Effects of astragaloside Ⅳ on apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation / 中国药理学通报

Aim To investigate the effects of astragalo-side IV on apoptosis of PC12 cells inducedby hypoxia/hypoglycemia and reoxygenation. Metheds PC12 cells were randomly divided into 4 groups: normal control group,hypoxia/hypoglycemia and reoxygenation group, astragaloside Ⅳ group and vehicle group. Hypoxia/hy-poglycemia and reoxygenation group, astragaloside Ⅳgroup and vehiclegroup were exposed to reoxygenation (12 h) after 3 h of oxygen and glucose deprivation, and astragaloside Ⅳ was added into cells at the same time. Inverted microscope was used to observe the morphological changes ofPC12 cells and MTT method to detect the activities of PC12 cells, and Annexin V-FITC/PI assay and TUNEL staining were used to meas-ure the apoptosis of PC12 cells. Results Compared with normal control group, cells became round or swol-len and its cellula processes were retracted or disap-peared in hypoxia/hypoglycemia and reoxygenation group;a large number of apoptotic cells could also be observed,whose nucleus were shrinkaged, fragmented or deep-stained. The activities of hypoxia/hypoglycemia and reoxygenation group were decreased markedly than those in normalcontrol group(P0. 05 ) . Conclusion Astragaloside IV can reduce the damage of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation, increase cell activity and inhibit cell apoptosis.

Evaluation of Bronchiolar and Alveolar Cell Injuries Induced by Short- and Long-term Exposure to Sidestream Smoke

BACKGROUND: We investigated effects of short- and long-term exposure to sidestream smoke on the bronchiolar and alveolar cells in Sprague-Dawley rats. METHODS: Rats were divided into five experimental groups: groups 1, 2, and 3 (1-month exposure to 3, 5, and 7 cigarettes a day, respectively), groups 4 and 5 (3- and 6 month exposure to five cigarettes a day, respectively). We examined the morphologic changes, the expressions of tumor necrosis factor alpha (TNF-alpha), tumor growth factor beta1 (TGF-beta1), interlekin (IL)-1alpha, IL-1beta, Ki-67, and cytokeratin 14 and in situ apoptosis in the bronchiolar and alveolar cells on light microscopy (LM) and electron microscopic (EM) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: LM showed the respiratory bronchiolar dilatation and alveolar wall collapse. In groups 3, 4, and 5, EM showed loss of the cilia and Clara cells with irregular size, more prominent alveolar wall collapse and dilation of alveolar duct than those of groups 1 and 2. Bronchiolar and alveolar cells showed increased expressions of TNF-alpha and TGF-beta in groups 4 and 5. LM and EM TUNEL stains showed increased apoptosis in groups 3, 4, and 5. CONCLUSIONS: Sidestream smoke causes a bronchiolar and alveolar cell injury and the severity correlates strongly the volume and duration of exposure to sidestream smoke.

p73 activation is involved in regulation of Ara-C-induced apoptosis in human lung adenocarcinoma cells / 解剖学报

Objective To study the apoptosis pathway of human lung adenocarcinoma cell line A549 induced by 1-β-D-arabinofuranosylcytosine (Ara-C) in vitro. Methods A549 cells were incubated with Ara-C for 72hours in vitro. Biological changes of apoptotic cells were studied by TUNEL staining. Morphological changes of the A549 cells treated with Ara-C were observed by transmission electron microscope. The expressions of p53 and p73 were investigated by Western blotting. Results 1.Apoptotic rates of A549 cells exposure to Ara-C studied by TUNEL staining were higher than that of the control (P＜0.01). 2.Apoptosis body was apparently observed by transmission electron microscope. 3.Endogenous p73 but not p53 was induced and activated in dose-dependent manner upon Ara-C treatment by Western blotting.Conclusion Ara-C can effectively induce apoptosis of A549 cells. DNA damage-induced apoptosis of A549 cells treated by Ara-C is independent of functional p53.Up-regulation of p73 may play an important role that enhances the sensitivity of A549 cells to Ara-C and be partly responsible for p53-independent apoptosis.

