黄曲霉毒素G_1诱发小鼠肺腺癌的组织发生及其对肺泡Ⅱ型上皮细胞影响的研究

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英文题名：Histogenesis of Lung Adenocarcinoma Induced by Aflatoxin G_1 in Mice and the Effects of AFG_1 on Alveolar TypeⅡ Cells 作者：申海涛 论文级别：博士 学科专业名称：病理学与病理生理学 中文关键词：黄曲霉毒素G_1 ; 肺腺癌 ; 上皮细胞 ; II型肺泡上皮细胞 ; 肺表面活性蛋白-C ; c-Jun氨基末端激酶 英文关键词：Aflatoxins G_1 ; lung adenocarcinoma ; epithelial cell ; alveolar typeⅡcell ; surfactant protein C ; c-Jun-NH2-terminalkinases 学位年度：2007 导师：张祥宏 学科代码：100104 学位授予单位：河北医科大学 论文提交日期：2007-04-01

摘要

黄曲霉毒素(Aflatoxins, AF)是黄曲霉菌(Aspergillus flavus)等真菌产生的具有致癌作用真菌毒素,包括AFB_1、B_2、G_1、G2和M_1等。黄曲霉毒素G_1 (Aflatoxin G_1,AFG_1)在我国胃癌、食管癌高发区居民饮食中的检出率很高,是当地粮食主要的污染真菌毒素。
     Ⅱ型肺泡上皮细胞(alveolar type II cell, AT-II)是肺泡上皮损伤修复的干细胞。外源性致癌因素如吸烟、粉尘、石英等作用于肺组织可引起AT-II损伤、修复和增生,在外源性致癌因素刺激长期反复作用下,AT-II细胞可发生癌变而导致肺癌发生。外源性致癌因素作用于靶细胞,直接和间接造成细胞损伤引起细胞结构和功能的改变是诱发癌变的重要基础。已有研究表明,多种外源性致癌因素诱发的实验动物肺腺癌均来源于AT-II。
     本研究室前期动物诱癌实验结果表明,AFG_1具有致肺癌性,经口给予AFG_1可诱发实验小鼠肺腺癌发生。目前有关AFG_1诱发肺腺癌研究还局限于长期动物诱癌实验及AFG_1诱发不同动物肺癌的验证方面,对AFG_1诱发实验动物肺腺癌的组织来源尚缺乏研究,AFG_1对实验动物肺组织及AT-II的短期影响也未见文献报道。
     为进一步探讨AFG_1诱癌机制,研究AFG_1诱发实验动物肺腺癌的组织学来源细胞类型,探讨AFG_1对肺组织和AT-Ⅱ的影响及其可能机制。本研究在前期动物诱癌实验研究的基础上,分析了AFG_1诱发肺腺癌组织AT-Ⅱ特异分化标志物SP-C和支气管无纤毛上皮细胞—Clara细胞特异分化标志物CC-10表达情况;观察了在整体水平和细胞水平上AFG_1急性作用对大鼠肺组织和AT-II结构和功能的影响及其致损伤生物学效应,同时探讨了JNK信号转导通路在AFG_1介导AT-II损伤中的作用。
     本研究论文共分为五个部分:
     1黄曲霉毒素G_1诱发小鼠肺腺癌组织发生的研究
     目的:探讨长期经口给予AFG_1诱发NIH小鼠肺腺癌的组织学来源以及AFG_1长期作用对小鼠肺泡上皮细胞SP-C和PCNA蛋白表达的影响。
     方法:采用本实验室存档的AFG_1长期灌胃NIH小鼠58周后存活小鼠肺组织取材石蜡包埋组织块标本24例作为研究对象,其中AFG_1诱发的肺腺癌9例。以同期实验中灌喂生理盐水的12例NIH小鼠正常肺组织作为对照。采用免疫组化方法检测9例肺腺癌组织中SP-C、CC-10、P53和RasP~(21)的表达情况。同时,采用免疫组化方法检测不同剂量AFG_1长期灌胃组和对照组肺组织肺泡上皮细胞SP-C和PCNA蛋白的表达情况。
     结果:
     1.1实验性肺腺癌组织中SP-C和CC-10免疫组化染色结果免疫组化染色结果表明,所有9例AFG_1诱发的肺腺癌组织SP-C均呈阳性表达,阳性率为100%,而CC-10均呈阴性表达。因此,本结果证实AFG_1诱发的NIH小鼠实验性肺腺癌来源于AT-II。
     1.2实验性肺腺癌组织P53和Ras P~(21)免疫组化染色结果实验性肺腺癌组织P53和Ras P~(21)免疫组化染色结果发现本组9例肺腺癌组织均未见突变型P53和Ras P~(21)在蛋白水平上的表达。
     1.3 AFG_1长期灌胃对肺组织肺泡上皮细胞SP-C和PCNA蛋白表达的影响不同剂量AFG_1处理组实验小鼠肺泡细胞SP-C阳性标记指数定量分析结果表明,AFG_13μg·kg~(-1)和AFG_1 30μg·kg~(-1)组动物肺脏SP-C阳性标记指数分别为31.63±5.51%和33.58±4.84%,明显高于对照组(19.72±1.92%,p<0.01),表明AFG_1长期灌胃NIH小鼠可诱发AT-II细胞增生。AFG_1小剂量组和大剂量组肺泡细胞PCNA标记指数明显高于对照组(p<0.01),进一步证明长期灌胃AFG_1可明显增高NIH小鼠肺泡细胞的增殖活性。
     2支气管内给予黄曲霉毒素G_1对大鼠肺组织的作用的研究
     目的:探讨一次性支气管内给予AFG_1对大鼠肺组织的影响。
     方法:按30μg/kg body weight剂量经支气管一次性给予雄性SD大鼠AFG_1处理。AFG_1处理1、3、7和14d后,分别处死实验动物。部分大鼠支气管插管收集支气管-肺泡灌洗液(BALF),离心10min收集上清,采用生化法检测乳酸脱氢酶(LDH)和碱性磷酸酶(AKP)活力。分别切取实验大鼠肺组织进行如下研究工作:取1mm~3大小的肺组织标本经2.5%戊二醛固定标本24h后,以扫描电镜方法观察大鼠肺组织超微结构变化;取部分肺组织4%多聚甲醛固定4h后,常规石蜡切片制备,采用原位杂交方法检测肺组织肿瘤坏死因子-α(TNF-α) mRNA的表达情况;取部分肺组织10%多聚甲醛固定,常规石蜡切片制备,按S-P法进行采用免疫组化方法检测核因子-κB(NF-κB)蛋白的表达;取部分肺组织用生理盐水制备成10%的肺匀浆,采用活性氧(ROS)测定法按相应试剂盒操作程序检测肺组中ROS(以H_2O_2为代表)含量。
     结果:
     2.1黄曲霉毒素G_1对支气管-肺泡灌洗液上清中LDH和AKP的影响给予AFG_1处理1、3和7d后,BALF上清中LDH和AKP酶活力明显升高,至d14时,LDH和AKP活力恢复至正常水平,提示从LDH和AKP酶活性的角度急性给予AFG_1处理对大鼠肺组织具有致损伤作用,但随着毒素在体内代谢,其损伤作用具有可逆性。
