巴豆醛亚慢性染毒致雄性大鼠肺部炎症反应作用机制
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摘要
目的观察巴豆醛亚慢性染毒致雄性大鼠肺部炎症反应、氧化应激与细胞凋亡情况,探讨其相关机制。方法将无特定病原体级雄性Wistar大鼠随机分为对照组和低、中、高染毒组,每组10只,分别予剂量为0. 0、2. 5、4. 5、8. 5 mg/kg体质量的巴豆醛溶液灌胃染毒,每天1次,连续120 d。染毒结束后处死大鼠,称量肺脏质量并进行组织病理学检查,采用比色法测定大鼠血清中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平,采用荧光探针检测肺组织中活性氧相对表达水平,采用Tunel染色法检测细胞凋亡率,采用免疫印迹法检测肺组织B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白的相对表达水平。结果与同时间点对照组比较,高剂量组大鼠体质量于染毒30 d开始出现下降(P <0. 05),低、中剂量组大鼠体质量于染毒90 d开始出现下降(P <0. 05);高剂量组大鼠体质量于染毒90 d起分别低于低、中剂量组(P <0. 05)。染毒结束后,3个剂量组大鼠肺组织出现不同程度的肺组织炎症性改变,呈剂量-效应关系。大鼠血清MDA水平随巴豆醛染毒剂量升高而升高(P <0. 01),SOD和GSH-Px活力均随巴豆醛染毒剂量升高而下降(P <0. 01)。大鼠肺组织中活性氧相对表达水平和细胞凋亡率均随巴豆醛染毒剂量升高而升高(P <0. 01),Bcl-2蛋白相对表达水平和Bcl-2/Bax比值均随巴豆醛染毒剂量升高而下降(P <0. 01),Bax和Caspase-3蛋白相对表达水平随巴豆醛染毒剂量升高而升高(P <0. 01)。结论巴豆醛亚慢性染毒通过改变大鼠肺组织氧化平衡状态,导致肺组织细胞凋亡,从而引起肺部炎症性病理改变。
Objective To observe the conditions of sub-chronic crotonaldehyde exposure-induced pulmonary inflammation,oxidative stress and apoptosis in male rats,and to explore the related mechanisms. Methods The specific pathogen free Wistar male rats were randomly divided into control group,low-,medium-and high-dose crotonaldehyde groups,with 10 rats in each group. Rats were treated with crotonaldehyde at the concentrations of 0. 0,2. 5,4. 5 and 8. 5 mg/kg body weight by intra-gastric administration,once per day for 120 consecutive days. After the end of treatment,rats were sacrificed,the lungs were weighed and histopathological examination was performed. The levels of malondialdehyde( MDA),superoxide dismutase( SOD) and glutathione peroxidase( GSH-Px) in the serum of rats were determined by colorimetry. The relative expression of reactive oxygen species in lung tissues was detected by fluorescence probe. The apoptosis rate was detected by Tunel staining. The relative expression of B-cell lymphoma( Bcl)-2,Bcl-2 associated X protein( Bax) and cysteine aspartic acid protease-3( Caspase-3) proteins in lung tissue was detected by western blotting.Results The body weight of the rats in the high-dose group began to decrease after 30 days of exposure( P < 0. 05),and the body weight in the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05),when compared with that of the control group at the same time. The body weight of the high-dose group was lower than that of the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05). After exposure,the lung tissue of the three doses groups showed different degrees of inflammatory change in a dose-effect relationship. The level of serum MDA in rats increased with the treatment of crotonaldehyde( P < 0. 01). The activities of serum SOD and GSH-Px decreased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of ROS and apoptosis rate in rat lung tissue increased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of Bcl-2 protein and the ratio of Bcl-2/Bax in the lung tissue of rats decreased with the treatment of crotonaldehyde( P < 0. 01). The relative protein expression of Bax and Caspase-3 increased with the treatment of crotonaldehyde( P < 0. 01). Conclusion Crotonaldehyde sub-chronic exposure can cause apoptosis in lung tissue by altering the oxidative balance,leading to inflammatory pathological changes in the lung.