Effect on Varying the Impact Velocity in the Controlled Cortical Impact Injury Model: Injury Severity and Impact Velocity

OBJECTIVE: A study of the histopathologic and neurobehavioral correlates of cortical impact injury produced by increasing impact velocity using the controlled cortical impact(CCI) injury model is studied. METHODS: Twenty-four Sprague-Dawley rats (200~250g) were given CCI injury using a pneumatically driven piston. Effect of impact velocity on a 3mm deformation was assessed at 2.5m/sec (n=6), 3.0m/sec (n=6), 3.5m/sec (n=6), and no injury (n=6). After postoperative 24hours the rats were evaluated using several neurobehavioral tests including the rotarod test, beam-balance performance, and postural reflex test. Contusion volume and histopathologic findings were evaluated for each of the impact velocities. RESULTS: On the rotarod test, all the injured rats exhibited a significant difference compared to the sham-operated rats and increased velocity correlated with increased deficit (P<0.001). Contusion volume increased with increasing impact velocity. For the 2.5, 3.0, and 3.5m/sec groups, injured volumes were 18.8+/-2.3mm3, 26.8+/-3.1mm3, and 32.5+/-3.5mm3, respectively. In addition, neuronal loss in the hippocampal sub-region increased with increasing impact velocity. In the TUNEL staining, all the injured groups exhibited definitely positive cells at pericontusional area. However, there were no significant differences in the number of positive cells among the injured groups. CONCLUSION: Cortical impact velocity is a critical parameter in producing cortical contusion. Severity of cortical injury is proportional to increasing impact velocity of cortical injury.

Placental apoptotic change in 2nd trimester and full-term of normal pregnancies / 대한산부인과학회잡지

OBJECTIVE: The study aims were to demonstrate apoptosis in the placenta of normal pregnancy, and to identify its change and quantify its incidence by gestational age. METHODS: Placenta samples were collected from 25 normal full-term pregnancies and 20 second trimester pregnancies undergoing termination due to medical and social reasons. Hematoxylin and eosin staining and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) staining were used to quantify the incidence of apoptosis and the electron microscopy was used to confirm it. Mann-Whitney U test and ANOVA test were used for statistical analysis. RESULTS: 1. Apoptosis was demonstrated by variable cytopathologic methods, and especially TUNEL staining and electron microscopy are found to be confirmatory methods. 2. In TUNEL staining, quantification of apoptosis was as follows: 2nd trimester (n=20) 1.05+/-0.69, full- term (n=25) 1.92+/-1.00. The incidence of apoptosis was significantly higher in full-term than in 2nd trimester (p0.05, Mann-Whitney U test). 4. There was no statistical significance in the incidence of apoptosis by maternal age, parity, cause of termination during 2nd trimester, and mode of delivery in each group. 5. In the electron microscopy, apoptotic cells were observed to have membrane blebbing, loss of microvilli, chromatin condensation and localization in the border of nuclear membrane, and cell shrinkage and increase in granularity. This method was conformatory in identifying apoptosis. CONCLUSION: Placental apoptosis increased significantly with increased gestational age, and this result suggests that it may play a role in the normal development and aging of the placenta.

A Comparative Study for Apoptosis on the Degree of the Amount of Photorefractive Ablation in Photorefractive Keratectomy

PURPOSE: This study was aimed to evaluate changes in the stromal keratocyte after ablation of 50 micrometer and 100 micrometer with use of photorefractive keratectomy(PRK). METHODS: At 4 hours, 24 hours, 72 hours, 7 days and 1 month after PRK, each group of rabbits including normal control group was treated with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling(TUNEL) staining using ApopTag(R) kit in vivo, then apoptotic keratocytes were evaluated with light microscope. RESULTS: There was no response with TUNEL staining of the epithelial cells, stromal keratocyte, and endothelium in normal cornea. In the ablation group, however, regardless of the depth of photorefractive ablation, the TUNEL signal was maximal after 4 hours, and it decreased with time. The signal was more intense in 100 micrometer ablation group than 50 micrometer ablation group, although the signal was not observed at the endothelial cells in both groups. The number of apoptotic stromal keratocytes at each time point of 4 hr, 24 hr, 72 hr, and 1 week was 57+/-8.9, 49+/-7.5, 36+/-5.1, and 12+/-1.3 cells/field in 100 micrometer ablation group, and 31+/-4.4, 28+/-4.6, 21+/-3.9, and 5+/-1.1 cells/field in 50 micrometer ablation group, and the difference between the two groups was statistically significant(P<0.05). CONCLUSION: The more the amount of ablation with photorefractive keratectomy, the stronger the apoptotic response. The postoperative apoptotic response was observed especially within 1 week. These findings suggest that early suppression of postoperative apoptosis within 1 week will influence on the prognosis of visual quality after photorefractive keratectomy, and more studies will be needed in the future.

