Cardiac glycosides from the seeds of Thevetia peruviana and their pro-apoptotic activity toward cancer cells.

Phytochemical investigation of the seeds of Thevetia peruviana resulted in the isolation of seven cardiac glycosides (1-7), including two new compounds (1 and 2). Cytotoxicity of them toward cancer cell lines P15 (human lung cancer cell), MGC-803 (human gastric cancer cells), SW1990 (human pancreatic cancer cells), and normal hepatocyte cell LO2 suggested that compound 1 could selectively inhibit the proliferation of cancer cell lines with IC50 from 0.05 to 0.15 µM. Pro-apoptotic activity revealed that it induced the apoptosis of MGC-803 cancer cells in a dose-dependent manner. Meanwhile, treatment of MGC-803 cancer cells with 1 resulted in diminution of pro-caspases 3 and 9 and activation of caspases 3 and 9, while it increased the Bax/Bcl-2 ratio in a dose-dependent manner. These meant that 1 induced the apoptosis of cancer cells by involving the intrinsic apoptotic pathway. In addition, the cell cycle distribution of MGC-803 cancer cells treated by 1 revealed that it could lead to cell cycle arrest at the G2/M phase. Altogether, this study suggested that compound 1 may exhibit anticancer activity by its capability of induction of intrinsic apoptosis and cell cycle arrest at G2/M phase.

Cardiac Glycosides from the Seeds of Thevetia peruviana.

Investigation of the seeds of Thevetia peruviana resulted in the isolation of 15 new (2-16) and 18 known (1 and 17-33) cardiac glycosides. Eight 19-nor-cardenolides (1-8), including two rare 19-nor-10-hydroperoxycardenolides, were obtained from T. peruviana for the first time. All the structures were characterized by NMR spectroscopy and chemical derivatization. The inhibitory effects of cardiac glycosides 1-33 against three cancer cell lines (human lung cancer cells, P15; human gastric cancer cells, MGC-803; and human pancreatic cancer cells, SW1990) and one normal hepatocyte cell line, LO2, were evaluated, and a preliminary structure-activity relationship is discussed. In addition, cardiac glycosides 1, 22, 26, and 28 were evaluated for their apoptosis-inducing activities in MGC-803 cells, showing IC50 values in the range 0.02-0.53 µM.

Protection of feruloylated oligosaccharides from corn bran against oxidative stress in PC 12 cells.

Feruloylated oligosaccharides (FOs) were prepared by autoclaving corn bran in oxalic acid (0.6%) solution, and their protection effects against oxidative stress in pheochromocytoma cells (PC 12) cells were investigated. The FOs samples, which comprised a mixture of feruloylated mono- and dipentoses with 4.88% bound ferulic acid (FA), as well as xylose, arabinose, galactose, and glucose amounting to 46.43, 40.46, 3.76, and 8.68% of the total sugars, respectively, were prepared by autoclaving the pretreated corn bran in 0.6% oxalic acid and then further separated. Antioxidant activity was tested by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) scavenging and oxygen radical absorbance capacity (ORAC) methods. Oxidative stress was induced by H2O2 in PC 12 neuronal cell culture model. The results showed that FOs exhibited higher antioxidant activity than free ferulic acid, with an IC50 value of 11 versus 128 µM for DPPH and an ORAC value of 4.77 versus 2.62 µmol Trolox/µmol. Tetrazolium blue assay showed that the addition of FOs with an FA concentration >50 µM significantly increased cell viability after treatment with H2O2. Flow cytometry analysis showed that the addition of FOs at concentrations of 800, 200, and 50 µM significantly decreased the apoptosis rate at the sub-G0 phase from 37.5 to 12.7, 16.2, and 20.9% (P < 0.01), respectively. FOs also significantly decreased the malonic dialdehyde content and lactate dehydrogenase (LDH) activity, but increased superoxide dismutase activity in PC 12 cells treated with H2O2 and prevented the damage of cellular membranes by decreasing the release of LDH to the cultures. The addition of FA at 800 µM showed an effect similar to that of FOs at 200 µM. Therefore, the FOs prepared from corn bran are potential functional ingredients for protection against oxidative stress.

[Study on anti-proliferation activity and the mechanisms of alkaloid monomers from Gelsemium elegans on HepG2 cell in vitro].

