AIM To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms. MLETHODS In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA,RNA and protein were analyzed by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine respectively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis.RESULTS Taxol was effective against SMMC 7721 human hepetoma cell growth in the ranges of 2.5 nmol/L - 10 nmol/L with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72 h. Taxol at 2.5 nmol/L reduced 3H-thymidine uptake to about 34% of the control value (P＜0.05). Increasing the dose of taxol to 20 nmol/L resulted in a greater decrease in 3Hthymidine incorporation to 60% of the control value (P＜0.01). At a concentration of 20 nmol/L, the 3H-uridine and 3H-leucine uptakes were reduced to 52% (P＜0.05) and 63%(P＜0.01), respectively. In vivo, taxol significantly inhibited SMMC-7721 tumor growth at 10 mg/kg, i.p., once daily for 10 d. A more than 90% decrease in tumor volume was observed by day 11 (P＜0.01) similarly with mitotic arrest and cell apoptosis.CONCLUSION Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently,apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol.

AIM Several triggering receptors have been described to be involved in natural killer (NK) cellmediated target cytotoxicity. In these studies, NK cells derived from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK-triggering molecules NKR-P1A, ANK61 antigen, and CD45 in pit cell-mediated killing of target cells. METHODS 51 Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. RESULTS Flow cytometric analysis showed that pit cells expressed CD45, NKR-P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody ( mAb ) to CD45 ( ANK74 ) not only inhibited CC531s or YAC-1 target lysis but also apoptosis induced by pit cells. The mAbs to NKRP1A (3.2.3) and ANK61 antigen (ANK61) had no effect on pit cell-mediated CC531s or YAC-1 target cytolysis or apoptosis, while they did increase the Fcγ receptor positive (FcγR+) P815 cytolysis and apoptosis. This enhanced cytotoxicity could he inhibited by 3,4-dichloroisocoumarin, an inhibitor of granzymes. CONCLUSION These results indicate that CD45 participates in pit cell-mediated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against FcγR+ P815 cells, which was mediated by the perforin/granzyme pathway.

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

AIM To evaluale the potential role of P-selectin and anti-P-selectin monoclonal antibody (mAb) in apoptosis during hepatic/renal ischemiareperfusion injury.METHODS Plasma P-selectin level, hepatic/renal P-selectin expression and cell apoptosis were detected in rat model of hepatic/ renal ischemia-reperfusion injury. ELISA, immunohistochemistry and TUNEL were used. Some ischemia-reperfusion rats were treated with antiP-selectin mAb.RESULTS Hepatic/ renal function insufficiency, up-regulated expression of P-selectin in plasma and hepatic/renal tissue, hepatic/renal histopathological damages and cell apoptosis were found in rats with hepatic/renal ischemiareperfusion injury, while these changes became less conspicuous in animals treated with anti-Pselectin mAb.CONCLUSION P-selectin might mediate neutrophil infiltration and cell apoptosis and contribute to hepatic/renal ischemia-reperfusion injury, anti-P-selectin mAb might be an efficient approach for the prevention and treatment of hepatic/renal ischemia-reperfusion injury.