Apoptotic Cell Death in Experimental Retinal Vein Occlusion

We used an animal model of laser-induced retinal vein occlusion to study the temporal and spatial patterns of neuronal necrosis and apoptosis. After photodynamic retinal vein thrombosis with argon-green laser, rats were sacrificed at 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and 14 days after vein occlusion. The temporal and spatial patterns of neuronal cell death were determined using light and electron microscopy, TdT-dUTP nick-end labeling[TUNEL]and DNA gel electrophoresis. The cells in retinal ganglion cell layer and inner nuclear layer showed both necrotic and apoptotic changes 12 hours after vein occlusion. The TUNEL positivity were detected at day 1 after vein occlusion and the number of positive cell increased until day 2, and decreased thereafter. DNA ladder pattern was observed 48 hours after vein occlusion by DNA gel electrophoreisis. These data demonstrated that retinal vein occlusion induces necrosis and apoptosis in the retinal ganglion cell and inner nuclear layer.

Apoptotic Cell Death in Experimental Retinal Vein Occlusion

We used an animal model of laser-induced retinal vein occlusion to study the temporal and spatial patterns of neuronal necrosis and apoptosis. After photodynamic retinal vein thrombosis with argon-green laser, rats were sacrificed at 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and 14 days after vein occlusion. The temporal and spatial patterns of neuronal cell death were determined using light and electron microscopy, TdT-dUTP nick-end labeling[TUNEL]and DNA gel electrophoresis. The cells in retinal ganglion cell layer and inner nuclear layer showed both necrotic and apoptotic changes 12 hours after vein occlusion. The TUNEL positivity were detected at day 1 after vein occlusion and the number of positive cell increased until day 2, and decreased thereafter. DNA ladder pattern was observed 48 hours after vein occlusion by DNA gel electrophoreisis. These data demonstrated that retinal vein occlusion induces necrosis and apoptosis in the retinal ganglion cell and inner nuclear layer.

STUDY ON PROLIFERATION AND APOPTOSIS IN DEVELOPING RENAL CORPUSCLES OF MOUSE / 解剖学报

Objective To study cell proliferation and apoptosis of renal corpuscle in normal developing mouse.Methods PCNA immunohistochemisty method,terminal deoxynucleotidyl transferase mediated dUTP-biotin nick endlabeling(TUNEL staining method)and light,electron microscopy were used to observe proliferation and apoptosis within corpuscle at different stages of development.Results Nearly all cells in Ⅰ and Ⅱ stages had nuclear PCNA immunolabeling.The number of PCNA immunolabeling cells during Ⅲ and Ⅳ stages was gradually decreased,and to less in Ⅴ stage.TUNEL positive cells could be easily observed before birth and identified in Ⅲ and Ⅳ stages.Electron microscope revealed that the mitotic activity was observed in all cells of Ⅰ and Ⅱ stages renal corpuscles.The mitotic phases of endothelial cells and podocytes could be seen in Ⅲ stage,but only endothelial mitotic activities in Ⅳ stage of renal corpuscle.Apoptotic activities of endothelial cell and Bowman's parietal cell were noted,predominantly in Ⅲ and Ⅳ stages.Conclusion Proliferation and apoptosis exist universally in the course of developing renal corpuscle,and supervise and control the regular development of renal corpuscle cooperatively.