     2.2黄曲霉毒素G_1对大鼠肺组织影响的病理形态学观察结果
     光镜下观察发现对照组、溶剂对照组大鼠肺组织肺泡间隔较薄,无水肿、炎症及纤维组织增生,肺泡腔内未见炎性渗出。AFG_1处理组肺泡壁可见炎性增厚,有淋巴细胞和巨噬细胞浸润,7和14d组还可见有纤维母细胞增生。
     扫描电镜观察可见,AFG_1处理后肺泡壁增厚且粗细不均,肺泡腔表面细胞有隆起,细胞肿胀,Ⅱ型肺泡上皮细胞表面微绒毛变短、消失,病变以AFG_1处理后7和14d为明显,证明支气管急性给予AFG_1对大鼠肺组织超微结构具有致损伤作用。
     2.3黄曲霉毒素G_1对大鼠肺组织TNF-αmRNA表达的影响
     原位杂交结果表明,AFG_1处理后1、3、7和14d组肺泡细胞TNF-αmRNA阳性表达细胞数分别为22.1±4.0%,20.3±4.2%,32.3±4.2%和24.3±3.8%,明显高于相应溶剂对照组(1.2±0.3%,1.1±0.2%,1.3±0.4%和1.0±0.4%,p<0.01),提示AFG_1急性作用后可激活处理大鼠肺组织TNF-αmRNA表达。
     2.4黄曲霉毒素G_1对大鼠肺组织NF-κB蛋白表达的影响
     NF-κB免疫阳性反应定位于细胞浆,呈棕黄色染色颗粒,对照组、溶剂对照组和AFG_1处理组大鼠肺支气管黏膜上皮细胞出现阳性反应,而在肺泡壁上皮细胞未见NF-κB阳性表达,说明给予AFG_1刺激后在蛋白水平上没有激活肺泡壁上皮细胞NF-κB的表达。
     2.5黄曲霉毒素G_1对大鼠肺组织抗活性氧能力的影响
     AFG_1处理后除d1外,d 3、7和14肺组织抗活性氧活力分别为53.4±12.4,42.7±10.8和47.2±11.0 mol·min~(-1)·g prot~(-1),明显低于溶剂对照3、7和14d组(61.7±4.2、66.3±5.7和64.3±5.4 mol·min~(-1)·g prot~(-1),p<0.05),表明AFG_1急性作用可以降低肺组织抗活性氧能力。
     3支气管内给予黄曲霉毒素G_1对大鼠肺组织肺泡Ⅱ型上皮细胞影响的研究
     目的:探讨一次性支气管给予AFG_1对大鼠肺组织Ⅱ型肺泡上皮细胞(AT-Ⅱ)结构和功能的影响
     方法:实验动物模型同前一部分。AFG_1处理1、3、7和14d后,分别处死实验动物,切取部分新鲜肺组织,分别进行如下研究,取1mm3大小的新鲜肺组织固定于4%戊二醛中,待制备透射电镜电镜标本,观察AT-II超微结构变化;取部分肺组织10%多聚甲醛固定,常规石蜡切片制备,参照试剂盒说明书按S-P法进行采用免疫组化方法检测SP-C蛋白的表达;部分肺组织固定于70%酒精,采用FCM检测SP-C蛋白的表达;取100mg肺组织加入500μL蛋白裂解液,提取肺组织总蛋白,Western blot方法检测SP-C蛋白的表达;部分肺组织液氮冻存,提取组织RNA,RT-PCR方法检测SP-C和SP-A mRNA的表达情况。
     结果:
     3.1黄曲霉毒素G_1对大鼠肺组织Ⅱ型肺泡上皮细胞的影响
     透射电镜观察可见AFG_1处理组Ⅱ型肺泡上皮细胞出现明显的损伤性变化,表现为板层小体半数空泡化,多数线粒体肿胀、完全空化,粗面内质网核糖体脱颗粒现象,部分细胞表面微绒毛明显减少或消失。
     3.2黄曲霉毒素G_1对肺组织肺泡上皮细胞SP-C蛋白表达的影响
     免疫组化染色表明,AFG_1处理1、3和14d实验动物肺泡上皮SP-C标记指数与相应对照组及溶剂对照组比较差异无显著性,但AFG_1处理7d时实验组大鼠肺泡上皮细胞SP-C蛋白阳性标记指数为9.10±1.28%,明显低于溶剂对照7d组(13.34±2.31%,p<0.05)。
     FCM结果显示AFG_1处理1、3和14d肺组织SP-C荧光指数与相应对照组比较差异无显著性,但AFG_1处理7d组肺组织SP-C蛋白荧光指数为0.77±0.03,明显低于溶剂对照7d组(0.95±0.06,p<0.05)。
     与免疫组化和FCM检测结果一致,Western blot半定量检测结果也提示AFG_1处理7d,AFG_1处理动物肺组织SP-C蛋白表达明显低于相应溶剂对照组。
     上述结果均提示,AFG_1可在一定程度上影响实验动物肺组织AT-Ⅱ特异分化标志物SP-C的表达,这种影响具有明显的时间特异性,支气管给予AFG_1处理7d可降低SP-C蛋白表达。
     3.3黄曲霉毒素G_1对肺组织中SP-C mRNA表达的影响
     RT-PCR SP-C mRNA表达的检测结果表明,给予AFG_1作用1和3d后,肺组织SP-C mRNA未见明显改变,AFG_1作用7d,肺组织SP-C mRNA的表达明显降低(p <0.01),至d14时SP-C mRNA表达又恢复至正常水平,提示给予AFG_1急性作用后可以时间特异性地降低肺组织SP-C mRNA的表达。给予AFG_1作用一定时间后,SP-C在蛋白和mRNA水平的表达趋势相一致,7d时表达下降,14d恢复至正常水平,进一步表明随着毒素在体内代谢一段时间,其损伤作用具有可逆性。
     3.4黄曲霉毒素G_1对肺组织中SP-A mRNA表达的影响
     RT-PCR检测SP-A mRNA的表达结果显示,AFG_1作用3和7d组SP-A mRNA表达量分别为0.72±0.09和0.43±0.12,明显低于相应溶剂对照3和7d组(1.03±0.21和1.06±0.10,p<0.01)。
     4黄曲霉毒素G_1对体外培养的大鼠肺泡II型上皮细胞影响的研究
     目的:进一步探讨AFG_1对体外培养的大鼠AT-Ⅱ致损伤作用。
     方法:以酶消化法原代分离、培养大鼠AT-Ⅱ,将10~15mL细胞悬液接种于0.1g·L~(-1)大鼠IgG处理后的平皿中纯化3h,纯化后AT-Ⅱ用20%DMEM调整细胞浓度为(1~2)×10~9L~(-1),接种于96孔板、24孔板和细胞培养瓶中培养24h。用10%DMEM换液后继续培养12h,实验组分别给予不同浓度(0.5, 1.0和2.0mg·L~(-1))的AFG_1处理,溶剂对照组和对照组给予DMSO(0.4mL·L~(-1))和生理盐水处理。AFG_1作用24h后,收集96孔板细胞采用噻唑蓝比色法(MTT)检测细胞存活率;收集24孔板培养基上清液,采用生物化学方法检测上清液乳酸脱氢酶(LDH)和碱性磷酸酶(AKP)活力;离心收集细胞于2.5%戊二醛中前固定24h,超薄切片(厚度50nm),醋酸铀-柠檬酸铅染色,日立H-7500透射电镜观察AT-Ⅱ超微结构的改变;收集24孔板细胞,Fluo-3/AM负载40min,采用激光扫描共聚焦显微镜(CLSM )检测细胞内[Ca~(2+)];收集24孔板细胞,采用免疫细胞化学、CLSM方法检测AT-II SP-C的表达;离心收集细胞于70%酒精固定,采用FCM方法检测AT-II SP-C蛋白表达。
