Effects of amino acids from selenium-rich silkworm pupas on human hepatoma cells

Selenium (Se) plays an important role in cancer-prevention. Silkworm pupas have been used as a Chinese traditional medicine since ancient time. In order to find effective carcinostatic agents, Se-rich amino acids were extracted from Ziyang silkworm pupas. The Se content of Ziyang pupas was measured to be 215 times higher than that of Luoyang normal ones, and the majority of Se was stored in proteins. Composition analysis showed that Se-rich amino acids from Ziyang pupas had higher amounts of selenomethionine, methionine, cystine, and tyrosine than normal amino acids from Luoyang pupas which were rich in amino acids containing alkyl side chains. When cultured with human hepatoma cells SMMC-7721, Se-rich amino acids at concentrations of 0.5, 1.5, and 2.5 micromol L(-1) Se significantly and dose-dependently inhibited cell viability, induced changes in cell morphology and cycle, and caused cell apoptosis. On the contrary, normal amino acids did not show any inhibitory effect on SMMC-7721 cells. Sodium selenite or selenomethionine at the same Se concentrations only slightly inhibited the hepatoma cells. Mechanism study showed that selenium-rich amino acids could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. Those results suggested that Se-rich amino acids were effective carcinostatic agents compared with sodium selenite and selenomethionine. The mechanism for their hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation.     

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.     

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 μM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10–25 μM on rat cultured hepatoma cells. Four hours after treatment with 25 μM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.     

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.     

The participation of nitric oxide in peritoneal exudate cell cytotoxicity of mice by Fusobacterium nucleatum

Previously we reported that mice infected recurrently with live Fusobacterium nucleatum(Fn) synthesize a significant amount of NO between 12 hr and 24 hr after Fn injection. Fn is a gram-negative rod periodontal pathogen. NO could not be induced by heat-killed Fn or in untreated mice. This NO, derived from the iNOS after infection of live Fn, was not involved in the Fn reduction because Fn clearance occurs within 6 hr. We investigated in this study whether this NO was involved in cytotoxicity in peritoneal exudate cells (PEC) in vivo. The mice were divided into two groups: those treated with live Fn (immune) and those left untreated (normal). PEC number, NO production, detection of apoptosis or death cells, and lactate dehydrogenase (LDH) release activity after injection of live Fn were compared in these groups. In the immune group, the increase of the total cell numbers caused by an increase in neutrophils, a significant NO production only after injection of live Fn at 24 hr and identification of iNOS positive macrophages were confirmed. The apoptotic rate was very low and did not increase at 24 hr in vivo. Therefore, apoptosis was seldom relevant to the NO. In the immune group, LDH activity was remarkable high at 24 hr, and dead cells and macrophages phagocytizing cell fragments increased at the same time. Pretreatment of L NMMA, an inhibitor of iNOS, suppressed LDH activity and cell death. Therefore, the NO derived from the iNOS is involved in the cytotoxicity. These results suggest that NO may contribute to the inflammatory response during Fn infection in periodontitis.     

硒对H2O2诱导的人甲状腺细胞凋亡和超微结构改变的影响

目的：观察硒对H2O2诱导的人甲状腺上皮细胞凋亡和超微结构改变的影响。方法：取良性甲状腺腺瘤旁正常组织进行细胞培养。加硒（10^-7mol／L）或不加硒后加入不同浓度H2O2（0～800μmol／L）刺激单层培养的甲状腺细胞,流式细胞术（FCM）检测甲状腺细胞凋亡率并在电镜下观察其超微结构的改变.结果：经过H2O2作用24h的人甲状腺细胞,随H2O2浓度升高,细胞凋亡率逐渐升高;电镜下甲状腺细胞超微结构呈损伤型改变,甚至出现凋亡、死亡。预先加入10^-7mol／L硒可降低细胞凋亡率,可明显减轻亚细胞结构损伤。结论：硒可减轻H2O2诱发的人甲状腺细胞的氧化损伤,拮抗其导致的细胞凋亡。     

Nitric oxide and cell death in liver cancer cells

Nitric oxide (NO) is a lipophillic, highly diffusible, and short-lived physiological messenger which regulates a variety of physiopathological responses. NO may exert its cellular action through cGMP-dependent and cGMP-independent pathways which includes different postranslational modifications. The effect of NO in cancer depends on the activity and localization of NOS isoforms, concentration and duration of NO exposure, cellular sensitivity, and hypoxia/re-oxygenation process. NO regulates critical factors such as the hypoxia inducible factor-1 (HIF-1) and p53 generally leading to growth arrest, apoptosis or adaptation. NO sensitizes hepatoma cells to chemotherapeutic compounds probably through increased p53 and cell death receptor expressions.     