Objective To observe the conditions of sub-chronic crotonaldehyde exposure-induced pulmonary inflammation,oxidative stress and apoptosis in male rats,and to explore the related mechanisms. Methods The specific pathogen free Wistar male rats were randomly divided into control group,low-,medium-and high-dose crotonaldehyde groups,with 10 rats in each group. Rats were treated with crotonaldehyde at the concentrations of 0. 0,2. 5,4. 5 and 8. 5 mg/kg body weight by intra-gastric administration,once per day for 120 consecutive days. After the end of treatment,rats were sacrificed,the lungs were weighed and histopathological examination was performed. The levels of malondialdehyde( MDA),superoxide dismutase( SOD) and glutathione peroxidase( GSH-Px) in the serum of rats were determined by colorimetry. The relative expression of reactive oxygen species in lung tissues was detected by fluorescence probe. The apoptosis rate was detected by Tunel staining. The relative expression of B-cell lymphoma( Bcl)-2,Bcl-2 associated X protein( Bax) and cysteine aspartic acid protease-3( Caspase-3) proteins in lung tissue was detected by western blotting.Results The body weight of the rats in the high-dose group began to decrease after 30 days of exposure( P < 0. 05),and the body weight in the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05),when compared with that of the control group at the same time. The body weight of the high-dose group was lower than that of the low-and medium-dose groups began to decrease at 90 days exposure( P < 0. 05). After exposure,the lung tissue of the three doses groups showed different degrees of inflammatory change in a dose-effect relationship. The level of serum MDA in rats increased with the treatment of crotonaldehyde( P < 0. 01). The activities of serum SOD and GSH-Px decreased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of ROS and apoptosis rate in rat lung tissue increased with the treatment of crotonaldehyde( P < 0. 01). The relative expression of Bcl-2 protein and the ratio of Bcl-2/Bax in the lung tissue of rats decreased with the treatment of crotonaldehyde( P < 0. 01). The relative protein expression of Bax and Caspase-3 increased with the treatment of crotonaldehyde( P < 0. 01). Conclusion Crotonaldehyde sub-chronic exposure can cause apoptosis in lung tissue by altering the oxidative balance,leading to inflammatory pathological changes in the lung.
引文
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[14]HUANG J F,ZHU D M,MA J F,et al.Acute respiratory distress syndrome due to exposure to high-concentration mixture of ethenone and crotonaldehyde[J].Toxicol Ind Health,2015,31(7):585-587.
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[16]候亚鹏,李悦,丁炎,等.巴豆醛对小鼠气管短路电流的影响及相关机制研究[J].毒理学杂志,2018,32(4):273-276.
[17]王利蒙,李翔,杨陟华,等.巴豆醛诱导人支气管上皮细胞(BEAS-2B)自噬及相关通路的时间效应变化[J].烟草科技,2017,50(10):35-41
[18]陈陵,赵学成,邓琼.等.纳米氧化铈急性染毒对雄性小鼠体重和脏器系数及血常规的影响[J].环境与健康杂志,2010,27(10):899-902.
[19]石鹏,齐莹莹,贾玉冰,等.纳米氧化镍致大鼠肺毒性实验研究[J].中国药物与临床,2016,16(8):1121-1124.
[20]TSIKAS D.Assessment of lipid peroxidation by measuring malondialdehyde(MDA)and relatives in biological samples:Analytical and biological challenges[J].Anal Biochem,2017,524:13-30.
[21]程浩,宋立成,陈旭昕,等.烟雾吸入后不同时间点SD大鼠肺损伤指标及氧化应激相关指标的变化趋势[J].实用医学杂志,2018,34(19):3189-3193.
[22]周程,吴南翔,范宏亮,等.TCS和PCB153联合暴露对斑马鱼肝脏SOD和MDA的影响[J].预防医学,2019,31(4):330-334.
[23]李文静,史聪,宋培,等.不同通量透析膜对维持性血液透析患者氧化应激状态及血清炎症因子的影响[J].中国临床研究,2018,31(4):486-489,493.
[24]陈廷玉,王东伟,张金波,等.异烟肼通过ROS/Caspase-3信号通路诱导L-02细胞凋亡及槲皮素的保护作用[J].癌变·畸变·突变,2019,31(1):53-57,68.
[25]朱晶,万丽,徐玮,等.人参首乌方对缺氧复氧诱导的皮质神经细胞凋亡的影响[J].广东药学院学报,2017,33(1):77-83.
[26]王聪,董春玲.氧化应激与肺纤维化肺泡上皮凋亡的研究进展[J].新医学,2018,49(1):15-18.
[27]李海龙,周勇,薛照芸,等.甘乐片对大鼠酒精肝损伤的保护作用及机制研究[J].热带医学杂志,2018,18(8):1032-1035.
