Protection of the hemopoietic system by Podophyllum hexandrum against gamma radiation-induced damage

A semi-purified extract of low-altitude Podophyllum hexandrum (REC-2001) containing a relatively low content of podophyllotoxin (3.25 %) exhibited potent antioxidant ability in lipid media (at 1000 microg/mLagainst 0.25 kGy) and significant (p < 0.05) hydroxyl ion scavenging potential (78.83 % at 500 microg/mL). In vitro investigations revealed the ability of REC-2001 to significantly (p < 0.05) reduce radiation-induced hemolysis (2 microg/mL; 46.184 %) and nitric oxide scavenging levels (IC (50): 792 +/- 1.25 microg/mL). Protection of the hemopoietic system of Strain 'A' mice administered 20 mg/kg BW REC-2001 30 min prior to lethal irradiation (10 Gy) was recorded and was mediated by free radical scavenging and lowering of lipid oxidation. Further studies investigating the effects of REC-2001 on stem cell modulation are warranted.     

Cytotoxic and radioprotective effects of Podophyllum hexandrum

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25-200μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.     

Radioprotection by Podophyllum hexandrum in the liver of mice: A mechanistic approach

To evaluate radiation protection offered by the extract of Podophyllum hexandrum, expression of various cytoprotective proteins was studied using liver of Swiss albino Strain 'A' male mouse by immunoblotting. Induction of heat-shock factor-1 (HSF-1), led to up-regulation of heat-shock protein-70 (HSP-70) upon P. hexandrum (200mg/kg b. wt.; i.p. 50% ethanolic extract) treatment 2h before irradiation (10Gy) as compared to sham-irradiated control. Translocation of free nuclear factor kappa B (NFκB) from cytoplasm to nucleus was found to be inhibited upon P. hexandrum treatment. An increase in Bcl-2, proliferating cell nuclear antigen (PCNA) along with a decrease in p53, caspase-3, apoptosis inducing factor (AIF) expression was observed in the mice treated with P. hexandrum. The present study indicated that P. hexandrum extract provides protection from radiation by modulation of expression of the proteins associated with apoptosis.     

Podophyllum hexandrum ameliorates endosulfan-induced genotoxicity and mutagenicity in freshwater cyprinid fish crucian carp

Context: Medicinal plants continue to act as a repository for novel drug leads with novel mechanisms of action. Podophyllum hexandrum Royale (Berberideceae) treats diverse conditions in folk medicine.

Objective: The antimutagenic potential of P. hexandrum was evaluated against endosulfan-induced clastogenicity in a piscine model by cytogenetic endpoints.

Materials and methods: Podophyllum hexandrum rhizomes were subjected to successive solvent extraction. Fish were exposed to hexane, chloroform, ethyl acetate, methanol and aqueous extracts (15?mg/L each) of plant and endosulfan (0.05?mg/L) alone followed by their combination for antimutagenicity estimates. Chromosomal aberrations (CA) were made from kidney cells and micronuclei (MN) slides from peripheral blood erythrocytes at 48, 72 and 96?h. Antioxidant activity was analyzed by the DPPH assay. Phytochemical analyses were carried out using chromatographic and spectroscopic techniques.

Results: Endosulfan induced significant (p?R^2?=^?.900) with antimutagenic activity, whereas EE-F4 seemed to act through a different mechanism.

Discussion and conclusion: This study confirms the antimutagenic potential of the subject plant with the identification of some novel compounds, justifying their use in folk medicine, and their corresponding benefit to mankind.     

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol

We have examinedin vitro andin vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of γ-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured γ-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5–8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to γ-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis.In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4±0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1±0.4 apoptotic cells per crypt was observed.In vitro andin vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.     

Protection of T cells from radiation-induced apoptosis by Cepharanthin®

Cepharanthin (CE) is a medicine that contains several biscoclaurine alkaloids. We examined the effects of CE on radiation-induced T cell apoptosis. Radiation induced apoptosis on T cells in a dose-dependent manner, while CE inhibited radiation-induced apoptosis. CE also attenuated the cytotoxic effects of radiation on the proliferative response of T cells. CE inhibited not only the loss of mitochondrial transmembrane potential but also the activation of caspase 3 in irradiated T cells. Radiation plus CE induced the up-regulation of Bax and the down-regulation of Bcl-2 in T cells in comparison with radiation alone. These results suggest that CE inhibits the signal transduction pathway of apoptosis induced by radiation, regardless of the expression of Bcl-2 or Bax.     

