was to investigate the possible mechanism underlying their neuroprotective effects on glutamate-induced apoptosis in PC12 cells and their digestive stability. Results showed that all these peptides could attenuate the reduction of cell viability caused by glutamate in PC12 cells, especially WSREEQEREE and ADIYTEEAGR. The addition of Arg residue in WSREEQEREE and ADIYTEEAGR might be the potential reason for their stronger protective effects. Additionally, these two peptides possibly protected PC12 cells against glutamate-induced apoptosis via activating intracellular antioxidant defence(superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px)) through Kelch-like ECH-associated protein 1(Keap1) inhibition, inhibiting ROS production, Ca;influx and mitochondrial membrane potential(MMP) collapse as well as regulating the expression of apoptosis-related proteins(Bax and Bcl-2). This might be due to the presence of Trp, Tyr and Arg in these two peptides. However, encapsulation of WSREEQEREE and ADIYTEEAGR should be considered based on their digestive sensibility during in vitro gastrointestinal digestion.

Mechanisms of rotenone-induced neurotoxicity in PC 12 cells

BACKGROUND： Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson＇s disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson＇s disease remains unclear. OBJECTIVE： To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING： A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS： PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS： PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco＇s modified eagle＇s medium （DMEM）, was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES： Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry （MALDI-TOF-MS） was used. RESULTS： In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition （P 〈 0.01）. Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition （P 〈 0.01）, as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition （P 〈 0.01）. By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION： γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.

Neuroprotective Effects of Sodium Ferulate and Its Antidepressant-Like Effect Measured by Acute and Chronic Experimental Methods in Animal Models of Depression

Antidepressants with novel targets and without side effects are in great demand. Ferulic acid (FA) is a ubiquitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have revealed that FA and SF show significant protective effect on excitotoxicity, we now test its potential neuroprotective and antidepressant-like effects. MTT assay and morphological analysis by fluorescence microscopy were adopted to measure the neuroprotective effects of SF;forced-swimming, tail-suspension, and chronic mild stress (CMS) tests were performed to assess its antidepressant-like activity. The results showed that SF had protection against H2O2-induced oxidative damage and dexamethasone (DXM)-induced neurotoxicity pheochromocytoma (PC12) cells. Acute administration of SF markedly decreased the duration of immobility during forced-swimming in rats and mice and tail-supension tests in mice. However, SF has no any effects on reserpine-induced hypothermia, 5-hydroxytryptophan-induced head-twitch response, and potentiation of noradrenaline toxicity in mice. Chronic administration of SF reversed the effects of CMS on consumption of food and sucrose solution, weight gain, and histopathology of hippocampus by light microscopy, and potently shortened the immobility time during forced-swimming test following CMS in rats. This study provides evidence that SF possesses obviously antidepressant-like activity, and the antidepressant-like effect may result from its neuroprotective effects.

A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways

The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san(SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT^(–1)3: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide(H_2O_2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways. The PC12 cells were exposed to H_2O_2(400 mmol·L^(–1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase(LDH), the intracellular content of malondialdehyde(MDA), the activities of superoxide dismutase(SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 μg·m L^(–1). SMXZF also inhibited H_2O_2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H_2O_2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H_2O_2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H_2O_2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

番茄红素对前列腺癌细胞PC-3增殖的影响

目的:观察番茄红素对体外培养的雄激素非依赖性前列腺癌细胞PC-3的的存活率、细胞周期及凋亡的影响。方法:采用MTT法观察番茄红素对癌细胞增殖的影响;流式细胞仪观察同步化的细胞经番茄红素作用后细胞周期及凋亡的变化。结果:番茄红素抑制PC-3细胞的增殖,1、2、4、8μmol/L最大抑制率分别为26.4%、40.7%、50.9%、60.7%,且诱导其凋亡,最大凋亡率达39.5%,并改变细胞周期分布。上述作用呈剂量效应关系。结论:番茄红素可诱导PC-3细胞凋亡、改变细胞周期分布,从而达到抑制肿瘤细胞增殖。

Neurogenesis-enhancing effect of sodium ferulate and its role in repair following stress-induced neuronal damage

