Protective Effect And Molecular Mechanism Of Pterostilbene Against Arsenic-induced Epithelial Cell Damage

Arsenic,one of a common constituent of the Earth's crust,has a certain degree of harm to human health.The inorganic form of arsenic,especially the trivalent oxidation state NaAsO₂ and As2O3,is generally considered highly toxic.Previous researches have shown that long-term exposure to drinking water with excessive levels of arsenic increases the risk of various types of cancer in the skin,lung,kidney and liver.In addition,exposure to arsenic can cause various skin injuries such as Bowen's disease and pigmentation disorders.Notably,skin damage caused by arsenic exposure occur after an incubation period of 5-10 years,and related diseases continue to develop decades after the cessation of arsenic exposure,indicating that long-term prevention of arsenic toxicity is required.Exposure to inorganic arsenic salts may induce oxidative stress,which leads to increased reactive oxygen species?ROS?,reduced cell viability,DNA damage,cell mutation,and apoptosis.Nrf2 is identified as a key transcription factor for cellular redox homeostasis and oxidative stress.Nrf2 regulates the expression of multiple antioxidant enzymes including heme oxygenase1?HO-1?,NADPH,quinone oxidoreductase 1?NQO-1?,and superoxide dismutase?SOD?.dismutase?SOD?.Earlier studies have shown that Nrf2 plays an important role in cellular anti-oxidative stress injury induced by inorganic arsenic.Activation of Nrf2 and Nrf2-regulated antioxidant and detoxification enzyme expression could protect Ha Ca T cells from arsenic-induced apoptosis and cytotoxicity.Therefore,the activating of Nrf2 expression is essential to protect against arsenic-induced cell damage.Previous studies have proved that the natural compound Pterostilbene?Pts?presented good antioxidant effects.Pterostilbene can reduce the activity of reactive oxygen species including hydrogen peroxide and superoxide anion.It can increase the expression of catalase in different cell lines,such as total glutathione,glutathione peroxidase,glutathione reductase,superoxide dismutase,etc.It may activate nuclearfactor-related factor 2 to exert its antioxidant effect through many interrelated mechanisms.Through the antioxidant effect,Pts can regulate and improve the antioxidant stress level of target cells,and protect cells from cell injury such as apoptosis and necrosis caused by various factors.Thus,in this study,we investigated the protective effect of Pts on NaAsO₂-induced cytotoxicity through cell viability assays.The DCFH-DA method was used to detect the alleviating effect of Pts on NaAsO₂-induced ROS generation.Intracellular lipid peroxidation?MDA?and superoxide dismutase?SOD?concentrations were measured to determine the antioxidant effect of Pts.JC-1 assays were performed to detect the mitochondrial membrane potential.To quantify the apoptotic cells,flow cytometry methods were performed using annexin V-FITC and propidium iodide?PI?double staining.Then,in in vivo assays,mice were supplied with drinking water containing NaAsO₂ for poisoning and then treated with Pts.By measuring the average weight changes and weight gain of mice during experiment,organ index changes,histopathological changes of liver and skin,changes in lymphocyte populations in splenocytes and thymocytes of mice,the protective effects of Pts on mice exposed to NaAsO₂ were studied.Furthermore,by western blot analysis,we determined the expression of apoptosis-related proteins in cells after Pts treatment.At last,we used Nrf2 si RNA to confirm the mechanism of Pts on antioxidant enzymes and NaAsO₂-induced cytotoxicity.Our data showed that: Pts alone had no significant effect on cell viability in the tested concentrations,but showed significant protective effects against NaAsO₂-induced cytotoxicity.Further,Pts decreased NaAsO₂-induced ROS generation,MDA formation,increased SOD content,ameliorated NaAsO₂-induced mitochondrial dysfunction.Furthermore,treatment with NaAsO₂ increased the release of cytochrome c from mitochondria into the cytoplasm clearly,while the effect was reversed by Pts pretreatment.And Pts inhibited NaAsO₂-induced cell apoptosis.In in vivo assays,we found that Pts alleviated the decrease in weight gain and liver index of mice caused by NaAsO₂ exposure.In histopathological examination,Pts alleviated liver damage and prevented skin keratinizing.Besides,Pts significantly reversed the changes of lymphocyte populations in splenocytes and thymocytes of mice induced by NaAsO₂ exposure,thereby protecting the immune function of mice.In addition,treatment of cells with Pts markedly increased AMPK and AKTphosphorylation,the translocation of Nrf2 from cytoplasm to nucleus and the expression of antioxidant enzymes in a dose-dependent manner.Moreover,arsenic treatment decreased Bcl-2 and Bcl-xl protein expression and increased Bax,Bad and Caspase 3 protein expression compared with the control,but Pts pretreatment dramatically blocked these changes in a dose-dependent manner.The protective effects of Pts on NaAsO₂-induced cytotoxicity and ROS generation were remarkably attenuated in Nrf2 si RNA transfected cells,indicating that Pts-mediated protective effects on NaAsO₂-induced cytotoxicity were dependent on Nrf2 activation.In conclusion,we found that Pts had a significant protective effect on NaAsO₂-induced cell damage.In vivo,Pts could alleviate the pathological damage caused by NaAsO₂,and protected the immune function of mice.In addition,we identified that Nrf2 was essential for Pts to play the above roles.Pts protected cells from NaAsO₂-induced cell damage by activating a Nrf2-dependent antioxidant response.Our data provided a basis for the antioxidant effects of Pts against NaAsO₂ exposure.Pts may have potential in the prevention and treatment of skin damage and other diseases caused by arsenic exposure.