AIM To investigate the clinical features of FADD and TRADD expressions in primary hepatocellular carcinoma ( HCC ) and to determine their relationship with hepatic apoptosis.METHODS FADD and TRADD expressions were detected by immunohistochemistry and hepatic apoptosis were determined by in situ endlabeling ( ISEL).RESULTS Ten (25.6%) cases of HCC were detected to express FADD protein. The positive rate in HCC is lower than that in non-cancerous adjacent liver tissues (62.5%) (P＜0.05). In those of grade Ⅰ - Ⅱ, 8 (38.1%) cases were FADD positive, while only 2/18 (11. 1%) cases of grade Ⅲ - Ⅳ had detectable FADD protein (P＜0.05). No relationship was found between FADD expression and other clinical features,such as gender, age, tumor size, differentiation or metastasis. ISEL positive cells can be seen in all cases of HCC. The hepatic apoptosis was associated with FADD expression as more apoptotic cells were detected in those cases which had moderately to strongly positive FADD, as compared with negative or weak positive FADD cases (P＜ 0.05). No relationship was found between FADD expression and hepatic apoptosis in non-cancerous adjacent liver tissues. Fifteen of 39 (38.5%) cases of HCC were found positive for TRADD protein, and similar positive rate (37.5%) in non-cancerous adjacent liver tissues (P ＞0.05). The expression of TRADD is correlated with HCC differentiation,as only 22.2% of moderately to highly differentiated HCC showed positive TRADD protein, while as high as 52.4% of poorly differentiated HCC had TRADD (P＜0.05). No relationship was found between TRADD expression and gender, age, tumor size or grade or metastasis, although 42.9% of HCC of grade Ⅰ/Ⅱ showed positive TRADD which was slightly higher than that of grade Ⅲ/Ⅳ (33.3%,P ＞ 0.05). Hepatic apoptosis was not related to TRADD expression in HCC or non-cancerous adjacent liver tissues.CONCLUSION Loss of FADD expression plays an important role in HCC carcinogenesis, and expression of TRADD also contributes to HCC development. The cell apoptosis in HCC is associated with FADD expression. However, the expression of TRADD does not correlate well with hepatic apoptosis in HCC.

AIM To study the effects of arsenic trioxide and HCPT on different degrees of differentiated gastric cancer cells (SGC-7901, MKN-45, MKN-28)with respect to both cytotoxicity and induction of apoptosis in vitro. ~ODS The cytotoxicity of As2O3 and HCPT on gastric cancer cells was determined by MTTassay. Morphologic changes of apoptosis of gastric cancer cells were observed by light microscopy and transmission electron microscopy. Apoptosis and cell cycle changes of gastric cancer cells induced by HCPT and As2O3 were investigated by TUNEL method and flow cytometry. RESULTS As2O3 and HCPT had remarkable cytotoxic effects on different degrees of differentiated gastric cancer cells. The IC50 of As2O3 on well differentiated gastric cancer cell MKN-28, moderately differentiated gastric cancer cell SGC-7901, and poorly differentiated gastric cancer cell MKN-28 were 8. 91 μmol/L, 10. 57 μmol/L, and 11.65 μmol/L, respectively. The IC50 of HCPT on MKN-28, SGC-7901, and MKN-45 were 9. 35 rg/L, 10. 21 rg/L, and 12. 63 mg/L respectively after 48 h treatment. After 12 h of exposure to both drugs, gastric cancer cells exhibited morphologic features of apoptosis, including cell shrinkage, nuclear condensation,and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The apoptotic rates of SGC7901, MKN-45, and MKN-28 were 13. 84%, 22.52%, and 9. 68%, respectively after 48 h exposure to 10 μmol/L As2O3. The apoptotic rates of SGC-7901, MKN-45, and MKN-28 were 21.88%, 12.35%, and 30. 26%, respectively after 48 h exposure to 10 mg/L HCPT. The apoptotic indice were 7% - 15% as assessed by TUNEL method. The effect of As2O3 on SGC-7901 showed remarkable cell cycle specificity, which induced cell death in G1 phase, and blocked G2/M phase. HCPT also showed a remarkable cell cycle specificity, by inducing cell death and apoptosis in G1 phase and arrest of proliferation at S phase. CONCLUSION AS2O3 and HCPT exhibit significant cytotoxicity on gastric cancer cells by induction of apoptosis. As2O3 and HCPT might have a promising prospect in the treatment of gastric cancer, which needs to be further studied.

