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Effects of esculin treatment on P2X7 receptor in experimental diabetic nephropathy
S110
A. Newman / Free Radical Biology and Medicine 128 (2018) S100–S114
2 purinergic receptors (P2RY2), which induce Ca2 þ -dependent signaling and activation of the NADPH oxidase dual oxidase 1 (DUOX1). Transient receptor potential (TRP) Ca2 þ channels such as TRP Vanilloid 1 (TRPV1) are important in e.g. pain responses, often in coordination with PAR2 and P2YR2 activation, and TRPV1 has recently been implicated in asthma pathology. Using a panel of pharmacological inhibitors and activators of P2YR2, PAR2, and TRPV1, we observed that each of these receptors can provoke activation of DUOX1 and secretion of IL-33 in primary human nasal epithelial (HNE) cells. Further mechanistic studies using primary mouse tracheal epithelial cells (MTECs) or human bronchial epithelial cells (HBE1) confirmed these findings, and showed that TRPV1 contributes importantly to innate responses to HDM and ALT, and the danger signal ATP which activates P2YR2, as shown by activation of DUOX1-dependent H2O2 production, activation of epidermal growth factor receptor signaling, and rapid secretion of IL-33, which were prevented using TRPV1 antagonists or TRPV1-siRNA. Strikingly, inhibition or siRNA silencing of P2YR2 also prevented innate responses to the TRPV1 activator capsaicin, highlighting a close functional relationship of these airway epithelial surface receptors in the innate allergen responses. Finally, airway IL-33 secretion and subsequent type 2 cytokine production in mice in response to acute airway HDM challenge were markedly impaired in TRPV1-deficient mice. Overall, our studies indicate a complex relationship between various receptor types in epithelial responses to environmental triggers, and highlight the importance of TRPV1 in such epithelial responses.
https://doi.org/10.1016/j.freeradbiomed.2018.10.265
259
LPS-induced systemic neonatal inflammation: blockage of P2X7R by BBG on neonatal phase decreases oxidative stress in hippocampus of adult rats Clivandir Silva 1,*, Michele Longoni Calio 1, Amanda Cristina Mosini 1, Jaime Moreira Pires 1, Débora Bandeira Rêgo 1, Luiz Eugênio Mello 2, Ana Teresa Leslie 1 1 2
Universidade Federal de São Paulo, Brazil Instituto D'Or de Pesquisa e Ensino, Brazil
The neonatal period is marked by intense maturation of the immune and central nervous systems and the exposure to inflammation during this phase of life may produce long-term effects on the neural system. Here, we examined the impact of neonatal lipopolysaccharide (LPS) exposure on anxiety and pain sensitivity, as well as the superoxide production in adult rats. We also assessed if the blockage of the P2X7R by its antagonist BBG could modulate the effects of LPS on the central nervous system. Wistar male rats were injected with saline (0.9% solution), LPS (1mg/kg, Escherichia coli) or LPS and BBG (50mg/Kg) on postnatal days (PNDs) 1, 3, 5 and 7. Anxiety was evaluated in the Elevated Plus Maze test and nociception in the Hot Plate and Tail Flick tests during adulthood at PNDs 80, 82 and 84 respectively. At PND89, euthanasia was performed and the brain collected for measuring superoxide levels by Dihydroethidium (DHE). No statistical differences were found in Elevated plus maze, Hot-plate nor Tail-flick tests. However, the persistent systemic neonatal inflammation lead to a higher production of superoxide anion in the hippocampus and the blockage of the P2X7R decreased its production (p o 0.001). These findings indicated that blocking the activation of the P2X7R during LPS exposure may have a potential therapeutic effect on the neural damage caused by the neonatal inflammation, in especial among the premature infants, who are susceptible to the exposure to the inflammation in the neonatal intensive care units.
