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.

Effects of phycocyanin on apoptosis and expression of superoxide dismutase in cerebral ischemia reperfusion injury

BACKGROUND ： The application of exogenous antioxidant is always the focus in the prevention and treatment of cerebral ischemia. Phycocyanin has the effects against oxidation and inflammation, but its role in the pathophysiological process of cerebral ischemia reperfusion injury still needs further investigation. OBJECTIVE： To observe the effects of phycocyanin on the expression of superoxide dismutase （SOD） apoptosis and form of the nerve cells in rats after cerebral ischemia reperfusion injury. DESIGN： A randomized control animal experiment SETTING ： Institute of Cerebrovascular Disease, Medical School Hospital of Qingdao University MATERIALS： Fifty-two healthy adult male Wistar rats of clean degree, weighing 220-260 g, were used. Phycocyanin was provided by the Institute of Oceanology, Chinese Academy of Sciences. METHODS： The experiments were carried out in Shangdong Key Laboratory for Prevention and Treatment of Brain Diseases from May to December 2005. ① All the rats were divided into three groups according to the method of random number table： sham-operated group （n=4）, control group （n=24） and treatment group （n=24）. Models of middle cerebral artery occlusion/reperfusion （MCAO/R） were established by the introduction of thread through external and internal carotid arteries in the control group and treatment group. After 1-hour ischemia and 2-hour reperfusion, rats in the treatment group were administrated with gastric perfusion of phy- cocyanin suspension （0.1 mg/g）, and those in the control group were given saline of the same volume, and no treatment was given to the rats in the sham-operated group. ②The samples were removed and observed at ischemia for 1 hour and reperfusion for 6 and 12 hours and 1, 3, 7 and 14 days respectively in the control group and treatment group, 4 rats for each time point, and those were removed at 1 day postoperatively in the sham-operated group. Forms of the nerve cells were observed with toluidine blue staining. Apoptosis after cerebral ischemia reperfusion was detected with TUNEL technique. SOD expression was detected with immunohistochemical technique.③ The intergroup difference was compared with the ttest. MAIN OUTCOME MEASURES： The apoptosis of the nerve cells and SOD expression were mainly observed in each group. RESULTS： Finally, 52 rats were involved in the analysis of results. ① Number of apoptotic cells： In the sham-operated group, a few apoptotic cells could be observed in brain tissue. The apoptotic cells at each time point in the control group and treatment group were obviously more than those in the sham-operated group （P 〈 0.05）. In the treatment group, the numbers of apoptotic cells at 12 hours, 1 and 3 days after reperfusion were significantly fewer than those in the control group, and those at 6 hours, 7 and 14 days were similar to those in the control group. ② Number of SOD positive cells： In the sham-operated group, there was weak expression of SOD in brain tissue, and the positive cells were extremely few, the positive cells at each time point were significantly more in the control group and treatment group than in the sham-operated group （P 〈 0.05）. In the treatment group, the numbers of positive cells at 6 and 12 hours, 1 and 3 days after reperfusion were significantly fewer than those in the control group, and those at 7-14 days were similar to those in the control group. ③ Cellular forms： In the control group, the karyopyknosis occurred in the nerve cells, which were irregularly distributed, nucleolus disappeared, and some scattered cell fragments were observed. The forms of the nerve cells in the treatment group were generally normal. CONCLUSION ： Phycocyanin plays a neuroprotective role in cerebral ischemia reperfusion injury by activating the SOD expression and inhibiting apoptosis.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Effect of ketamine on aquaporin-4 expression and neuronal apoptosis in brain tissues following brain injury in rats