Geraniin down regulates gamma radiation-induced apoptosis by suppressing DNA damage

Gamma ray irradiation triggers DNA damage and apoptosis of proliferating stem cells and peripheral immune cells, resulting in the destruction of intestinal crypts and lymphoid system. Geraniin is a natural compound extracts from an aquatic plant Nymphaea tetragona and possesses good antioxidant property. In this study, we demonstrate that geraniin rescues radiosensitive splenocytes and jejunal crypt cells from radiation-induced DNA damage and apoptosis. Isolated splenocytes from C57BL/6 mice treated with geraniin were protected against radiation injury of 2 Gy irradiation through the enhancement of the proliferation and attenuation of DNA damage. Also, geraniin inhibited apoptosis in radiosensitive splenocytes by reducing the expression level and immunoreactivity of proapoptotic p53 and Bax and increasing those of anti-apoptotic Bcl-2. In mice exposed to radiation, geraniin treatment protected splenocytes and intestinal crypt cells from radiation-induced cell death. Our results suggest that geraniin presents radioprotective effects by regulating DNA damage on splenocytes, exerting immunostimulatory capacities and inhibiting apoptosis of radiosensitive immune cells and jejunal crypt cells. Therefore, geraniin can be a radioprotective agent against γ-irradiation exposure.     

辐射致组织损伤凋亡机制的研究概况

辐射能够导致组织损伤,其机制通常与细胞凋亡、坏死、炎症等相关.其中细胞凋亡是辐射损伤的主要特征之一,但是凋亡发生的具体机制仍不清楚.研究表明,有多条通路涉及其中,代表性的通路有：聚ADP核糖聚合酶（PARP）、p53、自由基、神经酰胺、p38通路等.本文围绕上述蛋白质分子,综述了凋亡途径在辐射致组织损伤的作用.     

Podophyllotoxin content, above- and belowground biomass in relation to altitude in Podophyllum hexandrum populations from Kumaun region of the Indian Central Himalaya

The morphological features of Podophyllum hexandrum Royle, a 'critically endangered' medicinal herb and a source of podophyllotoxin, were studied in populations growing in different parts of the Kumaun region of the Indian Central Himalaya. Plant growth performance in terms of biomass accumulation and podopyllotoxin levels in the rhizomes collected from eleven natural populations (P1 to P11, altitude ranging from 2740 to 3350 m) were analyzed. Morphological features, e. g., plant height, stem diameter and leaf area were, in general, negatively correlated with an increase in the altitude. Maximum aboveground (8.46 g/individual) and belowground (48.18 g/individual) biomass values were recorded from a population (P9) at the lowest altitude (2740 m) and, in general, the species was found to perform better at the lower altitudes. The podophyllotoxin content of rhizomes ranged between 0.36-1.08% (on dry wt. basis) in different populations, and a positive correlation was observed between podophyllotoxin content and an increase in the altitude.     

Compound K suppresses ultraviolet radiation-induced apoptosis by inducing DNA repair in human keratinocytes

Ultraviolet (UV)-induced DNA damage is a crucial molecular trigger for sunburn cell formation and skin cancer. Nucleotide excision repair (NER) is the main mechanism in repairing UVB-induced DNA damage of mammalian cells. The purpose of this study is to investigate the functional role of ginsenoside compound K on HaCaT cells (a keratinocyte-derived permanent cell line) irradiated by UV. Hoechst 33258 staining were performed in analyzing UV-induced apoptosis on keratinocytes which were treated with compound K. ImmunoDotBlot assay was used in detecting cyclobutane pyrimidine dimers, the main DNA damage. Western blot analysis was applied for analyzing XPC and ERCC1, two of the NER proteins. Compound K inhibited UV-induced apoptosis of keratinocytes and caused a notable reduction in UV-specific DNA lesions which was due to induction of DNA repair. In agreement with this, compound K induced the expression of particular components of the NER complex, such as XPC and ERCC1. Our results demonstrate that compound K can protect cells from apoptosis induced by UV radiation by inducing DNA repair.     

In vitro propagation of Podophyllum peltatum

The lignan podophyllotoxin, occurring in Podophyllum emodi Wall, ex Royale and Podophyllum peltatum. L., is the starting compound for the semi-synthesis of the anticancer drugs etoposide and teniposide. In this study, we evaluate development of an in vitro propagation protocol to rapidly produce high yielding Podophyllum peltatum plants. Rhizome tips were inoculated on MS medium supplied with 4.4 microM N(6)-benzyladenine and 0.025% (w/v) activated charcoal. These explants formed terminal buds, similar to the ones found in nature. These buds were sources of in vitro bud cultures. These bud cultures were classified as: apical, axillary, and adventitious and the effects of various N(6)-benzyladenine concentrations on the three types of bud cultures were evaluated through bud, leaf, and root inductions. Cultures of axillary and adventitious buds were more proliferous for bud induction. Podophyllotoxin contents of in vitro rooted bud and plantlet cultures were similar to the content found in the wild. Plantlets and buds were acclimatized under controlled environment conditions.     

