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1. The objective of this study was to measure the effect of gallic acid on levels of ferric reducing antioxidant power, malondialdehyde, hydrogen peroxide, nitric oxide and the viability of broiler blood cells (BBCs) when exposed to high ambient temperature. 2. The BBCs were maintained at 41.5°C (control group, CG) or at ambient temperatures ranging from 41.5°C to 46°C. At 41.5°C to 46°C, BBCs were diluted with gallic acid at 0 (positive control group, PCG), 6.25, 12.5, 25 and 50 µM, respectively. Ferric reducing antioxidant power, malondialdehyde, hydrogen peroxide, nitric oxide and viability of BBCs were investigated. 3. Hydrogen peroxide, malondialdehyde and nitric oxide for the CG was lower than PCG (P < 0.05). However, the viability of CG was higher than PCG (P < 0.05). At 41.5 to 46°C, malondialdehyde, hydrogen peroxide, and nitric oxide of BBCs diluted with gallic acid were lower compared to PCG (P < 0.05). Viability of BBCs diluted with gallic acid was higher than PCG (P < 0.05). 4. These results indicated that gallic acid could reduce the adverse oxidative effects of high ambient temperature on BBCs, with an optimum dilution rate of 12.5 µM. [ABSTRACT FROM AUTHOR]

The aim of this experiment was to study the effect of N-Acetylcysteine (NAC) on oxidative and nitrosative stress, and on the viability of broiler blood cells (BBC) when exposed to high ambient temperature. The BBC were maintained at an ambient temperature of 41.5°C (normal broiler body temperature, NBBT) and compared with high ambient temperatures ranging from 41.5°C to 46°C (HAT). At HAT, BBC were diluted with NAC at concentrations of 0 (the positive control group, TPCG), 250, 500, 750 and 1000 μM. Total antioxidant power or ferric reducing antioxidant power (FRAP), malondialdehyde (MDA), hydrogen peroxide (H2O2), nitric oxide (NO), and the viability of the BBC were measured. It was found that MDA, H2O2, and NO of BBC at NBBT were lower than in the TPCG (P< 0.05). Cell viability of BBC at NBBT was higher than of the TPCG (P< 0.05). At HAT, MDA, H2O2, and NO of BBC diluted with NAC at the concentration of 250 μM were lower than in the TPCG (P< 0.05), and viability of cells of the TPCG was lower than in other groups (P< 0.05). These phenomena showed that NAC could decrease oxidative stress and NO, and could increase the viability of BBC under high ambient temperatures. Finally, a suitable concentration of NAC for BBC was 250 μM. [ABSTRACT FROM AUTHOR]

In broilers, the influence of high environmental temperatures (ET) leading to heat stress and oxidative stress was investigated. While almost all previous studies have been conducted in-vivo, the aim of this study is to investigate the effects of increasing temperature on expression of heat shock protein 60 (Hsp60), oxidative stress, antioxidants, apoptosis, and electrolyte changes of broiler blood cells (BBC) in-vitro. In-vitro studies can reduce the number of animals used for the experiment and avoid potential suffering of experimental animals. This study was divided into 2 trials. In Trial 1 the effect of increasing temperature (ranging from 41°C to 50°C) on Hsp60 expression were studied. Trial 2 was aimed to investigate effects of increasing temperature (ranging from 41°C to 50°C) on malondialdehyde, total antioxidant power, reduced glutathione, glutathione peroxidase, apoptosis and electrolyte changes in BBC, respectively. The following results were obtained: Trial 1, in BBC, Hsp60 was released at 46°C. Trial 2, reduced glutathione and glutathione peroxidase of BBC decreased when ET increased. ET, which caused approximately 50% apoptosis of BBC, was at 45°C. When ET increased, sodium and potassium ions increased, but, chloride ions decreased. These experiments showed that BBC maintained at a high ET were under heat stress, oxidative stress and undergoing apoptosis. Lastly, a high ET caused changes of electrolyte equilibrium of BBC in-vitro. From the present study, new information was obtained on the exact temperature that induces a significant secretion of Hsp60 and apoptosis of BBC, as well as the cause of the changes in the electrolyte balance of BBC when exposed to high ET invitro. This study may serve as a model for studying the effects of global warming on poultry. In the search for solutions to this problem, in-vitro studies should be carried out before animal experiments. