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The potential phosphorylation motifs of human SIRT3 were predicted by searching the data base using the combination of software Netphos2.0 server and Automotif server

Local mice xenograft tumor radiation settings showing the images of xenograft tumors derived from HCT116 cells harboring Thr150/Ser159 mutant or wild type SIRT3

Clonogenic survival ability of HCT116 cells via MnSOD activation

Western blot of mitochondrial acetylated proteins from irradiated MDA-231 or U87 cells harboring wild-type or Thr150/Ser159 mutant SIRT3.

MnSOD activity in MCF10A cells harboring mock, wild type or Thr150/Ser159 mutant SIRT3 following irradiation

Western blot analysis of SIRT3 and acetylated mitochondrial proteins of human breast cancer MDA-231 cells and glioblastoma U87 cells

Inhalation of high concentrations of phosgene often causes pulmonary edema, which obstructs the airway and causes tissue hypoxia. There is currently no specific antidote. This study was performed to investigate the effect behind pentoxifylline (PTX) treatment for phosgene-induced lung injury in rat models. Rats were exposed to phosgene. The protein levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and occludin proteins in lung tissue were determined. The effect of both prophylactic and therapeutic administration of PTX (50 mg/kg and 100 mg/kg) was evaluated. The lung permeability index and HIF-1α protein level increased, the arterial blood oxygenation index (PaO

Objective Chlorine is a chemical threat agent that can be harmful to humans. Inhalation of high levels of chlorine can lead to acute lung injury (ALI). Currently, there is no satisfactory treatment, and effective antidote is urgently needed. Pentoxifylline (PTX), a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, is widely used for the treatment of vascular disorders. The present study was aimed to investigate the inhibitory effects of PTX on chlorine-induced ALI in rats. Methods Adult male Sprague-Dawley rats were exposed to 400 ppm Cl2 for 5 min. The histopathological examination was carried out and intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. Subsequently, to evaluate the effect of PTX, a dose of 100 mg/kg was administered. The activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG) and lactate dehydrogenase (LDH) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expressions of SOD1, SOD2, catalase (CAT), hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), occludin, E-cadherin, bcl-xl, LC 3, Beclin 1, PTEN-induced putative kinase 1 (PINK 1) and Parkin. Results The histopathological examination demonstrated that chlorine could destroy the lung structure with hemorrhage, alveolar collapse, and inflammatory infiltration. ROS accumulation was significantly higher in the lungs of rats suffering from inhaling chlorine (PPPPPPP Conclusion PTX could ameliorate chlorine-induced lung injury via inhibition effects on oxidative stress, hypoxia and autophagy, thus suggesting that PTX could serve as a potential therapeutic approach for ALI.

Previous studies indicated that oxidative stress was involved in phosgene-induced acute lung injury (ALI) and many antioxidants had been used to prevent ALI. N-acetylcysteine (NAC) had been used to protect ALI induced by various types of oxidative stress. Considering the limited information of NAC on phosgene-induced ALI, the purpose of this study was to elucidate the molecular mechanisms of phosgene-induced ALI and the protective effects of NAC. This study discovered that intraperitoneal administration of NAC significantly alleviated phosgene-induced pulmonary edema, as confirmed by decreased lung wet to dry weight ratio and oxidative stress markers. The content of l-gamma-glutamyl-l-cysteinyl-glycine (glutathione; GSH) and the ratio of the reduced and disulfide forms (GSH/GSSG), significant indicators of the antioxidative ability, were apparently inhibited by phosgene exposure. However, both indicators could be reversed by NAC administration, indicating that dysregulation of redox status of glutathione might be the cause of phosgene-induced ALI. The nuclear factor (NF)-E2-related factor 2 (Nrf2), which has been proven to up-regulate the expression of glutathione reductase (GR), was obviously decreased by phosgene exposure. However, NAC administration elevated Nrf2 expression significantly. In conclusion, these data provided the first evidences showing that it was the transcriptional factor Nrf2 that connected phosgene-induced ALI with GSH metabolism. NAC protected against oxidative stress through acting on this newly disclosed Nrf2/GR/GSH pathway, by which NAC elevated the biosynthesis of protective GSH to repair and reconstitute the defense system destroyed by phosgene.