OBJECTIVE: To investigate the effects of alkaloid monomers from Gelsemium elegans on proliferation of HepG2 cell in vitro and the possible mechanism. METHODS: MTT assay was used to measure the inhibitory of three alkaloid monomers on HepG2 cell in vitro. The most effective fraction was chosen to test whether the effect was in time-and dose-dependent manner. The morphological changes were observed by the light microscope and the cell cycle alteration through the flow cytometric assay. The activity of Caspase-3, Caspase-8 and Caspase-9 were detected by a Caspases colorimetric assay kit. RESULTS: The results showed that koumine, Gelsemine and Gelsenicine could significantly inhibit the proliferation of HepG2 cell and Gelsenicine, the most effective fraction, was clearly in dose- and time-dependent manners, while exhibited low cytotoxicity to the Vero cell. The cell treated with Gelsenicine for 48 h showed distinctive morphological changes. The cells treated with 200 and 400 microg/mL shrinked and fell off from the bottom. At the same time, the cells were arrested at S phase and the apoptosis increased apparently. The activity of Caspase-3, Caspase-8 and Caspase-9 was increased in a dose-dependent manner. CONCLUSION: Three alkaloid monomers from Gelsemium elegans, especially Gelsenicine, could inhibit proliferation of HepG2 cell obviously. The mechanism may be related to cell cycle arrest and activation of Caspase-3, Caspase 8 and Caspase-9.

Effects of salvianolic acid B on proliferation, neurite outgrowth and differentiation of neural stem cells derived from the cerebral cortex of embryonic mice.

Salvianolic acid B is isolated from Salvia miltiorrhiza, the root of which is widely used as a traditional Chinese medicine to treat stroke. However, little is known about how salvianolic acid B influences growth characteristics of neural stem cells (NSCs). The purpose of the present study was to evaluate the effects of salvianolic acid B on proliferation, neurite outgrowth and differentiation of NSCs derived from the cerebral cortex of embryonic mice using MTT, flow cytometry, immunofluorescence and RT-PCR. It was found that 20 microg mL(-1) and 40 microg mL(-1) salvianolic acid B had similar effects on proliferation of NSCs, and a suitable concentration of salvianolic acid B increased the number of NSCs and their derivative neurospheres. The growth-promoting activity of salvianolic acid B was dependent on and associated with an accumulation in the G2/S-phase cell population. Salvianolic acid B also promoted the neurite outgrowth of NSCs and their differentiation into neurons. The mRNA for tau, GFAP and nestin were present in differentiating neurospheres induced by salvianolic acid B. However, high-level expression of tau mRNA and low-level expression of GFAP mRNA was detected in differentiated cells, in contrast to the control conditions. This collective evidence indicates that exogenous salvianolic acid B is capable of promoting proliferation of neurospheres and differentiation towards the neuronal lineage in vitro and may act in the proliferation of NSCs and may promote NSC differentiation into neuronal cells.

Retrograde transport of a traditional Chinese medicine, alpha-trichosanthin, and its selective neural toxicity.

OBJECTIVE: To investigate the peripheral neuronal toxicity of a traditional Chinese medicine, alpha-trichosanthin (TCS). METHODS: TCS and rhodamine-conjugated TCS were separately injected into the rat sciatic nerve. Saline and rhodamine were used alone and separately as control solutions. The motor neurons in the spinal cord and sensory neurons in the dorsal root ganglia were separately counted. The entry of TCS molecules into neurons was observed under the fluorescence microscope. The glial reactions were studied by lectin staining and immunohistochemical method. The muscles innervated by the sciatic nerve and distal to the injection sites, and the nerves proximal to the injection sites were also collected and examined. RESULTS: TCS was taken up and transported by peripheral axons, and at a dose of 1 nmol, killed more than 90% of the motor neurons in 5 days, but only one-third of the sensory neurons of the injected nerve. The loss of neurons was permanent, while the increase of glial activities was mild and transient. CONCLUSION: TCS is retrogradely transported by axons of the injected nerve. TCS shows a selective neurotoxicity on different types of neurons. Hence TCS is useful in producing neural lesion in research, and this use may also be of applicational value in treating chronic spasticity, hyperalgesia, and pain.

Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy.