[28]李帅,张炳东.细胞凋亡途径的研究进展[J].山东医药,2017,57(37):103-106.
[29]程宇凌,任艳萍.caspase-3和核因子-κB在细胞凋亡外途径中的双向作用研究进展[J].广东医学,2016,37(18):2837-2840.
[30]邹宝安,高胜兰,刘刚.线粒体凋亡途径在肺纤维化中的作用及机制研究进展[J].临床肺科杂志,2019,24(4):721-725.
[31]任玉伟,宿华威.Bcl-2基因家族研究进展[J].大连医科大学学报,2015,37(2):202-205.
[32]DAI Z,LAI J R.Isolation of synthetic antibodies against BCL-2-associated X protein(BAX)[J].Methods Mol Biol,2019,1877:351-357.
[33]李思,李宏玲,赵娜,等.1,2-二氯乙烷诱导SH-SY5Y细胞凋亡及对P53、BCL-2、BAX蛋白表达影响[J].中国职业医学,2017,44(2):164-169,175.
[34]梁顺,张鸿,杜玥,等.糖尿病肾病通过转录激活因子3诱导小鼠足细胞凋亡机制[J].实用医学杂志,2018,34(23):3883-3888.
[35]LOSSI L,CASTAGNA C,MERIGHI A.Caspase-3 mediated cell death in the normal development of the mammalian cerebellum[J].Int J Mol Sci,2018,19(12).doi:10.3390/ijms19123999.
[36]程宇凌,任艳萍.caspase-3和核因子-κB在细胞凋亡外途径中的双向作用研究进展[J].广东医学,2016,37(18):2837-2839,2840.
[2]李悦.巴豆醛对肺部作用的研究进展[J].医学综述,2016,22(7):1259-1262.
[3]LEE S E,PARK H R,PARK C S,et al.Autophagy in crotonaldehyde-induced endothelial toxicity[J].Molecules,2019,24(6).doi:10.3390/molecules24061137.
[4]BAGCHI P,GELDNER N,de CASTRO B R,et al.Crotonaldehyde exposure in U.S.tobacco smokers and nonsmokers:NHANES 2005-2006 and 2011-2012[J].Environ Res,2018,163:1-9.
[5]LI Y,CHANG J,CUI Y,et al.Novel mechanisms for crotonaldehyde-induced lung edema[J].Oncotarget,2017,8(48):83509-83522.
[6]WANG L,LI X,YANG Z,et al.Crotonaldehyde induces autophagymediated cytotoxicity in human bronchial epithelial cells via PI3K,AMPK and MAPK pathways[J].Environ Pollut,2017,228:287-296.
[7]WANG L,YANG Z,XU L,et al.Acute exposure to crotonaldehyde induces dysfunction of immune system in male Wistar rats[J].JToxicol Sci,2018,43(1):33-44.
[8]ZHANG B,LI S,MEN J,et al.Long-term exposure to crotonaldehyde causes heart and kidney dysfunction through induction of inflammatory and oxidative damage in male Wistar rats[J].Toxicol Mech Methods,2019,29(4):263-275.
[9]HECHT S S,KOH W P,WANG R,et al.Elevated levels of mercapturic acids of acrolein and crotonaldehyde in the urine of Chinese women inSingapore who regularly cook at home[J].PLo SOne,2015,10(3):e0120023.
[10]WANG L,LI X,YANG Z,et al.Autophagy induced by low concentrations of crotonaldehyde promotes apoptosis and inhibits necrosis in human bronchial epithelial cells[J].Ecotoxicol Environ Saf,2019,167:169-177.
[11]UL ISLAM B,AHMAD P,RABBANI G,et al.Neo-epitopes on crotonaldehyde modified DNA preferably recognize circulating autoantibodies in cancer patients[J].Tumour Biol,2016,37(2):1817-1824.
[12]LEE S E,PARK H R,PARK C S,et al.Autophagy in crotonaldehyde-induced endothelial toxicity[J].Molecules,2019,24(6).doi:10.3390/molecules24061137.
[13]UL ISLAM B,MOINUDDIN,MAHMOOD R,et al.Genotoxicity and immunogenicity of crotonaldehyde modified human DNA[J].Int JBiol Macromol,2014,65:471-478.