https://doi.org/10.1016/j.freeradbiomed.2018.10.266
260
Effects of esculin treatment on P2X7 receptor in experimental diabetic nephropathy Robson Serralha, Adelson Rodrigues, Inri Rodrigues, Deyse Lima, Camila Farias, Moises Nascimento, Giovana Punaro, Elisa Mieko Suemitsu Higa* Universidade Federal de Sao Paulo, Brazil
Introduction: Diabetes mellitus is a chronic disease, which progresses with many complications such as the diabetic nephropathy (DN). P2X7 receptor is expressed in pathological conditions, in response to high concentrations of extracellular ATP, as seen in hyperglycemia. When constantly activated, P2X7 can induce inflammatory responses, leading to oxidative stress (OS) and triggering toxic biochemical processes. Coumarin derivatives, such as esculin, are mainly antioxidants but their pharmacodynamics are not yet fully understood. The aim of this study is to evaluate the effects of esculin on P2X7 expression in the kidneys of diabetic rats. Methods: In male Wistar rats 7 weeks old, diabetes was induced by a single dose of streptozotocin (60 mg/kg; i.v.); CTL group received only the vehicle. Diabetes was considered in animals with fasting glycaemia greater than 200 mg/dL, 48 hours after induction. Then, the animals received daily doses of esculin (50 mg/kg, p.o.) forming CTLþESC and DMþESC groups. 24-hours urine and a small aliquot of blood via retro-orbital plexus were collected for biochemical analysis. The animals were euthanized under anesthesia, at the end of the eighth week of protocol and the kidneys were collected for Western blotting analysis, using antibodies against P2X7. The results are described as mean 7 SEM; significance for po0.05. Results: The protein content of P2X7 was significantly decreased in the renal cortex of diabetic animals that received esculin when compared to untreated diabetic animals (0.7670.11 vs 1.3370.21). These animals also presented lower proteinuria (38.7274.82 vs 45.4273.0) and urinary TBARS excretion (232.3728.83 vs 282.077.2), both po0.05 compared with untreated diabetic animals. Conclusion: Our data suggest that esculin treatment decreases the P2X7 receptor in diabetic animals, accompanied by reduced OS and proteinuria, an early marker of DN, pointing to a new treatment that can be applied to control the evolution of this disease.
https://doi.org/10.1016/j.freeradbiomed.2018.10.267
261
Activation of Z-DNA binding protein 1 by mitochondrial DNA damage links oxidative stress to inflammation in epithelial cells Bartosz Szczesny* The University of Texas Medical Branch at Galveston, USA
This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response. In cultured lung epithelial cells, prolonged, low-level oxidative stress preferentially damages the mtDNA but not the nuclear DNA, causing cellular depletion of the mtDNA together with a transient remodeling of cellular bioenergetics and morphology without decreasing of cell viability. Concurrently, released from mitochondria oxidatively damaged mtDNA binds specifically to cytoplasmic Z-DNA binding protein 1 (ZBP1) and triggers expression of pro-inflammatory markers via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Oxidatively damaged mtDNA is
A. Newman / Free Radical Biology and Medicine 128 (2018) S100–S114
2 purinergic receptors (P2RY2), which induce Ca2 þ -dependent signaling and activation of the NADPH oxidase dual oxidase 1 (DUOX1). Transient receptor potential (TRP) Ca2 þ channels such as TRP Vanilloid 1 (TRPV1) are important in e.g. pain responses, often in coordination with PAR2 and P2YR2 activation, and TRPV1 has recently been implicated in asthma pathology. Using a panel of pharmacological inhibitors and activators of P2YR2, PAR2, and TRPV1, we observed that each of these receptors can provoke activation of DUOX1 and secretion of IL-33 in primary human nasal epithelial (HNE) cells. Further mechanistic studies using primary mouse tracheal epithelial cells (MTECs) or human bronchial epithelial cells (HBE1) confirmed these findings, and showed that TRPV1 contributes importantly to innate responses to HDM and ALT, and the danger signal ATP which activates P2YR2, as shown by activation of DUOX1-dependent H2O2 production, activation of epidermal growth factor receptor signaling, and rapid secretion of IL-33, which were prevented using TRPV1 antagonists or TRPV1-siRNA. Strikingly, inhibition or siRNA silencing of P2YR2 also prevented innate responses to the TRPV1 activator capsaicin, highlighting a close functional relationship of these airway epithelial surface receptors in the innate allergen responses. Finally, airway IL-33 secretion and subsequent type 2 cytokine production in mice in response to acute airway HDM challenge were markedly impaired in TRPV1-deficient mice. Overall, our studies indicate a complex relationship between various receptor types in epithelial responses to environmental triggers, and highlight the importance of TRPV1 in such epithelial responses.