BACKGROUND： Aquaporin-4 （AQP-4） is closely related to the formation of brain edema. Neuronal apoptosis plays an important part in the conversion of swelled neuron following traumatic brain injury. At present, the studies on the protective effect of ketamine on brain have involved in its effect on aquaporin-4 expression and neuronal apoptosis in the brain tissues following brain injury in rats. OBJECTIVE： To observe the effect of ketamine on AQP-4 expression and neuronal apoptosis in the brain tissue following rat brain injury, and analyze the time-dependence of ketamine in the treatment of brain injury.DESIGN： Randomized grouping design, controlled animal tria SETTING ： Department of Anesthesiology, the Medical School Hospital of Qingdao University MATERIALS： Totally 150 rats of clean grade, aged 3 months, were involved and randomized into control group and ketamine-treated group, with 75 rats in each. Each group was divided into 5 subgroups separately at 6,12, 24, 48 and 72 hours after injury, with 15 rats at each time point. Main instruments and reagents： homemade beat machine, ketamine hydrochloride （Hengrui Pharmaceutical Factory, Jiangsu）, rabbit anti-rat AQP-4 polyclonal antibody, SABC immunohistochemical reagent kit and TUNEL reagent kit （Boster Co.,Ltd., Wuhan）. METHODS： This trial was carried out in the Institute of Cerebrovascular Disease, Medical College of Qingdao University during March 2005 to February 2006. A weight-dropping rat model of brain injury was created with Feeney method. The rats in the ketamine-treated group were intraperitoneally administered with 50 g/L ketamine （120 mg/kg） one hour after injury, but ketamine was replaced by normal saline in the control group. In each subgroup, the water content of cerebral hemisphere was measured in 5 rats chosen randomly. The left 10 rats in each subgroup were transcardiacally perfused with ketamine, then the brain tissue was made into paraffin sections and stained by haematoxylin and eosin. Neuronal morphology was observed. AQP-4 expression and neuronal apoptosis were measured with immunohistochemical method and TUNEL method respectively. MAIN OUTCOME MEASURES： Water content in brain tissue, neuronal morphology, the number of AQP-4 positive neurons and TUNEL positive neurons in rats of two groups at each time point after injury. RESULTS： Totally 150 rats entered the stage of result analysis. （1） Water content of brain tissue： The water content of brain tissue at each time point after injury in the ketamine-treated group was lower than that in the control group. There were very significant differences in water content at 12 and 24 hours after injury respectively between ketamine-treated group and control group [（77.34±2.35）% vs. （82.31 ±1.48）%; （78.01 ±2.21 ）% vs. （83.86±2.37）%, t=-4.001 6,4.036 7, both P 〈 0.01]. （2） Neuronal morphology： Pathological changes in traumatic region and peripheral region of injury in the ketamine-treated group were significantly lessened, and necrotic and apoptotic cells in the ketamine-treated group were also significantly reduced as compared with control group. （3） AQP-4 expression： AQP-4 positive neurons at each time point in the ketamine-treated group were significantly less than those in the control group. There were very significant differences in AQP-4 expression at 12 and 24 hours after injury between ketamine-treated group and control group [（34.17±4.74） /visual field vs. （43.42±5.65） /visual field;（40.83±3.17） /visual field vs. （58.88±6.23） /visual field,t=3.966 3,8.165 7, both P〈 0.01]. （4） Neuronal apoptosis： TUNEL positive neurons at each time point in the ketamine-treated group were less than those in the control group. There were very significant differences in the neuronal apoptosis at 12 and 24 hours after injury between ketamine-treated group and control group [（26.25±3.04） /visual field vs. （32.75±4.39） /visual field; （29.33± 4.02） /visual field vs. （39.83±5.61） /visual field,t=-3.849 3,5.169 2,both P 〈 0.01]. CONCLUSION： Ketamine can reduce brain edema, AQP-4 expression and neuronal apoptosis following brain injury in rats, and has obvious therapeutic effect on brain injury, especially at 12 and 24 hours after injury.

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin ( AM ) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group,IRI group, empty plasmid group and AM group. One week after re-

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Effect of Trichosanthin on Cell Cycle and Apoptosis of Murine Melanoma Cells

Objective:To study thei nhibitory effect of purified trichosanthin component on the proliferation of malignant melanoma.Methods:The effect of purified trichosanthin componenton the DNA synthesis,cell cycle and cell apoptos is of murinemelanomacellsweredetected by flow cytometry when culture dinvitro.Results:The significantG0/G1phasearrest was revealed by the inc rease of cellsinG0/G1 phase and decrease of cell sinS phase.The obviousapoptosis of melanoma cells was in duced bypuri fied trichos anthin component.G0/G1phase arrest was high lycorrelated withapop to sis(r=0.8705).Conclusion:The purified trichos anthin component can markedly inhibit melanoma cells by the suppression of DNA syn the sisin S phaseand cell mitosisas well as induction of cell apoptosis.

