自噬反应在饮水型砷中毒所致肺损伤过程中的作用及机制研究

Inner Mongolia is one of the most severe areas of water arsenic contamination. It has been found that arsenic exposure can cause lung damage, which is harmful to human health. Yet the mechanism of arsenism is still not clear. In this study, in order to discover the mechanism of lung damage due to the exposure of arsenic in drinking water, a rat lung damage model is set up with drinking water of different concentrations of sodium arsenite. Oxidative stress factors and inflammation related factors are measured respectively in serum and BALF after the rats are exposure to arsenic for 4w, and lung damage is graded as well. Mophological observation on autophagosome is undertaken using transmission electron microscope; Autophay related protein LC3, P62, NBR1 are measured by Western blotting and immunohistochemistry. Apoptosis related protein expression of Caspase-3 and -9 are measured by Western blotting. The expression of LC3 and Bcl-2 are measured by immunohistochemistry. Lung apoptotic index of rats is detected. Autophagy is intervened by applying autophagy specific inhibitor of 3-MA, which can preliminary clarify the role of autophagy in lung damage and its molecular mechanism. And PDTC, NF-κB inhibitor, is used to intervene NF-κB signal channel to observe the regulation of NF-κB signal channel on rat lung damage autophagy. Regulation mechanism of NF-κB signal channel is explored. The clarification of lung damage mechanism of exposure of arsenic in drinking water provides theoretical evidence for prevention and intervention of local arseniasis.

内蒙地区饮用水砷污染危害最为严重。已发现砷暴露可引起肺损伤，危害人群健康。但砷暴露致肺损伤发病机制尚未阐明。本研究拟采用SD大鼠饮用含不同浓度亚砷酸钠的水构建大鼠肺损伤模型并探讨其发病机制。染毒4w后分别测定血清和肺泡灌洗液中氧化应激反应因子和炎症因子含量；透射电镜对自噬体进行形态学观察；蛋白质印迹（WB）及免疫组化法测定肺组织中自噬相关蛋白LC3、P62、NBR1的表达，WB测定凋亡相关蛋白Caspase-3，-9表达，免疫荧光法测定LC3和Bcl-2的表达，TUNEL法检测肺组织凋亡指数。采用自噬特异性抑制剂 3-甲基腺嘌呤干预自噬发生，阐明自噬在肺损伤中作用及分子机制，并运用 NF-κB 抑制剂 PDTC 干预 NF-κB 信号通路，观察该通路对大鼠肺损伤自噬的调节，探讨 NF-κB 信号通路对自噬的调节机制，探讨饮水型砷暴露所致肺损伤发病机制，为地方性砷中毒预防和干预提供科学依据。

1.项目背景.环境中的砷主要通过饮水污染或食物污染经消化道吸收进入人体。通过饮用水所致的慢性砷暴露是一个全球关注的公共卫生问题。内蒙地区饮用水砷污染危害最为严重，已发现砷暴露可引起肺损伤，危害人群健康，但砷暴露致肺损伤发病机制尚未阐明。.2.主要研究内容.（1）建立饮水砷暴露所致大鼠肺损伤模型，观察NaAsO2所致大鼠肺损伤中自噬的表达及意义。 .（2）观察自噬抑制剂 3-MA 对大鼠肺损伤自噬及凋亡的影响，初步阐明自噬在肺损伤中作用及分子机制。 .（3） 细胞水平上运用 NF-κB 抑制剂 PDTC 干预 NF-κB 信号通道，探讨其在NaAsO2染毒致人胚肺成纤维细胞（HELF）损伤中的作用机制。 .3.重要结果.（1）砷通过饮水进入大鼠体内，肺组织中促炎因子增加和抗炎因子降低，细胞自噬水平下降，导致大鼠肺部损伤。.（2）NaAsO2可造成人胚肺成纤维细胞损伤，促使炎性因子增加，自噬相关蛋白表达水平增加，引起细胞自噬并激活NF-κB信号通路。.4.关键数据.（1）NaAsO2染毒28d后，各染毒组均观察到有自噬体的形成。.（2）随NaAsO2染毒剂量和时间的增加，大鼠BALF上清液中TNF-α、IL-1β、IL-6的水平增加；与对照组相比NaAsO2染毒组大鼠肺组织中p62的表达量增加，LC3Ⅱ蛋白表达量下降，且存在剂量-反应关系。.（3）随着NaAsO2染毒浓度的升高，HELF细胞上清液中TNF-、IL-6、IL-1β的浓度均呈上升趋势。.（4）与对照组和相同浓度PDTC干预组比较，NaAsO2染毒组HELF细胞的p-p65、p-IKKβ和LC3II/I蛋白表达水平、LC3蛋白荧光强度增强。.5.科学意义.本研究阐明饮水砷暴露所致大鼠肺损伤发生的过程中自噬所发挥的作用和机制，为下一步地方性砷中毒的预防和干预提供理论依据和技术支撑。