Ferulic acid (FA) is a ubiquitous phenolic acid of low toxicity, and sodium ferulate (SF) is its sodium salt. Our previous studies have revealed that FA shows neuroprotective effect and significant antidepressant- like effect. The aim of this study was to investigate its potential neurogenesis-enhancing effect and its role in repair following stress-induced neuronal damage. MTT assay was performed to measure the effect of SF on the growth of rat pheochromocytoma (PC12) cells;morphological and immunocytochemical meth- ods were used for assessing its differentiation-induc- ing action. Chronic mild stress (CMS) tests were per- formed to establish rat model of depression. The histopathology of animal brains was studied to ana- lyze CMS-induced morphological changes and the effect of SF on the repair of CMS-induced brain in- jury. The expressions of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the proliferation of neural stem cell/neural progenitor cells were assessed in the hippocampi of chronic mild stress (CMS)-induced depression-like model rats by immunohistochemistry and bromodeoxyuridine (BrdU)- incorporation assays, respectively. Our in vitro tests showed that SF promoted the proliferation of PC12 cells in the concentration range of 5 - 320 μM, and induced PC12 cells to differentiate to more mature cells with the morphological characteristics and mo- lecular marker of neuronal-like cells. In vivo tests showed that SF up-regulated the expressions of NGF and BDNF, and induced the proliferation of neural stem cell/neural progenitor cells in the hippocampi of CMS-induced depression-like model rats. This study provides evidences that SF shows neurogenesis-en- hancing effect, and its antidepressant-like effect of SF may be related directly and closely to its above-men- tioned effect.

大鼠脊神经节感觉神经特异蛋白35kD(SSP-35)的纯化及鉴定(英文)

以大鼠脊髓背根神经节及背根纤维组织为材料 ,通过离子交换层析 ,FPLC凝胶过滤等技术分离纯化了脊感觉神经特异蛋白 35kD(SSP 35) .经SDS PAGE分析 ,该蛋白特异地存在于脊感觉神经而不存在于脊运动神经 .非还原电泳结果表明 ,该蛋白分子内含有巯基 ,有二聚体存在 .根据HPLC测定 ,蛋白纯度达 90 %以上 .将此蛋白给予培养的PC 1 2细胞 ,观察到它具有神经营养作用 .

补骨脂素对H_2O_2诱导PC-12细胞损伤的保护作用

目的探讨补骨脂素对H_2O_2诱导PC-12细胞氧化损伤的保护作用,并考察其作用机制。方法 PC-12细胞分为对照组、模型组、Tempol组和补骨脂素组。对照组中未加入补骨脂素和H_2O_2;模型组中加入400μmol/L H_2O_2;Tempol组中加入100μmol/L Tempol及400μmol/L H_2O_2;补骨脂素组中加入400μmol/L H_2O_2及1、5、10、20μmol/L补骨脂素。CCK-8法测定PC-12细胞的存活率,考察对PC-12细胞形态的影响,PI/Annexin-V、Hoechst染色检测细胞凋亡率,蛋白质印迹法检测凋亡蛋白表达。结果 5、10、20μmol/L补骨脂素均可以显著提高PC-12细胞内的吸光度值(P<0.01),细胞存活率提高较明显,与模型组比较差异具有统计学意义(P<0.05)。补骨脂素1、5、10、20μmol/L组凋亡率明显低于模型组(P<0.05),并且随着补骨脂素浓度的增加,PC-12细胞的凋亡率逐渐下降。随着补骨脂素作用时间的增加,各组PC-12细胞凋亡率的增加幅度相近,但明显低于模型组(P<0.05)。随着补骨脂素浓度的增加,对磷酸化Bad、Bcl-XL水平的上调作用越明显。结论补骨脂素对H_2O_2诱导PC-12细胞损伤具有保护作用,其作用机制是通过上调磷酸化Bad和Bcl-XL表达来抑制细胞凋亡。

丝裂原活化蛋白激酶对佛波酯诱导滋养细胞MMP-9基因表达的影响

目的探讨佛波酯(PMA)诱导细胞滋养层细胞(CTB)MMP-9基因表达的调控机制。方法用细胞ELISA法测定CTB细胞的蛋白激酶活性变化;用反转录聚合酶链反应检测CTB中MMP-9的基因表达。结果100 nmol/L PMA能迅速激活CTB中丝裂原活化蛋白激酶(MAPK)家族中细胞外信号调节蛋白激酶(ERK)、c-jun氨基末端激酶(JNK)以及p38 MAPK激酶的活性。100 nmol/L PMA刺激CTB引起MMP-9 mRNA表达显著增加,能被ERK或p38 MAPK的特异性抑制剂所抑制。结论ERK和p38 MAPK可能是PMA诱导CTB中MMP-9基因表达增加的重要调节物质。

