【作者】 周俊峰；

【导师】 李珊山；

【作者基本信息】 吉林大学 ， 皮肤性病学， 2020， 博士

【摘要】 砷元素是地壳中的常见化学元素之一,对人类的健康具有一定程度的危害。砷元素的无机形态,尤其是三价氧化态的亚砷酸钠（NaAsO₂）和三氧化二砷（As2O3）等的毒性很强。以往的研究表明,长期摄入砷元素含量超标的饮用水会增加罹患皮肤癌、肺癌、肝癌、肾癌在内的多种类型癌症的发病风险。此外,人体接触砷元素还会引发如Bowen’s病、色素沉着异常等在内的多种皮肤损伤。值得注意的是,砷暴露引发的皮肤损伤具有5-10年的潜伏期,且在砷暴露停止后的数十年内相关的疾病仍旧会进一步加重,提示对于砷元素的毒性需要长期预防。当砷元素进入机体后,能够引起细胞氧化应激,进而导致活性氧（ROS）升高、细胞活力下降、引发DNA损伤及细胞突变、细胞凋亡。Nrf2是细胞维持氧化还原稳态和对抗氧化应激损伤的关键转录因子,能够调节包括血红素氧合酶1（HO-1）、还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、醌氧化还原酶1（NQO-1）和超氧化物歧化酶（SOD）在内的多种抗氧化酶的表达。有研究表明,Nrf2在细胞抗无机砷诱导的氧化应激损伤过程中发挥了关键作用,Nrf2的激活及其调节的细胞抗氧化物质可以保护Ha Ca T细胞免受砷诱导的细胞凋亡和细胞毒性损伤。因此,激活Nrf2的表达是对抗砷诱导细胞损伤的关键。一些前期研究证明了天然化合物紫檀芪（Pterostilbene,Pts）具有优秀的抗氧化作用。紫檀芪可以降低包括过氧化氢和超氧阴离子在内的活性氧活性;可以使得不同细胞系过氧化氢酶的表达增加,如总谷胱甘肽、谷胱甘肽过氧化物酶、谷胱甘肽还原酶,超氧化物歧化酶等。紫檀芪可能通过激活Nrf2等多个相互关联的机制发挥其抗氧化作用。通过抗氧化作用,Pts能够调节并改善靶细胞的抗氧化应激水平,保护细胞免于各种因素导致的细胞凋亡、坏死等细胞损伤。因此,在这项研究中,我们通过细胞活性试验研究了Pts对NaAsO₂诱导的细胞毒性的保护作用;通过DCFH-DA荧光检测方法检测了Pts对NaAsO₂诱导的细胞内ROS水平升高的缓解作用;通过检测细胞内丙二醛（MDA）和超氧化物岐化酶（SOD）水平,测定了Pts的抗氧化作用;通过JC-1法检测线粒体膜电位水平研究了Pts对NaAsO₂诱导的线粒体损伤的保护作用;通过免疫印迹法检测了Pts对细胞色素C（Cytochrome C）表达的调节作用;通过流式细胞术检测了Pts对NaAsO₂诱导的细胞凋亡的抑制作用。之后,在体内试验中,我们给予小鼠自由饮用含有NaAsO₂的饮水进行染毒,之后给予不同剂量的Pts对小鼠进行治疗,通过检测小鼠在实验期间的体重变化、脏器系数变化、肝脏及皮肤的病理组织学变化、脾脏及胸腺淋巴细胞亚群的变化等指标,研究了Pts对NaAsO₂诱导的小鼠亚慢性砷中毒模型的保护作用;进一步,我们通过研究Pts对NaAsO₂诱导的细胞内线粒体功能和凋亡相关蛋白的调节作用确定了Pts的作用靶点;最后通过si RNA转染技术敲低了Nrf2的表达,研究了Pts对NaAsO₂诱导细胞损伤的保护作用机制。结果表明:Pts在受试浓度范围内对细胞活力无显著影响,但对NaAsO₂诱导的细胞毒性具有极显著的保护作用。进一步研究发现,Pts可以降低由NaAsO₂诱导的细胞内ROS水平的升高、降低NaAsO₂诱导的细胞内MDA水平、提高细胞内的SOD水平、降低线粒体膜电位去极化率、抑制NaAsO₂诱导的线粒体中细胞色素C（Cytochrome C）向胞质部分的释放、显著抑制NaAsO₂诱导的细胞凋亡和坏死,进而对细胞产生保护作用。体内试验中,我们发现Pts能够缓解NaAsO₂染毒后造成的小鼠增重下降及小鼠肝脏系数下降、缓解NaAsO₂染毒后造成的小鼠肝脏损伤及皮肤角质化,并显著逆转NaAsO₂染毒造成的小鼠脾脏及胸腺淋巴细胞CD4/CD8的比值下降,保护小鼠的免疫功能。进一步研究发现,Pts可诱导腺苷酸激活蛋白激酶（AMP-activated kinase,AMPK）和AKT磷酸化,使Nrf2从细胞质向细胞核转移,上调Nrf2下游NQO1和HO-1的表达;Pts可以上调NaAsO₂抑制的抗凋亡蛋白Bcl-2和Bcl-xl的蛋白表达,下调NaAsO₂激活的促凋亡相关蛋白Bax、Bad和Caspase 3的蛋白表达。在Nrf2敲低的细胞中,Pts对NaAsO₂诱导的细胞毒性及ROS生成的抑制作用大幅减弱,说明Nrf2是Pts发挥抗氧化作用的关键蛋白,Pts通过激活Nrf2途径发挥对NaAsO₂诱导细胞损伤的保护作用。在本研究中,我们发现了Pts对NaAsO₂诱导的表皮细胞损伤具有显著的保护作用,能够缓解NaAsO₂染毒对小鼠造成的危害,保护小鼠的免疫功能。进一步研究发现,Nrf2是Pts发挥上述作用的关键转录因子,Pts通过激活Nrf2信号通路并调节抗氧化物质的表达,从而保护细胞免于NaAsO₂诱导的细胞损伤。我们的研究对Pts的抗氧化作用提供了研究基础。Pts在预防和治疗由砷暴露引起的皮肤损伤和其他疾病方面具有潜在的应用价值。更多还原

【Abstract】 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.更多还原