     结果:
     4.1肺泡Ⅱ型上皮细胞的培养和鉴定
     透射电镜观察可见,本研究纯化的细胞内具有AT-II特征性板层小体;纯化细胞涂片,碱性磷酸酶染色可见﹥95%细胞胞浆内有深蓝色酶阳性产物,证明原代分离细胞经纯化后主要为AT-II。
     4.2黄曲霉毒素G_1对AT-II细胞损伤影响
     MTT检测结果发现,AFG_1作用后体外培养AT-Ⅱ细胞存活率分别为88±3%、80±9%和72±8%,明显低于溶剂对照组(101±2%, p<0.01)。AFG_1处理组培养上清中LDH和AKP活力明显高于溶剂对照组(p<0.01),随着处理浓度的增加,LDH和AKP活力也增加,提示AFG_1对原代培养的AT-Ⅱ具有一定增殖抑制和致损伤作用,随着处理浓度的增加,其致损伤作用逐渐增强。
     4.3黄曲霉毒素G_1对AT-II超微结构的影响
     透射电镜观察可见AFG_1处理组AT-II细胞出现板层小体空化,线粒体肿胀、空泡化等损伤性变化,表明给予AFG_1处理对体外培养的AT-Ⅱ的超微结构具有明显的致损伤作用。
     4.4黄曲霉毒素G_1对AT-Ⅱ细胞内[Ca~(2+)]的影响
     激光扫描共聚焦显微镜方法检测发现,AFG_1 0.5、1.0和2.0mg·L~(-1)组细胞内平均钙离子浓度荧光强度分别为200±21、225±14和229±12,明显高于溶剂对照组(161±28,p<0.01),随着AFG_1处理浓度的增加,平均钙离子浓度荧光强度,呈剂量依赖关系(r=0.849, p<0.01),研究结果表明随着处理浓度的增加,AFG_1明显增加体外培养的AT-Ⅱ细胞内[Ca~(2+)]。
     4.5黄曲霉毒素G_1对AT-Ⅱ细胞SP-C蛋白表达的影响
     免疫细胞化学、激光扫描共聚焦显微镜方法检测发现各组AT-II中均可见SP-C蛋白表达,定量检测结果表明,AFG_1 0.5、1.0和2.0mg·L~(-1)组SP-C蛋白表达荧光强度分别为225±18、209±14和195±13,均明显低于溶剂对照组(243±8, p<0.01)。
     FCM定量检测结果发现,随着AFG_1浓度的增加,AT-Ⅱ细胞SP-C蛋白表达FI明显下降,AFG_1 0.5、1.0和2.0mg·L~(-1)组分别为0.91±0.04、0.88±0.06和0.76±0.05,均明显低于溶剂对照组(0.99±0.06,p<0.01)。表明AFG_1可以降低体外培养的AT-ⅡSP-C蛋白的表达。
     5 JNK信号转导通路在黄曲霉毒素G_1诱导A549细胞损伤中的意义
     目的:探讨JNK信号转导通路在AFG_1诱导A549细胞损伤中的作用及其机制。
     方法:
     5.1 AFG_1处理24h对A549细胞毒性作用、JNK激酶活性、SP-C表达及细胞内[Ca~(2+)]影响的检测
     A549细胞复苏培养后,取对数生长期A549细胞,用10%DMEM调整细胞浓度为(1~2)×10~8L~(-1),接种于96孔板。实验按AFG_1处理8、16、24和48h不同时间分为4个时间点,每个时间点按0.1、0.5、1.0和2.0mg·L~(-1)AFG_1依次给予AFG_1处理、溶剂对照组和对照组分别给予DMSO(0.4mL·L~(-1))和生理盐水处理。以MTT比色法检测AT-Ⅱ存活率。
     取对数生长期A549细胞,用10%DMEM调整细胞浓度为(1~2)×108L~(-1),接种于24孔培养板和细胞培养瓶中。细胞培养24h后,更换2%低血清DMEM培养基,实验组分别给予AFG_10.5、1.0和2.0mg·L~(-1)处理,溶剂对照组和对照组分别给予DMSO(0.4mL·L~(-1))和生理盐水处理,继续细胞培养24h。收集24孔板细胞,Fluo-3/AM负载40min,采用CLSM检测细胞内[Ca~(2+)];采用免疫细胞化学和CLSM方法检测A549细胞p-JNK水平;采用Western blot方法检测A549细胞JNK蛋白表达情况和p-JNK水平。采用RT-PCR方法检测A549细胞SP-C mRNA的表达情况。
     5.2 JNK抑制剂SP600125预处理对AFG_1诱导细胞毒性作用、JNK激酶活性和SP-C表达变化影响的检测
     取对数生长期A549细胞用10%DMEM调整细胞浓度为(1~2)×108L~(-1),接种于96孔培养板,随机分为9组:溶剂对照组、对照组、1μM SP600125组、0.5μM SP600125组、0.1μM SP600125组、1μM SP600125+ AFG_1 1.0 mg·L~(-1)组、0.5μM SP600125+ AFG_1 1.0 mg·L~(-1)组、0.1μM SP600125+ AFG_1 1.0 mg·L~(-1)组和AFG_1 1.0 mg·L~(-1)组。细胞培养24h后,更换2%低血清DMEM培养基,溶剂对照组和对照组给予DMSO(0.4mL·L~(-1))和生理盐水处理。其余各组(不包括1.0 mg·L~(-1)AFG_1组)加入不同浓度SP600125预处理,30min后含有AFG_1各组加入1.0 mg·L~(-1)AFG_1,A549细胞培养24h后,MTT方法检测细胞存活率。
     取对数生长期A549细胞用10%DMEM调整细胞浓度为(1~2)×10~8L~(-1),接种于培养瓶。按JNK特异阻断剂SP600125对AFG_1作用后A549细胞存活率实验结果,给予0.5μM SP600125预处理细胞。实验分为4组,即溶剂对照组、对照组、阻断剂组和AFG_1处理组。细胞培养24h后,更换2%低血清DMEM培养基,阻断剂组加入JNK特异性阻断剂SP600125, 0.5μM。30min后AFG_1组和阻断剂组分别给予1.0 mg·L~(-1)AFG_1,溶剂对照组和对照组给予DMSO(0.4mL·L~(-1))和生理盐水处理。A549细胞培养24h后离心收细胞,采用Western blot方法检测A549细胞p-JNK水平。采用RT-PCR方法检测A549细胞SP-C mRNA的表达情况。
     结果:
     5.1黄曲霉毒素G_1对A549细胞存活率的影响