槲皮素衍生物HPS5对酒精诱导的PC12细胞氧化损伤的保护作用

目的探讨槲皮素衍生物HPS5对酒精导致的PC12细胞氧化损伤的保护作用。方法酒精诱导PC12细胞建立模型,MTT法检测细胞的抑制率,比较治疗后乳酸脱氢酶(lactate dehydrogenase,LDH)活性,以及丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH)的水平,Western-Blot检测Bax和Bcl-2蛋白表达水平。结果与模型组比较,25、50、100 mmol/L HPS5能显著下调酒精对PC12细胞增殖的抑制作用[(28.87±2.34)%、(16.49±2.13)%、(10.31±1.17)%],减少LDH的漏出[(36.47±3.29)、(31.67±3.25)、(29.34±2.87)U/L],降低MDA水平[(1.89±0.14)、(1.61±0.51)、(1.48±0.41)nmol/mg],升高GSH[(81.25±8.33)、(92.14±9.34)、(96.24±11.24)mg/g]、SOD[(3.26±0.69)、(4.17±0.68)、(4.24±0.71)NU/mg]的水平,下调Bax蛋白[(0.31±0.03)、(0.29±0.03)、(0.27±0.03)]表达,升高Bcl-2蛋白[(0.34±0.01)、(0.31±0.02)、(0.28±0.02)]表达水平。尤其中高剂量组更加明显,与模型组比较,差异均有统计学意义(P<0.05)。结论 HPS5对酒精诱导的PC12细胞损伤具有保护作用,增强细胞的抗氧化能力以及上调Bcl-2和下调Bax蛋白的表达可能是其作用的机制之一。

栀子豉汤提取物对谷氨酸诱导PC 12细胞损伤的保护作用

目的研究栀子豉汤提取物对谷氨酸诱导PC 12细胞损伤的保护作用。方法通过谷氨酸诱导PC 12细胞建立细胞损伤模型,采用四甲基偶氮唑蓝(MTT)比色法检测细胞存活率;化学检测法测定乳酸脱氢酶(LDH)释放量;Annexin V-FITC/propidium iodide(PI)双染法流式细胞术(flow cytometry,FCM)检测细胞凋亡率;流式细胞仪检测PC 12细胞内活性氧(ROS)水平,酶标仪检测超氧化物歧化酶(SOD)和谷胱甘肽(GSH)的含量,初步探讨栀子豉汤提取物的抗氧化机制。结果栀子豉汤可显著降低由谷氨酸诱导的细胞损伤,提高细胞存活率,减少LDH释放量,增加SOD、GSH活性,抑制ROS的生成。结论栀子豉汤提取物具有减轻由谷氨酸诱导的PC 12细胞损伤的作用,其机制可能与抗氧化应激损伤有关。

Facile fabrication of drug-loaded PEGDA microcapsules for drug evaluation using droplet-based microchip

Droplet-based microfluidic technology can be utilized as a microreactor to prepare novel functional monodisperse microcapsules.In this study,a droplet-based microfluidic chip with surface modification,which allowed the one-step preparation of double emulsion microcapsules.An O/W/O double emulsion using polyethylene(glycol)diacrylate(PEGDA)solution as the intermediate water phase was prepared by regulating the hydrophilicity and hydrophobicity of the chip surface,with PEGDA microcapsules prepared using UV polymerization.And then anti-tumor drug paclitaxel and neurotoxin 6-OHDA were encapsulated in microcapsules for drug and toxicology evaluation,respectively.Compared to controls,drug-loaded mi-crocapsules caused a significant increase in the death rate of PC12 cells.This indicates that the obtained drug-loaded microcapsules could be used in drug evaluation and potentially in drug screening and deliv-ery.

雷公藤甲素自微乳对荷人前列腺癌裸鼠移植瘤的抑制作用研究

目的研究雷公藤甲素自微乳对荷人前列腺癌PC-3细胞裸鼠移植瘤的抑制作用。方法构建荷人前列腺癌PC-3细胞裸鼠移植瘤,分为模型组、雷公藤甲素组、雷公藤甲素自微乳组、空白辅料组,灌胃给药,每2天1次,连续18 d,每3天测定瘤体积;给药结束后处死裸鼠,比较各组裸鼠的体质量、肝脏与瘤块质量,计算抑瘤率,并对瘤块组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)进行免疫组化研究。结果与模型组相比,雷公藤甲素和雷公藤甲素自微乳均可抑制肿瘤体积的增长;与雷公藤甲素相比,雷公藤甲素自微乳可以提高抑瘤率,降低肿瘤组织中VEGF的表达水平,对肿瘤抑制作用更佳,且未发现明显的毒性反应;空白辅料组对肿瘤无抑制作用。结论自微乳给药系统可以提高雷公藤甲素的抗肿瘤作用,安全性良好。