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619-626). After culture for 72 h, cells were further cultured for 24-72 h in medium without FBS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The number of wild-type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (0.1 or 1 ng/ml) or thapsigargin (10(-7) or 10(-6) M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF-alpha (10 ng/ml) or thapsigargin (10(-5) M) caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity in wild-type cells was significantly increased by culture with TNF-alpha (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10(-5) M) caused a significant increase in Ca(2+)/calmodulin-dependent NO synthase activity in wild-type cells or transfectants. TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-nitro-L-arginine (10(-4) M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with thapsigargin (10(-6) M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin-induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF-alpha or thapsigargin.     

Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.     

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin induces NADPH cytochrome c (P-450) reductase in rat hepatoma cells in culture

The lack of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) (EC 1.14.14.1) induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a clone of rat hepatoma (HTC cl-1) cells is not caused by the lack of nuclear Ah receptor or by a deficiency in the activity of NADPH-cytochrome c (P-450) reductase. Treatment of HTC cl-1 cell line with TCDD for 18 h in culture resulted in a reproducible 500-600% increase in reductase activity without concomitant expression in AHH activity. These data suggests that TCDD induces cytochrome c reductase activity and that the lack of inducible AHH activity in rat hepatoma cells could reflect a defect in the structural gene (s) encoding for cytochrome P1-450, or an Ah receptor with a faulty DNA binding domain.     

The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na₂SeO₃, 2 mg/kg), Cd (CdCl₂, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl₂ and 2 mg/kg of Na₂SeO₃) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.     

Modulation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide)

Tiazofurin, a C-nucleoside, was cytotoxic in hepatoma 3924A cells grown in culture with an LC50 = 7.5 microM. In the culture, a closely linked dose-related response of tumor cell-kill and depletion of GTP pools was observed after tiazofurin treatment. In rats carrying subcutaneously transplanted hepatoma 3924A solid tumors, a single intraperitoneal injection of tiazofurin (200 mg/kg) caused a rapid inhibition of IMP dehydrogenase (EC 1.2.1.14) activity and depleted GDP, GTP, and dGTP pools in the tumor; concurrently, the 5-phosphoribosyl 1-pyrophosphate (PRPP) and IMP pools expanded 8- and 15-fold, respectively. Tiazofurin decreased tumoral IMP dehydrogenase activity and dGTP pools in a dose-dependent manner over a range of 50-200 mg/kg; by contrast, the depletion of GTP and the accumulation of IMP and PRPP pools were near maximum at 50 mg/kg. The increase in PRPP pools may be attributed to an inhibition by IMP of the activity of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8). The IMP dehydrogenase activity and the pools of ribonucleotides returned to the normal range by 24-48 h after the single injection of tiazofurin. However, the markedly depleted dGTP pools remained low for 72 h. Tiazofurin treatment resulted in significant anti-tumor activity in rats inoculated with hepatoma 3924A. The decrease in GTP levels and particularly the sustained depletion in the dGTP pools may explain, in part at least, the chemo-therapeutic action of tiazofurin on hepatoma 3924A. This is the first report showing that a marked therapeutic response was achieved against rapidly growing hepatoma 3924A by treatment with a single anti-metabolite.     

MAT2A基因小干扰RNA诱导人肝癌细胞凋亡的分子机制

为探讨甲硫氨酸腺苷转移酶2A(MAT2A)小干扰RNA对人肝癌细胞生长和细胞凋亡的影响及其机 制,采用脂质体转染法将MAT2A小干扰RNA质粒表达载体转染人肝癌细胞系Bel 7402细胞、HepG 2细胞和 HepG3B细胞.半定量RT PCR检测MAT2A mRNA表达,Western印迹检测MAT2A 蛋白质表达, M TT法观察MAT2A小干扰RNA对肝癌细胞生长的影响,流式细胞仪及DAPI染色检测siRNA对肝癌细 胞凋亡的影响.为探讨其作用机制, 进一步检测转染后肝癌细胞MAT的活性、MAT1A mRNA表 达及SAM、SAH含量.结果发现, MAT2A小干扰RNA特异性抑制人肝癌细胞MAT2A mRNA和蛋白质 的表达, 刺激MAT表达由MAT2A向MAT1A转变, 降低了肝癌细胞中MATⅡ活性（P<005） ,从而诱导肝癌细胞凋亡; MAT2A小干扰RNA诱导Bel－7402细胞、HepG 2细胞、 Hep 3B细胞凋亡 指数分别为19．3%±2．8%、22．8%±3．5%、21．8%±4．2%, 较对照组siRNA(凋亡指数为5 2%±19%)具有明显差异(P<005).DAPI染色显示, MAT2A小干扰RNA转染组可见多个细胞核 浓缩、碎裂成蓝色的小块状,染色质凝聚,形成典型的凋亡小体, 而对照siRNA转染组未发现典型的 凋亡小体.肝癌细胞的生长也受到抑制,MAT2A小干扰RNA转染Bel 7402细胞、HepG 2细胞 、HepG3B细胞72 h后,细胞生长抑制率达高峰,分别为39．62%、41．27%、38．84%.肝癌细胞 中SAM含量明显升高(P<001),而SAH含量改变不明显, SAM/SAH变化伴随SAM含量变化而改 变.提示靶向MAT2A基因的siRNA通过升高肝癌细胞中SAM含量,刺激MAT表达由MAT2A向MAT1A转变, 从而诱导肝癌细胞凋亡,抑制肝癌细胞生长.     