     MTT检测结果表明,AFG_1在小剂量和短时间内对A549细胞没有明显损伤作用,一定剂量的AFG_1只有作用一定时间才能对A549细胞产生明显的致损伤作用,降低细胞存活率。
     5.2黄曲霉毒素G_1对A549细胞p-JNK水平的影响
     5.2.1p-JNK表达免疫细胞化学和激光扫描共聚焦显微镜方法检测结果
     免疫细胞化学、激光扫描共聚焦显微镜方法检测发现随着AFG_1处理浓度的增加,A549细胞p-JNK荧光强度明显增强。半定量结果显示AFG_10.5、1.0和2.0mg·L~(-1)处理组p-JNK表达的FI值分别为126±11, 157±14和175±11,明显高于溶剂对照组(104±9,p<0.05),实验结果表明给予AFG_1处理可以激活A549细胞JNK激酶。
     5.2.2 p-JNK水平的Western方法检测
     采用图像分析系统对杂交条带进行定量分析,结果显示AFG_1 0.5、1.0和2.0mg·L~(-1)处理组相对p-JNK水平分别为0.40±0.02、0.51±0.08和0.58±0.05,明显高于溶剂对照组(0.29±0.05,p<0.05),进一步表明AFG_1提高A549细胞JNK磷酸化水平,AFG_1激活A549细胞JNK信号转导通路。
     5.3黄曲霉毒素G_1对A549细胞JNK蛋白表达的影响
     Western Blot结果显示,给予不同剂量AFG_1,体外培养的A549细胞JNK蛋白的表达与溶剂对照组比较差异无显著性,表明AFG_1对JNK蛋白的表达没有影响。
     5.4黄曲霉毒素G_1对A549细胞内[Ca~(2+)]的影响
     激光扫描共聚焦显微镜方法检测发现,随着AFG_1处理浓度的增加,AFG_1处理0.5、1.0和2.0mg·L~(-1)组,A549细胞内平均钙离子浓度荧光强度分别为169±13、204±16和228±11,明显高于溶剂对照组(134±12,p<0.01),AFG_1处理浓度和细胞内平均钙离子浓度荧光强度有剂量依赖关系(r=0.932, p<0.01),随着AFG_1浓度的增加,A549细胞内[Ca~(2+)]水平增加。
     5.5黄曲霉毒素G_1对A549细胞SP-C mRNA表达的影响
     A549细胞SP-C mRNA表达的RT-PCR检测结果发现, AFG_10.5、1.0和2.0mg·L~(-1)处理组A549细胞SP-C mRNA相对表达量分别为0.39±0.11,0.30±0.03和0.28±0.02,明显低于溶剂对照组(0.56±0.16,p<0.05),说明给予AFG_1作用可以降低A549细胞SP-C mRNA的表达。
     5.6 SP600125预处理对AFG_1作用后A549细胞存活率的影响
     0.1μM SP600125+1.0 mg?L~(-1)AFG_1组和0.5μM SP600125+1.0 mg?L~(-1) AFG_1组细胞存活率分别为87±5%和89±5%,明显高于1.0 mg·L~(-1)AFG_1组细胞78±8%,但低于溶剂对照组(98±10%,p<0.05),而1.0μM SP600125+1.0 mg?L~(-1)AFG_1组细胞存活率与AFG_1组比较无显著性差异(p>0.05)。提示小剂量SP600125对AFG_1细胞损伤效应具有部分阻断作用,0.5μM SP600125的阻断作用尤为显著,AFG_1通过JNK信号转导通路部分参与介导A549细胞死亡。
     5.7 SP600125预处理对AFG_1作用后A549细胞p-JNK激酶活性和SP-C mRNA表达的影响
     Western blot结果表明,1.0 mg·L~(-1)AFG_1处理A549细胞24h,处理组细胞相对p-JNK水平明显高于溶剂对照组,给予SP600125预处理后, p-JNK水平明显降低(p<0.05),说明SP600125对JNK的磷酸化具有特异阻断作用,进一步证实AFG_1可以激活JNK蛋白激酶。
     给予SP600125预处理后,1.0 mg·L~(-1)AFG_1处理A549细胞24h,RT-PCR分析发现,AFG_1组和阻断剂组SP-C mRNA相对表达量分别为0.37±0.08和0.35±0.05,明显低于溶剂对照组(0.66±0.11,p<0.05),阻断剂组SP-C mRNA相对表达量与AFG_1组比较差异无显著性。说明SP600125预处理对SP-C mRNA的表达没有抑制作用,AFG_1不通过JNK信号转导途径介导SP-C mRNA的表达。
     结论:
     1.长期经口灌胃NIH小鼠AFG_1所诱发的肺腺癌组织来源于AT-II,肺腺癌组织中未见突变型P53和Ras P~(21)蛋白表达;AFG_1长期经口灌胃NIH小鼠促进肺泡上皮细胞SP-C和PCNA的表达。
     2.急性支气管给予AFG_1可以增加处理大鼠BALF中LDH和AKP的活力,导致大鼠肺组织超微结构出现损伤性变化;激活肺泡细胞TNF-αmRNA的表达,降低肺组织抗活性氧能力,但对肺组织NF-κB蛋白的表达没有影响。
     3.急性支气管给予AFG_1可时间特异性地降低大鼠肺组织SP-C在蛋白水平上的表达,降低SP-C和SP-A在mRNA水平的表达。
     4. AFG_1对原代培养的大鼠AT-II具有明显的致损伤作用,可以增加大鼠AT-II细胞内[Ca~(2+)],降低原代培养的大鼠AT-II SP-C蛋白的表达。
     5. AFG_1作用于A549细胞可以增加A549细胞内[Ca~(2+)]、激活JNK蛋白激酶、降低A549细胞SP-C mRNA的表达;给予JNK特异性阻断剂-SP600125预处理可以部分抑制AFG_1对A549细胞的细胞毒性作用,但对AFG_1诱导SP-C mRNA表达降低没有影响。JNK信号转导通路可能部分参与介导AFG_1致AT-II损伤生物学作用。
Aflatoxin G_1 (AFG_1) is one of the members of aflatoxins, the toxic metabolites produced by Aspergillus flavus, etc. Studies showed that AFG_1 was the most frequently detected contaminating mycotoxins in the foodstuffs of the high incidence areas of esophageal and lung cancers in Taihang mountain area in north China.
