PM2.5气室注射染毒对鸡胚肝脏发育的毒性效应

Toxic effects of PM2.5 injection in air chamber on development of chicken embryo liver

[背景]大气细颗粒物(PM2.5)暴露对胚胎发育的影响及其毒性效应备受关注,其对肝脏发育毒性的机制研究尚不明确。[目的]探讨鸡胚暴露于不同剂量PM2.5对鸡胚肝脏发育的影响及潜在作用机制。[方法]将60只受精鸡蛋按照质量随机分为4组:空白对照组(0 mg·kg^-1 PM2.5)和0.2、0.5、2.0 mg·kg^-1 PM2.5染毒组。通过气室注射染毒建立鸡胚染毒模型,随后放入孵化箱孵化21 d,幼鸡孵出后称重记录,计算肝指数。断头处死后采用HE染色法观察孵化幼鸡的肝脏组织病理学变化,采用全自动生化分析仪对孵化幼鸡的血清肝功能进行检测,采用免疫组化法和Western blotting测定肝组织内核因子-κB(NF-κB)p65与诱导型一氧化氮合酶(iNOS)的蛋白表达情况。[结果]HE染色结果显示:0.5 mg·kg^-1 PM2.5暴露后,孵化幼鸡的肝细胞排列紊乱,出现少量炎症细胞浸润和轻微脂肪样变;2.0 mg·kg^-1 PM2.5暴露后,肝小叶结构破坏严重,出现大量炎症细胞浸润和肝细胞脂肪变性。肝功能血清学指标结果显示:0.5、2.0 mg·kg^-1组谷丙转氨酶(ALT)活性,0.2、0.5、2.0 mg·kg^-1组谷草转氨酶(AST)活性均较对照组升高(均P <0.05)。随着PM2.5剂量的增加,ALT和AST活性相应升高(r=0.731、0.720,P <0.05)。免疫组化结果显示:0.5、2.0 mg·kg^-1组中的NF-κB p65表达量高于对照组和0.2 mg·kg^-1组(P <0.05);2.0 mg·kg^-1PM2.5组NF-κB p65蛋白表达量高于0.5 mg·kg^-1组(P <0.05)。Western blotting检测结果显示:0.5、2.0 mg·kg^-1组iNOS蛋白表达量较对照组升高(P <0.05)。[结论]PM2.5暴露可引起孵化幼鸡的肝脏脂肪样变和炎症损伤。随着PM2.5浓度的增加,NF-κB p65和iNOS的蛋白表达也相应增加,炎症反应可能为PM2.5暴露导致鸡胚肝脏发育毒性的机制之一。

[Background]The impact of fine particulate matter(PM2.5)exposure on embryonic development and its toxic effects have attracted much attention.The mechanism of PM2.5 to liver development is not clear.[Objective]This study is designed to investigate the toxic effects of different doses of PM2.5 on the development of chicken embryo liver and the potential mechanism.[Methods]Sixty fertilized eggs were randomly divided into four groups:blank control group(0 mg·kg-1 PM2.5)and 0.2,0.5,and 2 mg·kg-1 PM2.5 groups with egg weight balanced.A chicken embryo exposure model was established by injecting different doses of PM2.5 into the air chamber,and then moving the eggs into the incubator for 21 d.After hatching,the chicks were weighed,calculated for liver index,and measured for selected biochemical indicators of liver function.HE staining was prepared for observing pathological changes of liver samples.Immunohistochemistry and Western blotting were used to detect the expressions of nuclear factor-κB(NF-κB)p65 and inducible nitric oxide synthase(iNOS)in liver tissues.[Results]The hatched chickens’liver samples with HE staining showed that the 0.5mg·kg-1 PM2.5 exposure led to disordered hepatocytes,with a small amount of inflammatory cell infiltration and slight fatty changes;the 2.0mg·kg-1 PM2.5 exposure resulted in severely damaged hepatic lobular structure,with a lot of inflammatory infiltration and hepatic cell steatosis.Regarding the biochemical indicators of liver function,compared with the control group,the activity of alanine aminotransferase(ALT)was increased in the 0.5 or 2.0 mg·kg-1 PM2.5 group(P<0.05),and the activity of aspartate aminotransferase(AST)was elevated in the 0.2,0.5,or 2.0mg·kg-1 PM2.5 group(P<0.05).The increase of PM2.5 concentration was correlated with higher activities of ALT and AST(r=0.731 and 0.720,P<0.05).The immunohistochemistry results showed that the expression levels of NF-κB p65 in the 0.5 and 2.0mg·kg-1 PM2.5 groups were significantly higher than those in the control group and the 0.2mg·kg-1 PM2.5 group(P<0.05);and the expression level in the 2.0mg·kg-1 PM2.5 group was higher than that in the 0.5mg·kg-1 PM2.5 group(P<0.05).The Western blotting results showed that compared with the control group,the expression levels of iNOS protein in the 0.5 and 2.0mg·kg-1 PM2.5 groups were increased(P<0.05).[Conclusion]Chicken eggs exposed to PM2.5 could cause liver steatosis and inflammatory injury in hatched chickens.The protein expression levels of NF-κB p65 and iNOS are increased with higher PM2.5 doses,suggesting that inflammatory response may be one of the mechanisms of PM2.5 induced developmental toxicity to chicken embryo liver.