Effects of arotinoid acid on induction of apoptosis and differentiation and telomerase activity and cell cycle in the HL 60 cell line

Objective To investigate the effects of arotinoid acid (Ro13 7410) on the morphological and functional alterations of leukemia HL 60 cell line and compared with those of RA Methods Differentiation of HL 60 cells was assessed by morphology and by NBT reduction Trypan blue exclusion was used to determine viability Apoptosis was assessed by changes in cell morphology and by measurement of fragmented DNA using the PCD assay kit Telomerase PCR ELISA kit tested telomerase activity The cell cycle was analyzed by flow cytometry Results Incubation of the HL 60 cells with 10 -6 10 -8 ?mol/L Ro13 7410 resulted in suppression of cell growth Apoptotic cells were detected following exposure to 10 -6 ?mol/LRo13 7410 for 3 hours by measurement of the “in situ” enzymatic labeling of DNA breaks with biotinylated dUTP Ultrastructural examination of Ro13 7410 treated samples showed cells with chromatin compaction and cytoplasm condensation and the presence of “apoptotic bodies” Cells induced into apoptosis were accompanied by Department of Hematology, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China (Liu XS, Lou LS, Zeng SR and Tang ZH) Department of Clinical Biochemistry, Chongqing University of Medical Sciences, Chongqing 400046, China (Jiang JK, Zhang Y, Xu XG, Liu BZ, He YJ and Kang GF) increase of intracellular free Ca 2+ Percentage of HL 60 cells reduced NBT following incubation with Ro13 7410 was lower than with all trans retinoic acid (RA) (27% vas 85%) Telomerase PCR ELISA assay showed that HL 60 cells cultured in the absence of inducing agents had significant telomerase activity Telomerase activity declined gradually after 10 -6 ?mol/L Ro13 7410 treatment, and changes becoming evident at 1 day The inhibition of telomerase activity at day 5 of treatment with Ro13 7410 was less effective than with RA DNA flow cytofluorimetric analysis revealed that Ro13 7410 caused partial cell arrest in the G 2/M phase after a 2 day treatment and the percentage of cells arrested in the G 2/M phase increased after 4 days treatment With RA treated cells, a reduction in the percentage of cells in the G 2/M phase was observed after 2 day of treatment Conclusion Our study shows that Ro13 7410 suppresses HL 60 cells growth mainly via the induction of apoptosis and is less effective than RA in induction differentiation Ro13 7410 dramatically inhibits telomerase activity during the course of induction and results in G 2/M arrest within 2 days These findings suggest that Ro13 7410 is worthy of further study for its effects on leukemic cells

Particulate matter(PM(2.5)) exposure season-dependently induces neuronal apoptosis and synaptic injuries

Epidemiological studies have shown that particulate matter 2.5（PM（2.5）） not only increases the incidence of cardiopulmonary illnesses but also relates to the development of neurodegenerative diseases. Considering that PM（2.5）is highly heterogeneous with regional disparity and seasonal variation, we investigated whether PM（2.5）exposure induced neuronal apoptosis and synaptic injuries in a season-dependent manner. The results indicated that PM（2.5）altered the expression of apoptosis-related proteins（mainly bax and bcl-2）, activated caspase-3 and caused neuronal apoptosis. Additionally, PM（2.5）decreased the levels of synaptic structural protein postsynaptic density（PSD-95） and synaptic functional protein N-methyl-D-aspartate（NMDA） receptor subunit（NR2B） expression. These effects occurred in a season-dependent manner, and PM（2.5）collected from the winter showed the strongest changes. Furthermore, the effect was coupled with the inhibition of phosphorylated extracellular signal-regulated kinase 1/2（p-ERK1/2） and phosphorylated c AMP-response element binding protein（p-CREB）. Based on the findings, we analyzed the correlations between the chemical composition of PM（2.5）samples and the biological effects, and confirmed that winter PM（2.5）played a major role in causing neuronal apoptosis and synaptic injuries among different season samples.