     Acting as progenitor cells to reform the alveolar epithelium after lung injury, alveolar type II cells (AT-II) contribute to the innate cellular immune response against airbrone pathogens. Repeated injury, repair and proliferation of AT-II induced by environmental carcinogenic agents such as smoking, quartz, fungi, etc, may finally result in the development of carcinoma in lung. Both direct and indirect mechanisms involved in injuries of lung tissues by environmental toxicants and carcinogens exposed to airway epithelium. The structural and functional changes of AT-II may form the basis for the carcinogenesis of lung tissue. It was confirmed that many adenocarcinomas of lung induced by environmental toxicants and carcinogens exposed to airway epithelium arised from alveolar type II cells.
     Our previous animal experiment studies showed that AFG_1 was carcinogenic. Oral administration of AFG_1 could induce lung adenocarcinomas in several experiment animals including mouse, rat, etc. Up to now, the studies of the effects of AFG_1 on carcinogenesis of lung have been limited to cancer inducing experiments and the further confirmation of the causative role of AFG_1 exposure to the carcinogenesis of lung tissue. No work has been done on the histogenesis of lung adenocarcinoma induced by oral administration of AFG_1. Few works have been involved in the acute effects of AFG_1 on the lung tissues and AT-II in vivo and in vitro.
     To further explore the putative mechanisms of carcinogenesis of AFG_1, the following works were carried out in this study. The histogenesis of the lung adenocarcinomas induced by AFG_1 was studied by determination of the phenotype of the adenocarcinoma cells with immunohistochemical staining of AT-II specific marker, SP-C and the Clara cell specific marker, CC-10 at protein level. The effects of acute AFG_1 treatment on the lung tissues and AT-II were studied both in vivo and in vitro. The effects of JNK signaling pathways on the injury of AT-II cell line-A549 cells induced by AFG_1 were also studied.
     The study includes the following five parts:
     1 Histogenesis of lung adenocarcinoma induced by oral adminstration of AFG_1 in mice
     Objective: To study the histogenesis of lung adenocarcinoma induced by AFG_1 and explore the effects of oral administration of AFG_1 on SP-C and PCNA expression of alveolar epithelial cells of lung in NIH mice.
     Methods: Paraffin embedded tissue blocks of 9 adenocarcinoma of lung induced by oral administration of AFG_1 as well as 15 lung tissues of the mice treated intragastrically by gavage with 3μg·kg~(-1) and 30μg·kg~(-1) AFG_1 for 58 weeks and 12 normal mice lung tissues in control group treated with normal saline were included in this study. The phenotype of the lung adenocarcinomas was determined by immunohistochemical expression of SP-C and CC-10 at protein level. The expression of P53 and Ras P~(21) in 9 cases of the lung adenocarcinomas was studied with immunohistochemical staining. The expression of SP-C and PCNA in lung tissues of 24 mice in AFG_1 group and that in lung tissue of 12 mice in control group was determined by immunohistochemical staining method.
     Results:
     1.1 The expression of SP-C and CC-10 in the lung adenocarcinomas induced by AFG_1
     The positive expression of SP-C was found in all the lung adenocarcinomas, while no expression of CC-10 could be seen in the 9 cases. Thus, the results in this study reveals that the lung adenocarcinomas induced by AFG_1 in NIH mice arise from alveolar type II cells
     1.2 The expression of P53 and Ras P~(21) in the lung adenocarcinomas induced by AFG_1
     Immunohistochemical results showed that no positive expression of mutant P53 and Ras P~(21) at protein level could be found in the lung adenocarcinomas.
     1.3 Effect of AFG_1 on the expression of SP-C and PCNA in alveolar epithelial cells in mice
     The immunohistochemical labelling index of SP-C of alveolar epithelial cells in AFG_1 3μg·kg~(-1) and AFG_1 30μg·kg~(-1) group were 31.63±5.51% and 33.58±4.84% respectively, which were significantly higher than that in control group (19.72±1.92%, p<0.01). The labelling index of PCNA of alveolar epithelial cells in AFG_1 3μg·kg~(-1) and AFG_1 30μg·kg~(-1) group were also significantly higher than that in control group (p<0.01). These results suggest oral administration of AFG_1 can significantly increase the expression of SP-C in AT-II and promote proliferation of alveolar epithelial cells in NIH mice.
     2 Effects of intratracheal administration of aflatoxin G_1 on lung tissues in rats.
     Objective: To explore the effects of single intratracheal administration of aflatoxin G_1 on rat lung tissues
     Methods: One hundred and twenty male SD rats weighting 110-130g were randomly divided into three groups: AFG_1 group, solvent control group and control group, 40 rats in each group. The experimental rats in the three groups were intratracheally administrated respectively with AFG_1 (30μg·kg~(-1)body weight), DMSO and saline. The rats were sacrificed 1, 3, 7 and 14d after AFG_1 treatment respectively. Bronchoalveolar lavage fluid (BALF) was collected for LDH and AKP release assay using Detection Kit with biochemical method. The lung tissues specimens were fixed in 2.5% glutaraldehyde and washed in PBS (0.1mol·L~(-1), pH7.2) overnight and the ultrastructural changes of lung tissues were studied with scanning electron microscopy (SEM). Representative tissues specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in paraffin and sectioned. The expression of TNF-αat mRNA level and NF-κB protein of lung tissues was studied with in situ hybridization and immunohistochemical staining method respectively. The concentrations of reactive oxygen species (ROS) of lung tissues were determined with biochemical method.
     Results:
     2.1 Effects of AFG_1 on LDH and AKP activity in BALF of rats
     No difference in LDH and AKP activity of BALF between the rats in solvent control group (DMSO) and control group was found. LDH and AKP activity of BALF in rats 1, 3and 7 d after AFG_1 treatment was significantly increased as compared to that in corresponding DMSO group (p<0.01). The LDH and AKP activity restored to control level 14 d after AFG_1 treatment. These results suggest that AFG_1 may cause reversible injury in rat lung tissues.
     2.2 Effects of AFG_1 on The pathologial changes of lung tissues
     No pathological changes were found in the rat lung tissues in control and DMSO group for each treat time point. The thickening of alveolar septum and lymphocyte and macrophage infiltration could be found in lung tissues after AFG_1 treatment.