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine the effect of increased ambient temperature on Hsp70 expression, superoxide dismutase (SOD) activity, nitric oxide (NO), malondialdehyde (MDA), caspase 8, caspase 9 and caspase 3 in relation to the intrinsic and extrinsic apoptosis pathway (IAP and EAP) of broiler blood cells (BBC). BBC were maintained at 42 °C and at 5 ranges of temperatures; 42-43, 44-45, 46-47, 48-49, and 50-51 °C. Then Hsp70 expression, SOD activity, NO, MDA, activity of caspase 8, caspase 9 and caspase 3, and living and apoptotic BBC were measured. It was found that Hsp70 expression and SOD were increased at 42-43 °C (P < 0.05), while the activity of caspase 9 and caspase 3 decreased (P < 0.05). At higher temperatures of 44-45 °C, caspase 9 and caspase 3 activities were increased and were higher than at 42 °C (P < 0.05). Apoptosis commenced in the temperature range 46-47 °C; during this range of temperature, NO increased (P < 0.05), but it decreased at 48-49 °C (P < 0.05). At the same time, activity of SOD in BBC was increased, achieving the highest levels (P < 0.05). In addition, activity of caspase 8 and caspase 3 at 48-49 °C was higher than that at 42 °C, and the apoptotic BBC was increased (P < 0.05). Therefore, temperature effects on BBC could be divided into three phases. The first phase was the temperature at 42 °C. The second phase, or pre-apoptotic phase, was in the temperature range from 42 to 45 °C. The third phase was the apoptosis phase, which manifested at 46 °C and above. These phenomena showed that in the pre-apoptosis phase, Hsp70 suppressed the IAP. On the other hand, in the apoptosis phase, apoptotic BBC is caused by the influence of NO, superoxide and MDA through the EAP., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The herbicide paraquat (PQ) is known to affect the immune system. Many reports have indicated that PQ impacts on the viability and functions of the immune cells, however, the underlying mechanism in detail is still unknown. The aim of this study was to evaluate the effects of PQ on the free radical production, oxidative stress, cell death and pro-inflammatory gene expression of murine bone marrow-derived macrophages (BMDMs) from C57BL/6NJcl mice in vitro. BMDMs were incubated with PQ at 0, 200 and 400 µM concentrations for 24 h. Intracellular reactive oxygen species (ROS) production, apoptosis, cell viability, nitric oxide, inducible nitric oxide synthase (iNOS), and IL-6 expression levels were measured. The results revealed that PQ treatments led to a decrease in the cell viability and induced apoptotic cell death in a dose-dependent manner. Additionally, PQ also induced the generation of ROS. The mRNA level of the pro-inflammatory mediator genes iNOS and IL-6 were also elevated, while the level of lipid peroxide (malondialdehyde) production remained unaltered. Interestingly, the PQ treatment led to a decrease in the nitric oxide production. These results indicate that the increased cellular ROS production, due to the PQ treatment, induces apoptosis and the herbicide triggers production of iNOS and IL-6 in BMDMs.

Chemotherapy is a palliative treatment for unresectable patients with cholangiocarcinoma (CCA). However, drug resistance is a major cause of the failure of this treatment. Derrischalcone (DC), a novel chalcone isolated from Derris indica fruit, has been shown pharmacologically active; though, the effect of DC on CCA is unknown. The present study investigated the cytotoxic, antiproliferative, anti-migration, and anti-invasion effects and underlying mechanisms of DC on CCA KKU-M156 and KKU-100 cells. Cytotoxicity and apoptosis were evaluated by acridine orange and ethidium bromide fluorescent staining. Reactive oxygen species (ROS) was measured by dihydroethidium assay. Cell proliferation and reproductive cell death were assessed by sulforhodamine B staining and colony-forming assay. Migration and invasion were determined by wound healing and transwell chamber assays. Protein expressions associated with cell death, proliferation, migration, and invasion were analyzed by western immunoblotting. We found that DC induced cytotoxicity and apoptosis in association with ROS formation and oxidative stress. Treatment with N-acetylcysteine suppressed ROS formation and attenuated DC-induced cytotoxic and apoptotic effects. DC increased the expression of p53, p21, Bax, and cytochrome c proteins in association with cell death. DC-induced antiproliferation, colony formation, anti-migration, and anti-invasion were associated with the suppression of Akt/mTOR/cyclin D1 and FAK signaling pathways. These findings suggest that the multi-targeting strategies with DC may be a novel treatment for cancer therapy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Metformin (Met), an essential antidiabetic agent, shows antitumor activity in some cancers. A previous study showed that Met enhanced cytotoxic activity of cisplatin (Cis) in cholangiocarcinoma (CCA) in association with the activation of AMP-activated protein kinase and suppression