OBJECTIVE: To characterize uterine natural killer (uNK) cells in nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice and investigate the potential role of these cells in pregnancy tolerance. DESIGN: An animal model-based study. SETTING: Academic research center in a university. ANIMAL(S): Syngeneic pregnant NOD/SCID mice were compared with non-immunodeficient BALB/c mice. INTERVENTION(S): Induction of Toll-like receptor (TLR) agonists. MAIN OUTCOME MEASURE(S): Flow cytometric analysis was performed to detect the percentage of cell subsets, and standard (51)Cr release assay was performed to determine cytotoxicity. RESULT(S): The dominant subset of uNK cells in NOD/SCID mice is DX5 (CD49b)(+), asialo ganglio-N-tetraosylceramide(+), CD25(+), CD122(+), Thy-1 (CD90)(hi), c-kit (CD117)(hi), and interleukin-10(+). In addition, the percentage of interferon-gamma(+) subset was slightly increased in response to selected TLR agonists in the NOD/SCID mice, whereas the corresponding percentage in BALB/c mice could be increased dramatically. Such an effect could be abrogated by inhibitors, including LY294002, SP600125, and PD98059. The significant increase of interferon-gamma(+) NK cell percentage in BALB/c mice was concomitant with the increase of the embryo resorption rate. In contrast, the resorption rate in NOD/SCID mice was not significantly increased upon the induction of polyinosinic polycytidylic acid or lipopolysaccharide. As expected, the NK cells from NOD/SCID mice display a detectable but lower cytotoxicity than BALB/c, as determined by standard (51)Cr release assay. In addition, the uNK cells from NOD/SCID mice also display a hyposensitivity to lipopolysaccharide-induced production of inducible nitric oxide synthase. CONCLUSION(S): A considerable percentage of immature NK cells were detected at the fetomaternal interface in NOD/SCID mice. These cells were hyposensitive to the stimulation of selected TLR agonists. Such a status seemed to be beneficial for the maintenance of pregnancy.

Immunomodulatory effects of Caulerpa racemosa var peltata polysaccharide and its selenizing product on T lymphocytes and NK cells in mice.

A polysaccharide, CrvpPS, was isolated from Caulerpa racemosa var peltata. It was reacted with nano-selenium in distilled water containing ascorbic acid (Vit C) to form a stable CrvpPS-nano-Se complex. The immunomodulatory effects of CrvpPS and CrvpPS-nano-Se on T lymphocytes subgroups and NK cells in mice were investigated. After intragastric administration for 10 days separately, both CrvpPS and CrvpPS-nano-Se showed significant stimulatory functions to thymus gland of mice. Moreover, the CrvpPS-nano-Se induced the percentage of CD3+, CD3+CD4+, NK cells and the CD4+/CD8+ value to increase significantly (P < 0.05) when analyzed by flow cytometry, which is better than the CrvpPS, sucrose-nano-Se, and even the positive drug levamisole.

TSLP-induced placental DC activation and IL-10(+) NK cell expansion: comparative study based on BALB/c x C57BL/6 and NOD/SCID x C57BL/6 pregnant models.

Thymic stromal lymphopoietin (TSLP)-TSLP receptor (TSLP-R) interactions activate CD11c(+) dendritic cells (DCs) and increase epithelial cell Th2-type cytokine production. We detected intracellular TSLP expression on CK7(+) trophoblast cells and TSLP-R expression on placental DCs from pregnant BALB/cxC57BL/6 and NOD/SCIDxC57BL/6 mice on gestational day 12.5. Murine recombinant TSLP activated DCs from BALB/c mice, with increased CD80 and CD83 expressions; TSLP-activated DCs induced IL-10-producing NK cell expansion. This was abrogated by anti-TSLP Ab or by culturing CD49b(+) NK cells alone. No TSLP-DC-induced IL-10(+)CD49b(+) cell expansion occurred when DCs and CD49b(+) cells were cultured separately. Although TSLP-induced DC activation occurred in NOD/SCID mice, the IL-10(+) NK cell percentage was unchanged. CK7(+) trophoblast cells may activate placental DCs via a TSLP-TSLP-R interaction and induce DC-dependent placental NK cell IL-10 production. TSLP-DC and NK cell contact appears necessary for IL-10(+)CD49b(+) cell expansion. Placental NK cells from NOD/SCIDxC57BL/6 mice appear less sensitive to TSLP-DC induction.

[Morphologic observation and 3D reconstruction of aortic endothelial cells in diabetic rats with the use of atomic force microscope].

This experiment was designed to observe the morphologic changes of aortic endothelial cells in diabetic rats and to investigate the influence of hyperglycemia. Diabetic rats were used and their aortic endothelial cells were observed under atomic force microscope, then these pictures were 3D reconstructed. The results showed that numerous microvilli and round window-hollow-like structures and insect- bitten-like caveloae were scattered on the surface of the vascular endothelia, and a few ball-like granules were adhering to the endothelial cells. In diabetic rats, microvilli decreased and the cells looked as if they were weather-beaten rocks. The adhering ball-like granules in the diabetic group were significantly more than those in the control group. They were most numerous at 12 weeks. The insect-bitten-like caveloae began deepening at 7 weeks, and their diameters markedly increased at 12 weeks, but they decreased in number. After 3D figures were reconstructed, the surface of cells manifested smoothness. However, in diabetic rats, the morphology of their endothelial cells was characterized by roughness and small surface projections. The quantitative results showed that the average roughness increased gradually as the experiment time prolonged. These indicate that excess sugar in the blood may destroy the endothelial structure, make the cells rougher, and hence cause the protein granules to adhere onto the endothelial cells. At the same time, the deepening and enlargement of the insect-bitten-like caveloae may indicate the increasing endocytosis.

