Gamma Radiation Induced Intestinal Proteomic Modulation in Mice: A Two Dimensional Electrophoretic Analysis

Exposure to high doses of radiation causes serious injuries in gastrointestinal tract, by affecting biomolecules of the tissue. To demonstrate the modulation of intestinal proteome by ionising radiations, we analysed changes in protein expression in 9 Gy irradiated C57BL/6 mice at 24 h and 72 h by using two dimensional electrophoresis technique. A total of 19 protein spots with statistical significance (fold change>1.5 and P<0.05) were found to be differentially expressed. Of these 07 spots were identified by MALDI-TOF MS and peptide mass fingerprinting techniques which matched with the known proteins documented in the online database. These proteins belong to biological-functional categories like cytoskeleton system, molecular chaperones, DNA damage response, and stress response. These identified radiation induced proteins can help in understanding the mechanisms behind the intestinal injuries and thus can become potential targets for therapeutics and also aid in drug development

A Combination of Podophyllotoxin and Rutin Alleviates Radiation-Induced Pneumonitis and Fibrosis through Modulation of Lung Inflammation in Mice

Pneumonitis and pulmonary fibrosis are predominant consequences of radiation exposure, whether planned or accidental. The present study, demonstrates radioprotective potential of a formulation, prepared by combining podophyllotoxin and rutin (G-003M), in mice exposed to 11 Gy thoracic gamma radiation (TGR). Treated mice were observed for survival and other symptomatic features. Formation of reactive oxygen species (ROS)/nitric oxide (NO) was measured in bronchoalveolar lavage cells. DNA damage and cell death were assessed in alveolar cells by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Total protein (TP), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were measured in bronchoalveolar lavage fluid (BALF)/serum of mice to assess lung vascular permeability. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), cluster of differentiation 45, inducible nitric oxide synthase (iNOS), and nitrotyrosine were also estimated in lungs/BALF of differentially treated mice. Our observations revealed 100% survival in G-003M-pretreated mice against 66.50% in 11 Gy TGR exposed. Other symptoms like reduction in graying of hair, weight loss, and breathing rate were also observed in pretreated groups. Significant decline in ROS/NO and cell death in formulation pretreated mice were also observed. Decreased level of TP, LDH, and ALP in BALF/serum samples revealed G-003M-induced inhibition in lung permeability. Level of IL-6, TNF-α, and TGF-β1 in the lungs of these mice was found corresponding to control group at 8 weeks posttreatment. On the contrary, these cytokines raised significantly in 11 Gy TGR-exposed mice. Lung pneumonitis and fibrosis were found significantly countered in these mice. The observations revealed that G-003M could regulate immune system by curtailing radiation-induced oxidative and inflammatory stress, which has helped in minimizing radiation-inflicted pneumonitis and fibrosis

A Combination of Podophyllotoxin and Rutin Attenuates Radiation Induced Gastrointestinal Injury by Negatively Regulating NF-κB/p53 Signaling in Lethally Irradiated Mice

<div><p>Development of an effective radio protector to minimise radiation-inflicted damages have largely failed owing to inherent toxicity of most of the agents examined so far. This study is centred towards delivering protection to lethally irradiated mice by pre-administration of a safe formulation G-003M (combination of podophyllotoxin and rutin) majorly through regulation of inflammatory and cell death pathways in mice. Single intramuscular dose of G-003M injected 60 min prior to 9 Gy exposure rescued 89% of whole body lethally irradiated C57BL/6J mice. Studies have revealed reduction in radiation induced reactive oxygen species (ROS), nitric oxide (NO) generation, prostaglandin E2 (PGE2) levels and intestinal apoptosis in G-003M pre-treated mice intestine. Restricted nuclear translocation of redox-sensitive Nuclear factor-κB (NF-κB) and subsequent downregulation of cyclo-oxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS; EC 1.14.13.39) and tumor necrosis factor (TNF-α) levels demonstrated the anti-inflammatory effect that G-003M exerts. Support to early hematopoietic recovery was exhibited through G-003M mediated induction of granulocyte colony stimulating factor (G-CSF) and interleukin (IL-6) levels in lethally irradiated mice. Considerable attenuation in radiation induced morphological damage to the intestinal villi, crypts and mucosal layers was observed in G-003M pre-treated mice. Additionally, our formulation did not reduce the sensitivity of tumor tissue to radiation. Altogether, these results suggest that G-003M ameliorates the deleterious effects of radiation exposure by minimising ROS and NO generation and effectively regulating inflammatory and cell death pathways. Mechanism of protection elucidated in the current study demonstrates that G-003M can be used as a safe and effective radio protective agent in radiotherapy for human application.</p></div

G-003M negatively regulates p53 signaling pathways and bax, bcl2 expression ratio in mice jejunum.