     Injury changes such as, alveolar septum thickening, alveolar wall swelling changes, disappearance of microvilli in AT-II could be seen in lung tissues of rats 3, 7 and 14d after AFG_1 treatment by microscopical observation using SEM. These results further suggest that intratracheal administration of AFG_1 may cause ultrastructural injury changes of rat lung tissues.
     2.3 Effects of AFG_1 on the expression of TNF-αmRNA in rats lung tissues
     In situ hybridization results showed that the positive expression percentages of TNF-αmRNA in the alveolar cells in lung tissues of rats 1, 3, 7 and 14d after AFG_1 treatment were 22.1±4.0%,20.3±4.2%,32.3±4.2% and 24.3±3.8% respectively, which were all significantly higher than those of their corresponding DMSO groups 1, 3, 7 and 14 d after DMSO treatment, (1.2±0.3%,1.1±0.2%,1.3±0.4% and 1.0±0.4%, p<0.01).
     2.4 Effects of AFG_1 on the expression of NF-κB protein in rats lung tissues
     Prominent staining for NF-κB in bronchi epithelial cells could be seen in control, DMSO and AFG_1 treated groups. No positive expression of NF-κB at protein level could be found in the alveolar cells after AFG_1 treatment. These results suggest that intratracheal administration of AFG_1 may have no effect on the expression of NF-κB in rat lung tissues.
     2.5 Effects of AFG_1 on concentration of reactive oxygen species in lung tissues
     Except the rats 1d after AFG_1 treatment, the concentrations of ROS of lung tissues in rats 3, 7 and 14d after AFG_1 treatment were (53.4±12.4),(42.7±10.8) and (47.2±11.0) mol·min~(-1)·gprot~(-1) , which were all significantly lower than those in their corresponding DMSO groups 3, 7 and 14 d, {(61.7±4.2), (66.3±5.7) and(64.3±5.4)mol·min~(-1)·gprot~(-1), p<0.05}. Intratracheal administration of AFG_1 may decrease the antioxidant ability in rat lung tissues.
     3 Effects of intratracheal administration of Aflatoxin G_1 on lung alveolar type II cells in rats
     Objective: To explore the effects of single intratracheal administration of Aflatoxin G_1 on structure and function of rat lung alveolar typeⅡcells.
     Methods: The rats in each group as described in part 2 were sacrificed 1, 3, 7 and 14d after AFG_1 treatment and parts of lung tissues specimens were used for the studies. The lung tissues specimens were fixed in 4 % glutaraldehyde and washed in PBS (0.1mol·L~(-1), pH7.2) overnight and the ultrastructural changes of AT-II in lung tissues were studied with transmission electron microscopy (TEM). Immunohistochemical stainning was uesd to detect the expression of SP-C in rat lung tissues. The lung tissues fixed in 70% ethanol were used for preparation of single cell suspension and flow cytometric analysis of the expression of SP-C. Fresh lung tissues (200mg) for Western Blot was first homogenized in 1ml lysis buffer and then the total protein was extracted from the lung tissues and stored at -80℃. The expression of SP-C was determined by Western blot. The expression of SP-C and SP-A at mRNA level was detected by semi-quantitative RT-PCR.
     Results:
     3.1 Effects of AFG_1 on the ultrastructural changes of AT-II in rats lung tissues
     Transmission electron microscopic observation revealed some injury changes in AFG_1 treated alveolar typeⅡcells, e.g. turgid and evacuated lamellar bodies, vacuolar degeneration of mitochondria with loss of crista structure, disappearance of microvilli, etc.
     3.2 Effects of AFG_1 on the expression of SP-C in the alveolar cells
     The results showed that 1d, 3d and 14d after AFG_1 treatment, no significant differences were found in SP-C labeling index of the alveolar cells between the rats in AFG_1 group and that in the corresponding DMSO group. But 7d after AFG_1 treatment, the immunohistochemical labelling index of SP-C of the alveolar cells on the rats in AFG_1 treatment Group was significantly lower than that of the DMSO group (9.10±1.28%, vs 13.34±2.31%, p<0.05).
     FCM results showed that 1, 3d and 14d after AFG_1 treatment, there was no difference in the expression of SP-C protein between AFG_1 treated groups and the corresponding DMSO groups. It was noted that 7d after AFG_1 treatment, the FI value of SP-C protein expression was significantly decreased in AFG_1 treated group (p<0.05).
     Similar to the results with immunohistochemical and FCM methods, Western blot also confirmed a significant decrease in SP-C expression at protein level 7d after AFG_1 treatment.
     Thus, the results in this study suggested that AFG_1 could cause decrease of SP-C expression in a time specific way.
     3.3 Effects of AFG_1 on the expression of SP-C mRNA in rats lung tissues
     The expression of SP-C mRNA in lung tissues of the rats 1 and 3d after AFG_1 treatment was not significantly changed as compared with that of conresponding DMSO rats. However, the expression of SP-C mRNA in lung tissues of rats was significantly decreased 7 days after AFG_1 treatment as compared with that in corresponding DMSO 7d group (p<0.01). The expression of SP-C mRNA in lung tissues of rats restored to normal level 7 days after AFG_1 treatment.
     3.4 Effects of AFG_1 on the expression of SP-A mRNA in rat lung tissues
     Except the rats 1d after AFG_1 treatment, the ratios of SP-A mRNA expression in AFG_1 treated groups 3 and 7 d after AFG_1 treatment were 0.72±0.09 and 0.43±0.12 respectively, which was significantly lower than those in the solvent groups at the same time points (1.03±0.21 and 1.06±0.1, p<0.01). The expression of SP-A mRNA in lung tissues of rats could also restore to normal level 14 days after AFG_1 treatment.
     The results further revealed AFG_1 treatment could cause reversible injury in a time specific way.
     4 Effects of aflatoxin G_1 on the primarily cultured rat lung alveolar type-II cells
     Objective: To further explore the putative effects of aflatoxin G_1 on the possible target cells, primarily cultured AT-II from SD rats.
     Methods:
     The primarily cultured cells were resuspended in 10~15mL DMEM medium supplemented with 20% FCS. Then AT-II cells were purified as follows, the cells were transferred to 0.1g·L~(-1) rat IgG-coated (diluted in Tris buffer solution) Petri dishes (10cm) and AT-II was purified by adherence of macrophages to the dishes for 3 h of incubation. After 3h incubation with rat IgG, the cells were removed and centrifuged and then were seeded at (1~2)×109L~(-1) in culture flasks (8 ml), 24-well plates and 96-well plates with DMEM medium supplemented with 20% FCS at 37℃and 5% CO2. The medium of AT-II was replaced by new DMEM medium supplemented with 10% FCS 24 later and cultured for 12 h.