Objective: To study the effect of electromagnetic pulse (EMP) on apoptosis of human lung carcinoma cell line GLC-82. Methods: The injury changes in GLC-82 cells after irradiated by EMP (electric field intensity was 60 kV/m with 5 pulses/2 min) were analyzed by cytometry, MTT chronometry and flow cytometry. The immuno- histochemical SP staining was used to determine the expressions of bcl-2 protein and p53 protein. The stained positive cells were analyzed by CMIAS-II image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. Results: EMP could obviously inhibited lung carcinoma cell line GLC-82 proliferation and increased the number of non-adherent cells. The absorbance value (A570) of MTT decreased immediately, at 0 h, 1 h and 6 h after the GLC-82 cells irradiated by EMP as compared with control group. The highest apoptosis rate was found to reach 13.38% by flow cytometry at 6 h after EMP irradiation. Down-regulation of bcl-2 expression and up-regulation of bax expression were induced by EMP. Conclusion: EMP promoted apoptosis of GLC-82 cells. At same time, EMP can down-regulate bcl-2 expression and up-regulate p53 expression in GLC-82 cells. The bcl-2 and the p53 protein may involve the apoptotic process.

Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we construct-ed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1αgene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1αrepresses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results conifrmed that rAAV-HIF-1αsigniifcant-ly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1αadministration also induced robust and prolonged HIF-1αproduction in rat hippocampus. Single rAAV-HIF-1αadministration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer’s disease rat model established by intrace-rebroventricular injection of aggregated amyloid-beta protein (25-35). Our in vitro and in vivo ifndings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurode-generative diseases using gene therapy.

There are few studies on the neuroprotective effects of syringaldehyde in a rat model of cerebral ischemia. The study aimed to elucidate the mechanisms underlying the neuroprotective effects of syringaldehyde on ischemic brain cells. Rat models of cerebral ischemia were intraperitoneally administered syringaldehyde. At 6 and 24 hours after syringaldehyde administration, cell damage in the brain of cerebral ischemia rats was obviously reduced, superoxide dismutase activity and nuclear respiratory factor 1 expression in the brain tissue were markedly increased, malondi-adehyde level was obviously decreased, apoptosis-related cysteine peptidase caspase-3 and -9 immunoreactivity was obviously decreased, and neurological function was markedly improved. These ifndings suggest that syringaldehyde exerts neuroprotective effects on cerebral ischemia injury through anti-oxidation and anti-apoptosis.

Our previous study showed that when glutamate receptor (GluR)6 C terminus-containing pep-tide conjugated with the human immunodeficiency virus Tat protein (GluR6)-9c is delivered into hippocampal neurons in a brain ischemic model, the activation of mixed lineage kinase 3 (MLK3) and c-Jun NH2-terminal kinase (JNK) is inhibited via GluR6-postsynaptic density pro-tein 95 (PSD95). In the present study, we investigated whether the recombinant adenovirus (Ad) carrying GluR6c could suppress the assembly of the GluR6-PSD95-MLK3 signaling module and decrease neuronal cell death induced by kainate in hippocampal CA1 subregion. A seizure model in Sprague-Dawley rats was induced by intraperitoneal injections of kainate. The effect of Ad-Glur6-9c on the phosphorylation of JNK, MLK3 and mitogen-activated kinase kinase 7 (MKK7) was observed with western immunoblots and immunohistochemistry. Our findings revealed that overexpression of GluR6c inhibited the interaction of GluR6 with PSD95 and prevented the kainate-induced activation of JNK, MLK3 and MKK7. Furthermore, kainate-mediated neuronal cell death was signiifcantly suppressed by GluR6c. Taken together, GluR6 may play a pivotal role in neuronal cell death.

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a pre-liminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H2O2-induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H2O2 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis poly-saccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood lfow after ischemia. Our ifndings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.