Tumor adaptive resistance to therapeutic radiation remains a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of NAD+-dependent protein deacetylases in mitochondria, promotes metabolic homeostasis through regulation of mitochondrial protein deacetylation and plays a key role in prevention of cell aging. Here, we demonstrate that SIRT3 expression is induced in an array of radiation-treated human tumor cells and their corresponding xenograft tumors, including colon cancer HCT-116, glioblastoma U87, and breast cancer MDA-MB231 cells. SIRT3 transcriptional activation is due to SIRT3 promoter activation controlled by the stress transcription factor NF-κB. Posttranscriptionally, SIRT3 enzymatic activity is further enhanced via Thr150/Ser159 phosphorylation by cyclin B1–CDK1, which is also induced by radiation and relocated to mitochondria together with SIRT3. Cells expressing Thr150Ala/Ser159Ala-mutant SIRT3 show a reduction in mitochondrial protein lysine deacetylation, Δψm, MnSOD activity, and mitochondrial ATP generation. The clonogenicity of Thr150Ala/Ser159Ala-mutant transfectants is lower and significantly decreased under radiation. Tumors harboring Thr150Ala/Ser159Ala-mutant SIRT3 show inhibited growth and increased sensitivity to in vivo local irradiation. These results demonstrate that enhanced SIRT3 transcription and posttranslational modifications in mitochondria contribute to adaptive radioresistance in tumor cells. CDK1-mediated SIRT3 phosphorylation is a potential effective target to sensitize tumor cells to radiotherapy. Mol Cancer Ther; 14(9); 2090–102. ©2015 AACR.

Oxidative stress is associated with insulin resistance (IR) and is thought to contribute to the development and progression toward type 2 diabetes (T2DM). This study was undertaken to isolate the bioactive polysaccharide (SMPW1) from Salvia miltiorrhiza Bunge and investigated its protective effects on IR model in rats induced by tert-butyl hydroperoxide (t-BHP). In vivo animal experiments showed that SMPW1 (50 and 100mg/kg) possessed high antioxidative and protective capacity against the injury induced by t-BHP, as reflected in the increased expression or activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the decreased formation of malondialdehyde (MDA) in serum and liver homogenates. In addition, SMPW1 (50 and 100mg/kg) also attenuated IR and the morphological injury of liver and pancreas induced by t-BHP, and improved insulin sensitivity index. In conclusion, SMPW1 can protect against the development of T2DM and improve IR via reduction of oxidative stress.

Phosgene is a poorly water-soluble gas penetrating the lower respiratory tract which can induce acute lung injury characterized by a latent phase of fatal pulmonary edema. Pulmonary edema caused by phosgene is believed to be a consequence of oxidative stress and inflammatory responses. Ethyl pyruvate (EP) has been demonstrated to have anti-inflammatory and anti-oxidative properties in vivo and in vitro. The potential therapeutic role of EP in phosgene-induced pulmonary edema has not been addressed so far. In the present study, we aim to investigate the protective effects of EP on phosgene-induced pulmonary edema and the underlying mechanisms. Rats were administered with EP (40 mg kg(-1)) and RAW264.7 cells were also incubated with it (0, 2, 5 or 10 µm) immediately after phosgene (400 ppm, 1 min) or air exposure. Wet-to-dry lung weight ratio (W:D ratio), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, cyclooxygenase2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and mitogen-activated protein kinases activities (MAPKs) were measured. Our results showed that EP treatment attenuated phosgene-induced pulmonary edema and decreased the level of NO and PGE(2) dose-dependently. Furthermore, EP significantly reduced COX-2 expression, iNOS expression and MAPK activation induced by phosgene. Moreover, specific inhibitors of MAPKs reduced COX-2 and iNOS expression induced by phosgene. These findings suggested that EP has a protective role against phosgene-induced pulmonary edema, which is mediated in part by inhibiting MAPK activation and subsequently down-regulating COX-2 and iNOS expression as well as decreasing the production of NO and PGE(2).