Effect of crocin on experimental atherosclerosis in quails and its mechanisms

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg x L(-1)) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 microg x L(-1) Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca2+]i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca2+, crocin concentration-dependently inhibited the [Ca2+]i elevation induced by 120 mg x L(-1)Ox-LDL, In the absence of extracellular Ca2+, crocin could inhibit the [Ca2+]i elevation induced by CHCl3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca2+]i elevation in smooth muscle cell, Ca2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.     

Putative tumor suppressor YueF affects the functions of hepatitis B virus X protein in hepatoma cell apoptosis and p53 expression

Previously, we identified YueF as a novel Hepatitis B virus X protein (HBx)-interacting protein. Herein, we studied the functions of YueF and HBx in hepatocarcinogenesis. YueF was expressed at high levels in normal human hepatic cells and tissues, but scarcely found in hepatoma cells or other tumor tissues. Over-expression of YueF, or YueF and HBx could induce cell apoptosis and enhance p53 expression in hepatoma cells, whereas over-expression of HBx alone behaved contrarily. These results indicate that YueF has tumor suppressor activity and affects the functions of HBx in cell apoptosis and p53 expression in hepatoma cells.     

Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo

The anticryptosporidial effect of sodium selenite (selenium) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system and an immunosuppressed C57BL/6N adult mouse model. Parasite numbers in cell culture were significantly reduced (p<0.01) following treatment with selenium (Se) at concentrations of 6, 9, and 12 μM at 48 h postinoculation (PI) and at 1.5, 3, and 6 μM at 96 h PI. Parasite reduction was greater than 50% at 48 h PI when 9 and 12 μM Se was used, and at 96 h PI when 6 μM Se was used. Such Se-induced reduction of Cryptosporidium parvum infection was significantly (p<0,0001) blocked when using free-radical scavengers such as mannitol (20 mM). A combined solution of mannitol (20 mM) and reduced glutathione (0.5 mM) enhanced the blockage to almost 100%. Adult C57BL/6N mice were immunosuppressed with dexamethasone phosphate administered ad libitum (16 μg/mL) in drinking water and inoculated with 10⁵ oocysts/mouse. Significantly fewer (p<0.001) oocysts were shed in the feces of mice treated with Se administered ad libitum (12 μM) in drinking water than in untreated mice. The survival time of mice was also significantly increased (p<0.001) following Se treatment. Collectively, these results indicate that Se plays an important role in cryptosporidiosis, and oxidative stress caused by Se is probably a major mechanism in inhibition of C. parvum infection.     

The morphological and immunocytochemical evaluation of primary rat hepatocytes undergoing spontaneous cell death: modulation by the nitric oxide donor S-nitroso-N-acetylpenicillamine

Nitric oxide (NO) is one of the smallest molecules synthesised in the human body. It is produced by three distinct nitric oxide synthase isoenzymes (NOS) and plays a number of physiological functions in many organs and tissues. Among its numerous properties is the ability to influence programmed cell death. NO can either inhibit or induce apoptosis depending on the context of its production. In the liver, NO is produced in greater amounts especially during inflammation. The effect of NO in liver physiology and pathophysiology can be both beneficial and detrimental. Therefore, the aim of our study was to examine NO effect on cell viability and cell death in primary rat hepatocyte culture. By using NO donor, S-nitroso-N-acetylpenicillamine (SNAP), the potential of exogenously delivered NO to influence spontaneous cell death in culture was examined. The morphological approach was used in order to discriminate between apoptotic and necrotic cell death. The nitrite level, urea production and alanine aminotransferase leakage were determined in the culture medium. The immunocytochemical detection of three apoptotic markers: cleaved caspase-3, cleaved caspase-9 and lamin A, was performed. Immunocytochemical analysis of hepatocyte apoptosis revealed different labelling pattern for each method, while the detection of cleaved caspase-3 best correlated with defined phenotypical criteria. Our data showed that under present conditions NO improved the viability of primary rat hepatocytes compared to untreated cells. This was manifested by the increase of viable hepatocytes in contrast to the decrease of necrotic and apoptotic hepatocytes as assessed by the morphological examination of cell culture. The NO effect was dose-dependent in the range of SNAP concentration between 200-800 microM.     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。