Ganoderma lucidum is a traditional Chinese medicine, which has been shown to have both an-ti-oxidative and anti-inlfammatory effects, and noticeably decreases both the infarct area and neuronal apoptosis of the ischemic cortex. This study aimed to investigate the protective effects and mechanisms of pretreatment with ganoderma lucidum (by intragastric administration) in cerebral ischemia/reperfusion injury in rats. Our results showed that pretreatment with ganoder-ma lucidum for 3 and 7 days reduced neuronal loss in the hippocampus, diminished the content of malondialdehyde in the hippocampus and serum, decreased the levels of tumor necrosis fac-tor-αand interleukin-8 in the hippocampus, and increased the activity of superoxide dismutase in the hippocampus and serum. hTese results suggest that pretreatment with ganoderma lucidum was protective against cerebral ischemia/reperfusion injury through its anti-oxidative and an-ti-inlfammatory actions.

Previous studies have indicated that electrical stimulation of the cerebellar fastigial nucleus in rats may reduce brain infarct size, increase the expression of Ku70 in cerebral ischemia/reperfusion area, and decrease the number of apoptotic neurons. However, the anti-apoptotic mechanism of Ku70 remains unclear. In this study, fastigial nucleus stimulation was given to rats 24, 48, and 72 hours before cerebral ischemia/reperfusion injury. Results from the electrical stim-ulation group revealed that rats exhibited a reduction in brain infarct size, a signiifcant increase in the expression of Ku70 in cerebral ischemia/reperfusion regions, and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Double immunofluorescence staining revealed no co-localization of Ku70 with TUNEL-positive cells. However, Ku70 partly co-localized with Bax protein in the cytoplasm of rats with cerebral ischemia/reperfusion injury. These ifndings suggest an involvement of Ku70 with Bax in the cy-toplasm of rats exposed to electrical stimulation of the cerebellar fastigial nucleus, and may thus provide an understanding into the anti-apoptotic activity of Ku70 in cerebral ischemia/reperfu-sion injury.

Flavonoids from the stems and leaves of Scutellaria baicalensis Georgi, an antioxidant, marked-ly improve memory impairments and neuronal injuries. In the present study, primary cortical neurons of rats were exposed to potassium cyanide to establish a model of in vitro neural cell apoptosis. Inhibition of apoptosis by lfavonoids from the stems and leaves of Scutellaria baical-ensis Georgi at concentrations of 18.98, 37.36, and 75.92μg/mL was detected using this model. These lfavonoids dramatically increased cell survival, inhibited cell apoptosis and excessive pro-duction of malondialdehyde, and increased the activities of superoxide dismutase, glutathione peroxidase, and Na+-K+-ATPase in primary cortical neurons exposed to potassium cyanide. The lfavonoids from the stems and leaves of Scutellaria baicalensis Georgi were originally found to have a polyhydric structure and to protect against cerebral hypoxia in in vitro and in vivo models, including hypoxia induced by potassium cyanide or cerebral ischemia. The present study suggests that lfavonoids from the stems and leaves of Scutellaria baicalensis Georgi exert neuroprotective effects via modulation of oxidative stress, such as malondialdehyde, superoxide dismutase, gluta-thione peroxidase and Na+-K+-ATPase disorders induced by potassium cyanide.

The microglia-mediated inlfammatory reaction promotes neuronal damage under cerebral isch-emia/hypoxia conditions. We therefore speculated that inhibition of hypoxia-induced microglial activation may alleviate neuronal damage. To test this hypothesis, we co-cultured ginsenoside Rb1, an active component of ginseng, and cortical neurons. Ginsenoside Rb1 protected neuronal morphology and structure in a single hypoxic culture system and in a hypoxic co-culture system with microglia, and reduced neuronal apoptosis and caspase-3 production. The protective effect was observable prior to placing in co-culture. Additionally, ginsenoside Rb1 inhibited levels of tumor necrosis factor-αin a co-culture system containing activated N9 microglial cells. Ginse-noside Rb1 also signiifcantly decreased nitric oxide and superoxide production induced by N9 microglia. Our ifndings indicate that ginsenoside Rb1 attenuates damage to cerebral cortex neu-rons by downregulation of nitric oxide, superoxide, and tumor necrosis factor-αexpression in hypoxia-activated microglia.