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia. Although the clear mechanisms of DM and insulin resistance are still to be cleared, it has been well documented that reactive oxygen species (ROS) play a pivotal role in DM and multiple types of insulin resistance. For the past few years, natural substances have been shown to have the potential to treatment DM. Attention has been especially focused on plants rich in triterpenoids, which generally show antioxidant and antiglycation effect. In our previous studies, it was shown that oleanolic acid (OA), a natural triterpenoid and an aglycone of many saponins, is a potent antioxidant acting as not only a free radical-scavenger through direct chemical reactions but also as a biological molecule, which may enhance the antioxidant defenses. The present study aimed to investigate the potential antidiabetic effect of OA. Oleanolic acid showed a significant blood glucose-lowering and weight-losing effect in diabetic animals induced by streptozotocin (STZ). In the insulin resistant model, it was also shown that OA may promote insulin signal transduction and inhibit oxidative stress-induced hepatic insulin resistance and gluconeogenesis, in which process the phosphorylation of ERK and the protective effect on mitochondrial function may be involved. These findings may significantly better the understanding of the pharmacological actions of OA and advance therapeutic approaches to DM.

Since biological functions of the elements are generally different, depending on their chemical forms, chemical speciation analysis is really important in metallomics research. Thus, multielement analysis and chemical speciation of the elements in serum were carried out in the present work. A hyphenated technique was developed for high-throughput speciation analysis of the copper, iron and zinc in serum by molecular biology technology and flame atomic absorption spectrophotometry (AAS). Here, Cu, Fe and Zn in serum were classifyied as the forms of combination and non-combination. The serum protein was precipitated by 60% concentration of ethanol under hypothermy. The forms of combination of Cu, Fe and Zn in serum which combined with proteins were in precipitations, and the forms of non-combination of Cu, Fe and Zn in serum, which were free ions, were in supernatant. The total amount of Cu, Fe and Zn in serum and the amount of the forms of non-combination of Cu, Fe and Zn were analyzed by AAS. The amount of the forms of combination of Cu, Fe and Zn was obtained by calculation. The detection limit of Cu in serum by the method is around and 9.84 x 10(-3) microg x mL(-1). For Fe and Zn, the detection limit is about 2.76 x 10(-2) microg x mL(-1) and 1.06 x 10(-3) microg x mL(-1), respectively. The percentage recovery of trace elements Cu, Fe and Zn by the proposed procedure is in the range 95.0%-101.0%, 95.0%-102.0% and 95.0%-103.0%, respectively. The relative standard deviation (RSD) of trace elements Cu, Fe and Zn in the serum is in the range 1.88%-2.26%, 0.56%-1.59% and 0.34%-1.36%, respectively. Speciation of trace elements Cu, Fe and Zn in the serum of SD rat were analyzed by the method.

To study apoptosis of pulmonary epithelial cells and endothelial cells in mice with pulmonary edema induced by phosgene exposure.Thirty-two mice were divided into normal group and phosgene group with 16 mice in each group. The mice in phosgene group were exposed to phosgene (11.9 mg/L) for 5 min and those in the control group to air. Four hours after exposure, alveolar type II cells were isolated and cultured to observe their apoptosis by electron microscope and flow cytometry. The lung tissues were also taken for DNA gel electrophoresis and TUNEL assay.Apoptotic bodies were observed in alveolar type II cells under electron microscope in phosgene group, which had higher cell apoptosis rate than the control group [(40.26+/-7.74)% vs (1.58+/-1.01)%, P0.001] as determined by flow cytometry. Ladder-like DNA fragmentation pattern was observed in DNA gel electrophoresis in phosgene group with apoptosis of the pulmonary epithelial and endothelial cells observed by TUNEL.Phosgene can induce pulmonary epithelial and endothelial cell apoptosis in mice, suggesting that the mechanism of phosgene-induced pulmonary edema involves apoptosis of the lung cells.