[Distribution of neuronal nitric oxide synthase-immunopositive neurons in rat corpus striatum and their ultrastructures].

OBJECTIVE: To observe the distribution of neuronal nitric oxide synthase (nNOS)-immunopositive neurons in rat corpus striatum and their ultrastructural features. METHODS: Brain tissue specimens were obtained from normal SD rats, in which nNOS-immunopositive neurons were visualized by ABC immunocytochemistry and observed under immunoelectron microscope with pre-embedding staining. RESULTS: Under light microscope, nNOS-immunopositive neurons appeared brown with distinct profiles of the cell body and processes. These neurons, mostly medium-sized and small cells, were located mainly in the lateral region of the corpus striatum. Only a few immunopositive neurons were detected in the medial region of the corpus striatum. Immunohistochemistry and transmission electron microscopy identified the nNOS-immunopositive neurons as interneurons possessing large nuclei with small amount of cytoplasma. The immunopositive granules were visualized as black plaques, and the larger ones distributed mainly in the cell bodies, some with monolayer membrane encapsulation. The small granules did not have the encapsulation, scattering in perinuclear regions and under the cell membrane, but not in the cell body. The immunopositive granules were also found in the axons and dendrites, but not in the vesicles of the synapses. In addition, many immunopositive terminals were found close to the blood vessels. CONCLUSIONS: nNOS-immunopositive neurons in rat corpus striatum are mainly medium-sized and small cells as is typical of the interneurons. The immunopositive granules locate in the cytoplasma, axons and dendrites, and larger granules have membrane coating while small ones do not, possibly in relation to their functions.

[Changes of neuronal nitric oxide synthase-immunopositive neurons in the central amygdaloid nucleus of spontaneously hypertensive rats].

OBJECTIVE: To observe the changes of neuronal nitric oxide synthase (nNOS)-immunopositive neurons in the central amygdaloid nucleus of spontaneously hypertensive rats (SHRs). METHODS: Thirty SHRs and 30 Wistar-Koyto rats (WKYs) were sacrificed at the ages of 90, 180, and 360 days respectively to observe the changes of nNOS-immunopositive neurons with ABC immunocytochemical assay. RESULTS: No significant changes were observed in the blood pressure of WKY rats at the specified time points, when SHRs maintained significantly higher blood pressures from 20.8+/-1.1 and 26.3+/-1.0 kPa (P<0.05), gradually increasing during the development of hypertension. The nNOS-immunopositive neurons in the central amygdaloid nucleus were of moderate sizes with long intersected nerve fibers. No significant changes were found in WKY rats. The number of the positive neurons decreased with age in SHRs, especially obvious at 360 days (P<0.05), which was significantly different from that in the WKYs (P<0.05). CONCLUSION: The reduction of nNOS-immunopositive neurons in the central amygdaloid nucleus of SHRs might accelerate the development of hypertension by modulating the cardiovascular function and sensory transmission.

Changes of neuronal nitric oxide synthase-immunoreactive neurons in the paraventricular hypothalamic nucleus of diabetic rats.

OBJECTIVE: To explore the role of nitric oxide in the development of diabetes mellitus by observing the changes of neuronal nitric oxide synthase (nNOS)-immunoreactive (nNOS-ir) neurons in the paraventricular hypothalamic nucleus (PVN) of diabetic rats. METHODS: Diabetic rat models were established by means of intraperitoneal streptozotocin injections. At the end of 2, 7 and 12 weeks after model establishment, tissue sampling was performed and the number of nNOS-ir neurons in PVN were counted and quantitatively analyzed. RESULTS: The number of nNOS-ir neurons in PVN of diabetic rats remained normal 2 weeks after model establishment (P >0.05) but was significantly increased by the time of 7 weeks (P <0.05); at the 12th weeks, the number of nNOS-ir neurons of diabetic group was still greater than that of the control group (P <0.05). CONCLUSION: The number of nNOS-ir neurons in PVN increases significantly in middle and advanced stages of diabetes, suggesting the possible relations between the nNOS activity changes in PVN and the changes in cerebral neuroendocrine and adrenal activity in diabetic condition.