<p>(A) G-003M was administered 60 min prior to 9 Gy dose of gamma radiation and protein extracts (30 μg) were loaded onto 12% SDS-polyacrylamide gel, electrophoresed and transferred to nitrocellulose membrane and probed with p53, PUMA, MDM-2, p21 antibody. The western blot is representative results of three independent experiments. Quantification of p53, PUMA, MDM-2, p21 expression was normalised to β-actin expression, using IMAGE lab software for densitometry. (B) G-003M treatment resulted in up regulation of bcl2 and downregulation of bax expression at both 6 hr and 24 hr post-irradiation in mice jejunum as evident by western blot analysis. Each data point represents mean of triplicates ± SD. A value of p < 0.05 is considered statistically significant. ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p

G-003M regulates cytokines (IL-6, TNF-α and G-CSF) levels.

<p>After isolation of blood serum at different time points (6 hr, 24 hr) following 9 Gy γ-irradiation, serum levels of (A) IL-6 (B) TNF-α and (C) G-CSF cytokines (pg/ml) in different treatment groups was determined by flow cytometry using respective BD^TM Cytometric Bead Array (CBA) Flex Set. Each data point represents mean of triplicate ± SD. ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p

G-003M treatment minimised radiation-induced histological alterations during intestinal injury.

<p>H&E-stained sections of C57BL/6J jejunum obtained at (B) (C) (D) 5^th day, (E) (F) 10^th day and (G) 30^th day after exposure to 9 Gy TBI with or without G-003M pretreatment. Representative micrographs were taken at 20X magnification.</p

G-003M blocked nitric oxide (NO) generation in intestinal epithelial cells (IEC) and peritoneal macrophages.

<p>(A) Flow cytometry histogram showing changes in NO levels in IEC of mice (n = 3) that were left untreated (U/t), G-003M alone, exposed to 9 Gy TBI 60 min after injection of DMSO (9 Gy) or G-003M (G-003M+9Gy). Bar diagram showing % change in intracellular NO production at different time intervals post irradiation compared to untreated mice and in different treatment groups is also shown. (B) Depicts NO levels in peritoneal macrophages after exposure to 9 Gy TBI with or without G-003M treatment. Quantification of NO level is presented as percent change in mean fluorescence intensity of different treatment groups. Cells from untreated mice were used as controls. All the figures are representative results of three independent experiments and expressed as mean±SD. A value of p<0.05 is considered statistically significant. ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p

G-003M attenuates radiation-induced apoptosis in mice IEC.

<p>(A) IEC were collected from different treatment groups at 6 hr after irradiation, stained with propidium iodide and Annexin V-FITC and analyzed by flow cytometry. Representative diagrams of distribution of stained cells are shown. Bar graphs of (B) early apoptotic and (C) late apoptotic cells expressed as a percent of total cells for each treatment with SD from three independent experiments are shown. A value of p < 0.05 is considered statistically significant ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p

G-003M reduces lethal radiation induced ROS level in mice intestinal epithelial cells (IEC).

<p>(A) Flow cytometry histogram showing changes in the ROS levels of IEC in mice (n = 3) that were left untreated (U/t), given G-003M without 9Gy radiation (G-003M), or exposed to 9 Gy TBI 60 min after injection of DMSO (9 Gy) or G-003M (G-003M+9 Gy). Bar diagram showing % change in intracellular ROS production at different time intervals post irradiation compared to untreated mice and in different treatment groups is also shown. (B) Fluorescent microscope (magnification; 40X) imaging of ROS levels in IEC was observed in mice injected with G-003M or DMSO 60 min before 9 Gy TBI. The ROS levels were measured in IEC after 1 hr of treatment by staining with 2',7'-dichlorofluorescein diacetate (DCF-DA) dye. All the figures are representative of three independent experiments and expressed as mean± SD. A value of p<0.05 is considered statistically significant. ns = nonsignificant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.</p