Simultaneous quantification of eight active compounds in Sinopodophyllum hexandrum by HPLC-DAD

Sinopodophylli Fructus is the commonly used traditional Tibetan medicinal herb. In the present study, we established a reversed-phase high performance liquid chromatography method to simultaneously determine three lignans and five flavonoid constituents, namely podophyllotoxin, desoxypodophyllotoxin, 4′-demethyldesoxypodophyllotoxin, 8-prenylkaemferol, quercetin, kaempferol, 8,2'-diprenylquercetin 3-methylether and 8-prenylquercetin, in Sinopodophylli Fructus. The chromatographic separationwas achieved on a C₁₈ analytical column with a gradient mobile phase consisting of acetonitrile and 0.05% phosphoric acid at a flowrate of 1.0 mL/min. UV detection was set at 290 nm and 370 nm, and the column oven was set at 35 °C. This method provided a good reproducibility, and its overall intra- and inter-day precision was less than 3% and 4%, respectively. The recovery of the method was 98.29%–101.60%, and a good linearity (R²≥0.9992) was obtained for all the analytes over a relatively wide range of concentration. A total of 17 samples of S. hexandrum (12 fruits, 5 roots and rhizomes) were collected from different areas and then successfully quantified. The results indicated that the contents of eight compounds significantly varied (the sum content rangedfrom 16.90 to 55.68 mg/g), and prenylated flavonoids could be used as marker constituents in the identification and quality control of Sinopodophylli Fructus.     

Enhancement of human eosinophil apoptosis by fluticasone propionate, budesonide, and beclomethasone

Beclomethasone, budesonide, dexamethasone, and fluticasone propionate enhanced human eosinophil apoptosis in a concentration-dependent manner in vitro as assessed by flow cytometric analysis and morphological analysis. The order of potency was fluticasone propionate (EC(50) 3.7+/-1.8 nM) approximately budesonide (EC(50) 5.0+/-1.7 nM)>beclomethasone (EC(50) 51+/-19 nM)>dexamethasone (EC(50) 303+/-40 nM). Hydrocortisone, prednisolone, and prednisone (up to 1 microM) did not induce any significant increase in eosinophil apoptosis. The apoptosis promoting effects of glucocorticoids on eosinophils were reversed by an antagonist of glucocorticoid receptor mifepristone. The survival-prolonging effect of tumor necrosis factor (TNF)-alpha was reversed by dexamethasone and fluticasone (1 microM). In contrast, fluticasone, and dexamethasone (1 microM) did not reverse the survival-prolonging effects of interleukins-3 and -5 or granulocyte-macrophage colony-stimulating factor (GM-CSF). The results suggest that fluticasone and budesonide induce eosinophil apoptosis at clinically achievable drug concentrations via an effect on glucocorticoid receptor.     

Down-regulation of ceramide production abrogates ionizing radiation-induced cytochrome c release and apoptosis

Previous work has demonstrated that down-regulation of ceramide production after selection of cells with N-oleoylethanolamine (OE), an inhibitor of ceramidase, results in resistance to DNA damage-induced apoptosis. We report here that acute exposure of WEHI-231 cells (murine B-cell lymphoma) to OE activates neutral sphingomyelinase, induces ceramide production and increases intracellular reactive oxygen species. OE exposure also induces mitochondrial permeability, cytochrome c release, and apoptosis. Cells selected for resistance to OE exhibit little if any change in reactive oxygen species and cytochrome c release when exposed either to OE or to toxic doses of ceramide. Importantly, the OE resistant cells are also resistant to ionizing radiation-induced cytochrome c release and apoptosis. These findings demonstrate that down-regulation of neutral sphingomyelinase activity is associated with decreased DNA-damage-induced apoptosis. In addition, the data suggests that agents that modify extranuclear targets responsible for ceramide production select for cells resistant to ionizing radiation-induced apoptosis through alterations in mitochondrial function.     

Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein.

Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 gamma-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus.     

Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells

We examined the effects of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, on human HepG2 hepatoma cells to better understand its effect on apoptosis and apoptosis-related genes. Our study has demonstrated that DADS affects cell proliferation activity and viability and elicits typical apoptotic morphologic changes (chromatic condensation and nuclear fragmentation) in human HepG2 hepatoma cells. Also, treatment with DADS induces a temporary increase in phosphorylated p38 MAPK (phospho-p38) and phosphorylated p42/44 MAPK (phospho-p42/p44) in a time- and concentration-dependent manner. Inhibition of activated/phosphorylated mitogen-activated protein kinase (MAPK) with phospho-p38 or phospho-p42/44 specific inhibitors, SB203580 or U0126, induces apoptosis without DADS treatment, indicating that at least the endogenous activated forms of p38 MAPK and p42/p44 MAPK markedly exert cytoprotective roles from cell apoptosis in the HepG2 hepatoma cells. Combined treatment with these inhibitors followed by DADS further enhances the DADS-induced apoptosis. Taken together, these results show that both DADS and the specific inhibitors of MAPKs could induce apoptosis in HepG2 hepatoma cells and that the MAPKs inhibitors further enhance the apoptotic effect in DADS-treated HepG2 hepatoma cells.