of Akt-mTOR. However, these effects do not entirely explain the observed chemosensitizing effect. The present study investigated the interaction of Met and Cis over the enhanced antitumor effect., Main Methods: KKU-100 and KKU-M156 cells were used in the study. Cytotoxicity was assessed by acridine orange-ethidium bromide staining. Reactive oxygen species (ROS) and mitochondrial transmembrane potential (Δψ m ) were measured by dihydroethidium and JC-1 fluorescent methods. Cellular glutathione (GSH) and redox ratio were analyzed by enzymatic coupling assay. Proteins associated with antioxidant system and cell death were evaluated by western immunoblot., Key Findings: Cytotoxicity of Cis was enhanced by Met in association with ROS formation and GSH redox stress. The antioxidants, N-acetylcysteine and TEMPOL, and MPTP inhibitor, cyclosporine, attenuated cytotoxicity in association with suppression of ROS formation and the losses of Δψ m . Met in combination with Cis suppressed expression of Nrf2 and altered the expression of Bcl2 family proteins., Significance: The chemosensitizing effect of Met in combination with Cis is causally associated with increased oxidative stress-mediated mitochondrial cell death pathway. Met may improve the efficacy of Cis in the treatment of cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: AMP-activated protein kinase (AMPK) functions as a cellular energy sensor regulating various aspects of cellular metabolism. Metformin (Met), an activator of AMPK, has been reported to reduce the cancer risk and enhance antitumor effects in certain cancers. Cholangiocarcinoma (CCA) is an aggressive malignancy which rarely responds to chemotherapeutic agents. We investigated the chemosensitizing effects of Met in CCA cells., Materials and Methods: KKU-100 and KKU-452 cells were used in the study. Antiproliferation of Met and cisplatin (Cis) was analyzed by sulforhodamine B and colony forming assays. Apoptotic cell death was analyzed by acridine orange and ethidium bromide staining method. Cell cycle analysis was performed by flow cytometric method. Effects on cell migration and invasion were analyzed by wound healing assay and transwell chamber method. Expression of proteins was examined by western blot analysis., Key Findings: Met enhanced the antiproliferation of Cis, and conferred antimigration and anti-invasion in CCA cells, where Cis alone did not have two latter effects. This chemosensitizing effect is related to the activation of AMPK and suppression of Akt, mTOR and p70S6K. Met and Cis increased expression of p53 and p21 and suppressed expression of cyclin D1. This effect was associated with cell cycle arrest at S phase. The anti-invasion effect was casually associated with the suppression of FAK expression. The cytotoxic effect of the drug combination was mimicked by AICAR, an AMPK agonist., Significance: Met may be a novel agent to increase the efficacy of Cis to treat CCA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Transcription factor Nrf2, which regulates the expression of cytoprotective and antioxidant enzymes, contributes to proliferation and resistance to chemotherapy in cancer. The inhibition of Nrf2 can sensitize cholangiocarcinoma (CCA) cells to the cytotoxicity of several chemotherapeutic agents. In this study, we investigated the mechanism of this chemosensitizing effect., Main Methods: KKU-100 cells were used in the study. Nrf2 expression was knocked down by siRNA and expression was validated by reverse transcription and polymerase chain reaction. Cytotoxicity was assessed by sulforhodamine B method. Intracellular reactive oxygen species (ROS) was examined by fluorescent dye, dichlorofluorescin diacetate method and mitochondrial transmembrane potential was assessed by JC1 dye assay., Key Findings: Cytotoxicity of cisplatin (Cis) in KKU-100 cells was enhanced by knockdown of Nrf2 expression. The enhanced cytotoxic effect was abolished by treatment with N-acetylcysteine, TEMPOL and MnTBAP. Cells with Nrf2 knockdown or Cis treatment increased production of ROS, and ROS was markedly enhanced by a combination of Nrf2 knockdown and Cis. The increased ROS formation was associated with a decrease in mitochondrial transmembrane potential (Δψ m ), where this decrease was prevented by antioxidant compounds. The loss of Δψ m and cell death were prevented by cyclosporine, an inhibitor of mitochondrial permeability transition pore (MPTP). Luteolin inhibited Nrf2 and markedly enhanced cytotoxicity in combination with Cis., Significance: Inhibition of Nrf2 is a feasible strategy in enhancing antitumor activity of chemotherapeutic agents and improving efficacy of chemotherapy in CCA., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)