     After culture for 12 h, the primarily cultured cells were randomly divided into 5 groups: control, solvent control, AFG_1 0.5 mg·L~(-1), AFG_1 1.0 mg·L~(-1) and AFG_12.0 mg·L~(-1). AFG_1 were added to the medium at final concentrations of 0.5, 1.0 and 2.0mg·L~(-1) as AFG_1 treated groups, DMSO was added to the medium of solvent control group and saline was added to the medium of control group. The cells were incubated with AFG_1, DMSO, or saline for 24 h. Cell viability for AT-II cells in vitro was assessed with MTT assay. The activities of lactate dehydrogenase (LDH) and alkaline phosphotase (AKP) in supernatant of AT-II cells in 24-well plates were evaluated by biochemical method. The cultured cells were harvested and fixed in 2.5% glutaraldehyde and ultrathin sections were prepared and stained by uranyl acetate-leadcitrate, the ultrastructural changes of AT-II were observed under transmission electron microscopy (TEM). The concentrations of intracellular free [Ca~(2+)] ([Ca~(2+)]i) of AT-II in 24-well plates loaded with Fluo-3/AM were observed under confocal laser scanning microscopy (CLSM). The immunocytochemical expression of SP-C of AT-II was determined by CLSM. The cultured cells were centrifuged and fixed in 70% ethanol for preparation of single cell suspension and used for flow cytometric analysis of the expression of SP-C.
     Results:
     4.1 Identification of AT-II in vitro
     The specificity of the isolated and cultured AT-II was evaluated using TEM and alkaline phosphatase (AKP) histochemical staining. The specific ultrastructural structure - lamellar bodies could be seen in the cultrued alveolar type-II cells. AKP was positive in the cultured cells. Thus, the results showed that the isolated cells in culture in this study were AT-II.
     4.2 Effects of AFG_1 on the injuries of AT-II in vitro
     MTT assay showed that survival rates of AFG_1 0.5, 1.0 and 2.0 mg·L~(-1) group was 88±3%,80±9% and 72±8% respectively, which was significantly lower than that in DMSO group (101±2%, p<0.01). The activities of LDH and AKP in supernatant were significantly increased after AFG_1 treatment. The results suggest that AFG_1 may cause proliferation inhibition and injuries in cultured rat lung alveolar type-II cells.
     4.3 Effects of AFG_1 on the ultrastructure of AT-II in vitro
     Injury changes at ultrastructural level, such as, turgid and evacuated lamellar bodies, vacuolar degeneration of mitochondria were observed in AFG_1-treated cells under TEM. Exposure of AFG_1 could cause direct injury at structural level in cultured rat AT-II cells.
     4.4 Effects of AFG_1 on the concentrations of [Ca~(2+)]i of AT-II in vitro
     The [Ca~(2+)]i was measured by CLSM and a significant elevation of [Ca~(2+)]i was observated in AFG_1-treated cells. The concentrations of [Ca~(2+)]i in 0.5, 1.0 and 2.0 mg·L~(-1) AFG_1 group were 200±21, 225±14 and 229±12 respectively, which were significantly higher than that in DMSO group (161±28, p<0.01). A significant dose- depended response correlation could be found between AFG_1concentrations and [Ca~(2+)]i (r=0.849, p<0.01).
     4.5 Effects of AFG_1 on the expression of SP-C protein in AT-II in vitro
     Green fluorescence of SP-C protein expression was visualized by CLSM in ecah immunocytochemical stained sample. Fluorescence intensity of SP-C expresson in all AFG_1-treated groups (0.5, 1.0 and 2.0 mg?L~(-1))was significantly lower than that in DMSO group (225±18,209±14 and 195±13 vs 243±8, p<0.01).
     FCM analysis showed that FI of SP-C protein expression in 0.5, 1.0 and 2.0 mg?L~(-1) AFG_1 group was 0.91±0.04, 0.88±0.06 and 0.76±0.05 respectively, which was significantly lower than that in DMSO group (0.99±0.06, p<0.01). The results indicated that exposure of AFG_1 could decrease the expression of SP-C protein in the cultured AT-II.
     5 Effects of JNK signaling pathways on cell injury of A549 cells induced by AFG_1
     Objective: To explore the effects of JNK signaling pathways on the injury of AT-II cell line-A549 cells induced by AFG_1.
     Methods:
     5.1 Determination of the effects of AFG_1 on cell injury, activation of JNK, the concentrations of [Ca~(2+)]i and the expression of SP-C mRNA in A549 cells
     After initial culture for 24h, A549 cells were harvestd, centrifuged and resuspended in fresh DMEM medium supplemented with 10% FCS at the concentration of (1~2)×10~8cells·L~(-1) in 96-well culture plates. The cells were randomly divided into 6 groups: control, solvent control, AFG_1 0.1 mg·L~(-1), AFG_1 0.5 mg·L~(-1), AFG_1 1.0 mg·L~(-1) and AFG_12.0mg·L~(-1) at each treat time point. The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS at 37℃and 5% CO2 24h later. Then the cells in different groups were incubated with different concentrations of AFG_1 (final concentrations of AFG_1 0.1, 0.5, 1.0 and 2.0mg·L~(-1)), or DMSO and saline for 8, 16, 24 and 48h. Cell viability for A549 cells was assessed with MTT assay.
     A549 cells were harvestd, centrifuged and resuspended in DMEM medium supplemented with 10% FCS at the concentration of (1~2)×10~8 cells·L~(-1) in culture flasks (8 ml) and 24-well plates. The cells were randomly divided into 5 groups: control, solvent control, AFG_1 0.5 mg·L~(-1), AFG_1 1.0 mg·L~(-1) and AFG_12.0mg·L~(-1). The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. Then the cells in AFG_1 groups were respectively treated with AFG_1, 0.5 mg·L~(-1), 1.0mg·L~(-1) and AFG_12.0mg·L~(-1), while these in solvent control and control group were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The concentrations of [Ca~(2+)]i of A549 cells in 24 well plates loaded with Fluo-3/AM were observed under CLSM. The phospho-JNK (p-JNK) levels of A549 cells treated with AFG_1 were determined by the immunocytochemical stainning and Western blot. The expression of SP-C mRNA of A549 cells was detected by RT-PCR.
     5.2 Determination of the effects of AFG_1 on cell injury, activation of JNK, and the expression of SP-C mRNA in A549 cells pretreated with SP600125
     The cells in 96-well culture plates were randomly divided into 9 groups: control, solvent control, JNK inhibitors SP600125 0.1μM, SP600125 0.5μM, SP600125 1μM, 0.1μM SP600125+AFG_1 1.0mg·L~(-1), 0.5μM SP600125+AFG_1 1.0mg·L~(-1), 1μM SP600125+AFG_1 1.0mg·L~(-1) and AFG_1 1.0mg·L~(-1). The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of solvent control and control were incubated with DMSO and saline respectively. The cells in other groups (except AFG_1 1.0mg·L~(-1) group) were pretreated for 30 min with different concentrations of SP600125 and then 1.0mg·L~(-1) AFG_1 were added into the medium of 0.1μM SP600125+AFG_1 1.0mg·L~(-1), 0.5μM SP600125+AFG_1 1.0mg·L~(-1), 1μM SP600125+AFG_1 1.0mg·L~(-1) and AFG_1 1.0mg·L~(-1) group. Cell viability for A549 cells was assessed with MTT assay after the cells were cultured for another 24h.