To study the protective effect of hyperoxia solution on acute lung injury caused by phosgene poisoning by observing the changes of PaO2 and malondialdehyde (MDA) contents, superoxide dismutase (SOD) activity in serum and Glutathione (GSH/GSSG) contents in lung tissues.The rabbits were divided into normal control group, hyperoxia solution (H0) and balance salt (BS) groups. Group HO and Group BS inhaled phosgene and the former was given intravenously hyperoxia solution (which was replaced by balance salt solution in Group BS). The content of MDA and the activity of SOD in serum were observed at different time points, the amount of GSH and GSSG in lung tissue were also measured.(1) The serum MDA contents increased and PaO2, SOD activity decreased significantly in Group HO and Group BS along with time increasing as compared with control group. The contents of GSH in lung tissue decreased in two groups compared with that in control group, however the contents of GSSG ascended instead. (2) At 3 and 8 h of the experiment, PaO2 of Group HO [(9.91 +/- 0.49), (9.15 +/- 0.46) mm Hg respectively] were significantly higher than those of Group BS [(9.03 +/- 0.76), (8.11 +/- 0.57) mm Hg respectively] (P0.01). The contents of MDA of Group HO (3.66 +/- 0.35), (5.31 +/- 0.15) micromol/L respectively] were lower than those of Group BS [(4.32 +/- 0.26), (7.4 +/- 0.33) micromol/L respectively] (P0.01). SOD activity in Group HO [(237.37 +/- 29.96), (208.10 +/- 18.80) NU/ml respectively] were higher than those of Group BS [(195.02 +/- 21.44), (144.87 +/- 21.26) NU/ml respectively] (P0.05 or P0.01). The content of GSSG lung tissue in Group HO (423.67 +/- 38.21) micromol/L were lower than those of Group BS (523.85 +/- 43.14) mol/L (P0.01). There were no significant differences in the content of GSH in lung tissues between Group HO and group BS.Hyperoxia solution can reduce acute lung injury of rabbits following phosgene poisoning.

To study the apoptosis of alveolar type II cells, alterations of vascular endothelial growth factor (VEGF), VEGF receptor (Flt1) in serum and lung and expression of VEGF mRNA in lung in pulmonary edema mice induced by phosgene.Twenty-six BALB/C mice were randomly divided into 2 groups: control group, exposed group (13 mice in each group). Mice of exposed group were intoxicated by inhalation of phosgene 11.9 mg/L for 5 minutes. Mice of control group were treated as the same way by inhalation of air. Isolation of mice alveolus type II cells 4 h after intoxication was carried out to observe their apoptosis under electron microscope. Contents of VEGF and Flt1 in lung and serum by ELISA, and expression of VEGF mRNA were determined.Alveolar type II cells were identified by tannic acid staining and electron microscopy. After exposed to 11.9 mg/L of phosgene for 5 minutes, the apoptotic body in alveolus type II cells was found in exposed group. The contents of VEGF in serum and lung and Flt1 in lung of exposed mice [(134.07 +/- 120.26), (477.76 +/- 98.06), (1,2818.48 +/- 2,304.15) pg/ml] were significantly lower than those of control group [(445.57 +/- 173.30), (1,026.87 +/- 474.56), (21,976.51 +/- 7,421.01) pg/ml, P0.05] but the content of Flt1 in serum [(2,369.56 +/- 381.70) pg/ml] was higher than that in control group [(1,898.00 +/- 453.69) pg/ml, P0.05]. The expression of VEGF mRNA in pulmonary edema mice was decreased.Phosgene can induce apoptosis of alveolar type II cells, and decrease in the content of VEGF and Flt1, and expression of VEGF mRNA in lung.