In the central nervous system, Asiaticoside has been shown to attenuatein vitro neuronal damage caused by exposure toβ-amyloid.In vivo studies demonstrated that Asiaticoside could attenu-ate neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion animals. In addition, Asiaticoside showed anxiolytic effects in acute and chronic stress animals. However, its potential neuroprotective properties in glutamate-induced excito-toxicity have not been fully studied. We investigated the neuroprotective effects of Asiaticoside in primary cultured mouse cortical neurons exposed to glutamate-induced excitotoxicity invoked by N-methyl-D-aspartate. Pretreatment with Asiaticoside decreased neuronal cell loss in a con-centration-dependent manner and restored changes in expression of apoptotic-related proteins Bcl-2 and Bax. Asiaticoside pretreatment also attenuated the upregulation of NR2B expression, a subunit of N-methyl-D-aspartate receptors, but did not affect expression of NR2A subunits. Additionally, in cultured neurons, Asiaticoside significantly inhibited Ca2+ influx induced by N-methyl-D-aspartate. These experimental ifndings provide preliminary evidence that during excitotoxicity induced by N-methyl-D-aspartate exposure in cultured cortical neurons, the neu-roprotective effects of Asiaticoside are mediated through inhibition of calcium inlfux. Aside from its anti-oxidant activity, down-regulation of NR2B-containing N-methyl-D-aspartate receptors may be one of the underlying mechanisms in Asiaticoside neuroprotection.

Previous experimental studies have shown that cerebral infarction can be effectively reduced following treatment with scutellaria baicalensis stem-leaf total lfavonoid (SSTF). However, the mechanism of action of SSTF as a preventive drug to treat cerebral infarction remains unclear. In this study, Sprague-Dawley rats were pretreated with 50, 100, 200 mg/kg SSTF via intragastric ad-ministration for 1 week prior to the establishment of focal cerebral ischemia/reperfusion injury. The results showed that pretreatment with SSTF effectively improved neurological function, re-duced brain water content and the permeability of blood vessels, ameliorated ischemia-induced morphology changes in hippocampal microvessels, down-regulated Fas and FasL protein expres-sion, elevated the activity of superoxide dismutase and glutathione peroxidase, and decreased malondialdehyde content. In contrast to low-dose SSTF pretreatment, the above changes were most obvious after pretreatment with moderate-and high-doses of SSTF. Experimental ifndings indicate that SSTF pretreatment can exert protective effects on the brain against cerebral isch-emia/reperfusion injury. The underlying mechanisms may involve reducing brain water content, increasing microvascular recanalization, inhibiting the apoptosis of hippocampal neurons, and attenuating free radical damage.

Mitochondria play an important role in neuronal apoptosis caused by cerebral ischemia, and the role is mediated by the expression of mitochondrial proteins. This study investigated the involvement of mitochondrial proteins in hippocampal cell apoptosis after transient cerebral ischemia-reperfusion injury in aged rats using a comparative proteomics strategy. Our exper-imental results show that the aged rat brain is sensitive to ischemia-reperfusion injury and that transient ischemia led to cell apoptosis in the hippocampus and changes in memory and cognition of aged rats. Differential proteomics analysis suggested that this phenomenon may be mediated by mitochondrial proteins associated with energy metabolism and apoptosis in aged rats. This study provides potential drug targets for the treatment of transient cerebral isch-emia-reperfusion injury.

Evidence suggests that autophagy may be a new therapeutic target for stroke, but whether acti-vation of autophagy increases or decreases the rate of neuronal death is still under debate. This review summarizes the potential role and possible signaling pathway of autophagy in neuronal survival after cerebral ischemia and proposes that autophagy has dual effects.

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression signiifcantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibit-ed, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.