[14]HUANG J F,ZHU D M,MA J F,et al.Acute respiratory distress syndrome due to exposure to high-concentration mixture of ethenone and crotonaldehyde[J].Toxicol Ind Health,2015,31(7):585-587.
[15]PARK S L,CARMELLA S G,CHEN M,et al.Mercapturic acids derived from the toxicants acrolein and crotonaldehyde in the urine of cigarette smokers from five ethnic groups with differing risks for Lung cancer[J].PLo S One,2015,10(6):e0124841.
[16]候亚鹏,李悦,丁炎,等.巴豆醛对小鼠气管短路电流的影响及相关机制研究[J].毒理学杂志,2018,32(4):273-276.
[17]王利蒙,李翔,杨陟华,等.巴豆醛诱导人支气管上皮细胞(BEAS-2B)自噬及相关通路的时间效应变化[J].烟草科技,2017,50(10):35-41
[18]陈陵,赵学成,邓琼.等.纳米氧化铈急性染毒对雄性小鼠体重和脏器系数及血常规的影响[J].环境与健康杂志,2010,27(10):899-902.
[19]石鹏,齐莹莹,贾玉冰,等.纳米氧化镍致大鼠肺毒性实验研究[J].中国药物与临床,2016,16(8):1121-1124.
[20]TSIKAS D.Assessment of lipid peroxidation by measuring malondialdehyde(MDA)and relatives in biological samples:Analytical and biological challenges[J].Anal Biochem,2017,524:13-30.
[21]程浩,宋立成,陈旭昕,等.烟雾吸入后不同时间点SD大鼠肺损伤指标及氧化应激相关指标的变化趋势[J].实用医学杂志,2018,34(19):3189-3193.
[22]周程,吴南翔,范宏亮,等.TCS和PCB153联合暴露对斑马鱼肝脏SOD和MDA的影响[J].预防医学,2019,31(4):330-334.
[23]李文静,史聪,宋培,等.不同通量透析膜对维持性血液透析患者氧化应激状态及血清炎症因子的影响[J].中国临床研究,2018,31(4):486-489,493.
[24]陈廷玉,王东伟,张金波,等.异烟肼通过ROS/Caspase-3信号通路诱导L-02细胞凋亡及槲皮素的保护作用[J].癌变·畸变·突变,2019,31(1):53-57,68.
[25]朱晶,万丽,徐玮,等.人参首乌方对缺氧复氧诱导的皮质神经细胞凋亡的影响[J].广东药学院学报,2017,33(1):77-83.
[26]王聪,董春玲.氧化应激与肺纤维化肺泡上皮凋亡的研究进展[J].新医学,2018,49(1):15-18.
[27]李海龙,周勇,薛照芸,等.甘乐片对大鼠酒精肝损伤的保护作用及机制研究[J].热带医学杂志,2018,18(8):1032-1035.
[28]李帅,张炳东.细胞凋亡途径的研究进展[J].山东医药,2017,57(37):103-106.
[29]程宇凌,任艳萍.caspase-3和核因子-κB在细胞凋亡外途径中的双向作用研究进展[J].广东医学,2016,37(18):2837-2840.
[30]邹宝安,高胜兰,刘刚.线粒体凋亡途径在肺纤维化中的作用及机制研究进展[J].临床肺科杂志,2019,24(4):721-725.
[31]任玉伟,宿华威.Bcl-2基因家族研究进展[J].大连医科大学学报,2015,37(2):202-205.
[32]DAI Z,LAI J R.Isolation of synthetic antibodies against BCL-2-associated X protein(BAX)[J].Methods Mol Biol,2019,1877:351-357.
[33]李思,李宏玲,赵娜,等.1,2-二氯乙烷诱导SH-SY5Y细胞凋亡及对P53、BCL-2、BAX蛋白表达影响[J].中国职业医学,2017,44(2):164-169,175.
[34]梁顺,张鸿,杜玥,等.糖尿病肾病通过转录激活因子3诱导小鼠足细胞凋亡机制[J].实用医学杂志,2018,34(23):3883-3888.
[35]LOSSI L,CASTAGNA C,MERIGHI A.Caspase-3 mediated cell death in the normal development of the mammalian cerebellum[J].Int J Mol Sci,2018,19(12).doi:10.3390/ijms19123999.
[36]程宇凌,任艳萍.caspase-3和核因子-κB在细胞凋亡外途径中的双向作用研究进展[J].广东医学,2016,37(18):2837-2839,2840.