To study the effect of acute phosgene inhalation on the antioxidant enzymes, nitric oxide (NO) and nitric oxide synthase (NOS) in rats.Phosgene was produced by decomposing bis (trichdomethyl) carbonate in the presence of N,N-dimethyl formamide. SD rats were randomly divided into two groups: control and phosgene exposure groups. In a special experimental device with equipment modulating the gas flow, phosgene exposed rats inhaled phosgene quantitatively for five minutes. Two hours later, all the rats were sacrificed and the ratio of wet weight to dried weight of lung (WW/DW) was calculated. Peripheral blood, serum and liver were collected to examine the activities of antioxidant enzymes including glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), NOS, and NO level. The total content of proteins were also determined.The WW/DW ratio of lung in phosgene exposure group was much higher than that in control group (P0.01). The activities of GST in serum and liver of phosgene exposure group increased significantly (P0.05). The activities of SOD, CAT, GSHPx and NOS in serum or blood and liver of phosgene exposure group were also increased significantly (P0.05). But the content of NO was significantly decreased (P0.01).Acute phosgene inhalation may cause a dramatically changes of several antioxidant enzyme activities, and acute injury of liver to some extent in rats. The latter is related to reactive oxygen species. But the elevation of antioxidant enzyme activities suggests that antioxidative treatment for acute phosgene poisoning should not be considered first.

To observe the pro-apoptotic effect of RCasp-6 gene in Hela cells.RCasp-6 gene was amplified by PCR and cloned into the pIND vector. Hela cells were transfected with pIND-RCasp-6 and then inducced with ecdyson analogue. Expression of target gene was detected by immunocytochemical staining. Changes of the morphology and growth of Hela cells subjected to transfection of target gene were observed by HE staining and viable cell counting.RCasp-6 gene was cloned by PCR and its eukaryotic expression vector was successfully reconstructed. Expression of RCasp-6 gene in the transfected Hela cells leads to the morphological changes of the cells. Many of the transfected Hela cells shrunk and some cells were died.The expression of reconstructed human caspase-6 gene can efficiently accelerate death of Hela cells.

To observe the significance of ricin (RT) with chemical nidification to reduce the hepatotoxicity in mice and its anticancer effect.Mice were exposed to RT and RT-PDP [ricin chemically modified by N-succinimidyl 3-(2-pyridyldithio)-propionate, (SPDP)] respectively, and their serum activity of glutathione-S-transferase (GST) and liver glutathione (GSH) content were determined. The ultramicro-structure under electron microscope was also observed.The GST activity increased with doses, and the increase in ricin group was higher than that in RT-PDP group; the activities of GST in RT group at 12.5, 15.0 micro g/kg [(93.65 +/- 12.30), (153.71 +/- 26.64) IU/L respectively] were higher than those in RT-PDP group [(62.97 +/- 11.22), (78.20 +/- 15.71) IU/L] (P0.05). The contents of GSH were decreased with doses; but the contents of GSH in RT-PDP group at 2.5, 5.0, 7.5, 10.0, 15.0 micro g/kg [(6.34 +/- 1.43), (4.14 +/- 1.82), (3.54 +/- 0.64), (2.73 +/- 1.82), (1.82 +/- 0.62) micro mol/L respectively] were still higher than those in RT group [(3.53 +/- 0.95), (2.12 +/- 0.54), (1.82 +/- 0.71), (1.52 +/- 0.34), (0.81 +/- 0.36) micro mol/L] (P0.01). Electron microscopic examination showed that the injury of liver cells in RT group was more severe than that in RT-PDP group.The hepatotoxicity of ricin in mice may be reduced by chemical modification.