https://doi.org/10.1016/j.freeradbiomed.2018.10.265
259
LPS-induced systemic neonatal inflammation: blockage of P2X7R by BBG on neonatal phase decreases oxidative stress in hippocampus of adult rats Clivandir Silva 1,*, Michele Longoni Calio 1, Amanda Cristina Mosini 1, Jaime Moreira Pires 1, Débora Bandeira Rêgo 1, Luiz Eugênio Mello 2, Ana Teresa Leslie 1 1 2
Universidade Federal de São Paulo, Brazil Instituto D'Or de Pesquisa e Ensino, Brazil
The neonatal period is marked by intense maturation of the immune and central nervous systems and the exposure to inflammation during this phase of life may produce long-term effects on the neural system. Here, we examined the impact of neonatal lipopolysaccharide (LPS) exposure on anxiety and pain sensitivity, as well as the superoxide production in adult rats. We also assessed if the blockage of the P2X7R by its antagonist BBG could modulate the effects of LPS on the central nervous system. Wistar male rats were injected with saline (0.9% solution), LPS (1mg/kg, Escherichia coli) or LPS and BBG (50mg/Kg) on postnatal days (PNDs) 1, 3, 5 and 7. Anxiety was evaluated in the Elevated Plus Maze test and nociception in the Hot Plate and Tail Flick tests during adulthood at PNDs 80, 82 and 84 respectively. At PND89, euthanasia was performed and the brain collected for measuring superoxide levels by Dihydroethidium (DHE). No statistical differences were found in Elevated plus maze, Hot-plate nor Tail-flick tests. However, the persistent systemic neonatal inflammation lead to a higher production of superoxide anion in the hippocampus and the blockage of the P2X7R decreased its production (p o 0.001). These findings indicated that blocking the activation of the P2X7R during LPS exposure may have a potential therapeutic effect on the neural damage caused by the neonatal inflammation, in especial among the premature infants, who are susceptible to the exposure to the inflammation in the neonatal intensive care units.
https://doi.org/10.1016/j.freeradbiomed.2018.10.266
260
Effects of esculin treatment on P2X7 receptor in experimental diabetic nephropathy Robson Serralha, Adelson Rodrigues, Inri Rodrigues, Deyse Lima, Camila Farias, Moises Nascimento, Giovana Punaro, Elisa Mieko Suemitsu Higa* Universidade Federal de Sao Paulo, Brazil
Introduction: Diabetes mellitus is a chronic disease, which progresses with many complications such as the diabetic nephropathy (DN). P2X7 receptor is expressed in pathological conditions, in response to high concentrations of extracellular ATP, as seen in hyperglycemia. When constantly activated, P2X7 can induce inflammatory responses, leading to oxidative stress (OS) and triggering toxic biochemical processes. Coumarin derivatives, such as esculin, are mainly antioxidants but their pharmacodynamics are not yet fully understood. The aim of this study is to evaluate the effects of esculin on P2X7 expression in the kidneys of diabetic rats. Methods: In male Wistar rats 7 weeks old, diabetes was induced by a single dose of streptozotocin (60 mg/kg; i.v.); CTL group received only the vehicle. Diabetes was considered in animals with fasting glycaemia greater than 200 mg/dL, 48 hours after induction. Then, the animals received daily doses of esculin (50 mg/kg, p.o.) forming CTLþESC and DMþESC groups. 24-hours urine and a small aliquot of blood via retro-orbital plexus were collected for biochemical analysis. The animals were euthanized under anesthesia, at the end of the eighth week of protocol and the kidneys were collected for Western blotting analysis, using antibodies against P2X7. The results are described as mean 7 SEM; significance for po0.05. Results: The protein content of P2X7 was significantly decreased in the renal cortex of diabetic animals that received esculin when compared to untreated diabetic animals (0.7670.11 vs 1.3370.21). These animals also presented lower proteinuria (38.7274.82 vs 45.4273.0) and urinary TBARS excretion (232.3728.83 vs 282.077.2), both po0.05 compared with untreated diabetic animals. Conclusion: Our data suggest that esculin treatment decreases the P2X7 receptor in diabetic animals, accompanied by reduced OS and proteinuria, an early marker of DN, pointing to a new treatment that can be applied to control the evolution of this disease.
https://doi.org/10.1016/j.freeradbiomed.2018.10.267
261
Activation of Z-DNA binding protein 1 by mitochondrial DNA damage links oxidative stress to inflammation in epithelial cells Bartosz Szczesny* The University of Texas Medical Branch at Galveston, USA
This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response. In cultured lung epithelial cells, prolonged, low-level oxidative stress preferentially damages the mtDNA but not the nuclear DNA, causing cellular depletion of the mtDNA together with a transient remodeling of cellular bioenergetics and morphology without decreasing of cell viability. Concurrently, released from mitochondria oxidatively damaged mtDNA binds specifically to cytoplasmic Z-DNA binding protein 1 (ZBP1) and triggers expression of pro-inflammatory markers via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Oxidatively damaged mtDNA is