     A549 cells seeded at (1~2)×10~8 cells·L~(-1) in culture flasks were randomly divided into 4 groups: control, solvent control, JNK inhibitors and AFG_1 1.0 mg·L~(-1). The medium of A549 cells was replaced by new DMEM medium supplemented with 2% FCS 24h later. The cells of inhibitors group were pretreated for 30 min with 0.5μM SP600125. Then the cells in inhibitors and AFG_1 group were treated with AFG_1 1.0 mg·L~(-1), while these in solvent control and control groups were incubated with DMSO and saline respectively. The cells were cultured for 24 h after treatment and harvested for detection. The JNK activation of A549 cells treated with AFG_1 were determined with Western blot using antibodies specific for p-JNK. The expression of SP-C mRNA of A549 cells was detected by RT-PCR.
     Results:
     5.1 Effects of AFG_1 on cell viability for A549 cells
     Eight hours after treatment, no significant differences were found in the survival rates of A549 cells between all AFG_1 treatment groups and the solvent DMSO group. While 16, 24 and 48h after AFG_1 treatment, the survival rates of A549 cells in AFG_1 treated groups were significantly lower than those in corresponding DMSO groups (p<0.05). The changes in survival rate were not seen in 0.1 mg·L~(-1)AFG_1 group 16 and 24 h after AFG_1 treatment.
     5.2 Effects of AFG_1 on the phospho-JNK levels of A549 cells
     Blue fluorescence of p-JNK was visualized by CLSM in ecah immunocytochemical stained sample. Fluorescence intensity of p-JNK in 0.5, 1.0 and 2.0 mg·L~(-1)AFG_1 group was 126±11, 157±14 and 175±11 respectively, which was significantly higher than that in DMSO group (104±9, p<0.05).
     Western blot results showed that the p-JNK levels in AFG_1 treated A549 cells were increased as compared with that in DMSO group. The relative p-JNK levels in 0.5, 1.0 and 2.0 mg·L~(-1) AFG_1 treated groups were 0.40±0.02, 0.51±0.08 and 0.58±0.05 respectively, which was all significantly higher than that in DMSO group (0.29±0.05, p<0.05).The results showed that AFG_1 could cause the activation of JNK in A549 cells.
     5.3 Effects of AFG_1 on the expression of JNK protein in A549 cells
     No difference in the expression of JNK among the all AFG_1 treated groups and the DMSO group was found by Western blot using antibodies specific for JNK.
     5.4 Effects of AFG_1 on the concentrations of [Ca~(2+)]i of A549 cells
     The concentrations of [Ca~(2+)]i in0.5, 1.0 and 2.0 mg? L~(-1) AFG_1-treated groups were significantly higher than that in DMSO group(169±13, 204±16 and 228±11 respectively, vs 134±12, p<0.01). A significant dose-depended response correlation could be found between concentrations of [Ca~(2+)]_i and AFG_1 concentrations (r=0.932, p<0.01).
     5.5 Effects of AFG_1 on the expression of SP-C mRNA in A549 cells
     The effects of AFG_1 on the decrease of SP-C mRNA expression were investigated by RT-PCR. The relative expressions of SP-C mRNA in A549 cells in 0.5, 1.0 and 2.0 mg? L~(-1) AFG_1 treated groups were 0.39±0.11, 0.30±0.03 and 0.28±0.02 respectively, which were all significantly lower than that in DMSO group (0.56±0.16, p<0.05).
     5.6 Effects of AFG_1 on the cell survival rates of A549 pretreatment with different concentrations of SP600125
     The survival rates of 1.0 mg·L~(-1) AFG_1 treated A549 cells pretreated with 0.1, 0.5μM SP600 125 and 1.0 mg·L~(-1) were 87±5% and 89±5% respectively, which significtantly higher than that in only 1.0 mg·L~(-1) AFG_1 treated group 78±8%, but lower than that in DMSO group (98±10%, p<0.05). The results suggest the pretreatment with low concentrations of SP600125 may partly reduce the cell injury of AFG_1 and the activation of JNK pathway in AFG_1-treated cells may partly modulate the AFG_1 related cell injury.
     5.7 Effects of AFG_1 on the p-JNK levels and SP-C mRNA expression in A549 pretreatment with 0.5μM SP600125
     Pretreatment of A549 cell with 0.5μM SP600125 could significantly decrease the relative p-JNK level as compared with that of 1.0 mg·L~(-1) AFG_1 treatment alone. The effect of activation of JNK in AFG_1-treated cells was inhibited in the presence of SP600125.
     RT-PCR results showed that there was no difference in the expression of SP-C mRNA in A549 cells between SP600 125 pretreatment group and only 1.0 mg·L~(-1) AFG_1 treatment group. The relative expression of SP-C mRNA in P600 125 pretreatment group and 1.0 mg·L~(-1) AFG_1 treatment groups was 0.37±0.08 and 0.35±0.05 respectively, which was significant lower than that in DMSO group (0.66±0.11, p<0.05). The presence of SP600125 did not affect the expression of SP-C mRNA in A549 cells treated with AFG_1. The results suggest that activation of the JNK pathway in AFG_1-treated cells was not involved in the modulation of SP-C mRNA.
     Conclusions:
     1. The lung adenocarcinomas induced by AFG_1 in NIH mice arised from alveolar type II cells. No positive expression of mutant P53 and Ras P21 was found in the lung adenocarcinomas. Oral administration of AFG_1 for long time could significantly increase the expression of SP-C in AT-II and promote proliferation of alveolar epithelial cells in NIH mice.
     2. Intratracheal administration of AFG_1 in rats could increase the activities of LDH and AKP in BALF and cause ultrastructural injury changes of lung tissues in acute stage. AFG_1 could increase the expression of TNF-αmRNA in alveolar cells, decrease the antioxidant ability but have on effect on the expression of NF-κB in rat lung tissues.
     3. Intratracheal administration of AFG_1 in rats could cause ultrastructural injury changes of AT-II and decrease the expression of SP-C and SP-A mRNA in lung tissues.
     4. AFG_1 could cause injuries in cultured rat lung alveolar type-Ⅱcells. At the same time, AFG_1 could increase the concentration of [Ca~(2+)]i and decrease the expression of SP-C in AT-II in vitro.
     5. AFG_1 could elevate the concentration of [Ca~(2+)]i, cause the activation of JNK and decrease the expression of SP-C mRNA in A549 cells. Pretreatment of JNK inhibitor, SP600125 may partly reduce the cell injury by AFG_1 but have no effects on the expression of SP-C mRNA. The activation of the JNK pathway in AFG_1-treated cells may partly modulate the cell injury.

引文

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