多种重金属联合毒性的拮抗药物筛选及拮抗分子机制研究

发布时间：2018-11-27 12:13

【摘要】：目的:探讨实际暴露比例下,镍、锌、铅、铬、镉、铜、汞、锰八种重金属的联合毒性,筛选出有效的拮抗药物,并进一步研究拮抗药物的拮抗机制,为预防膳食暴露途径所致的重金属慢性危害提供科学依据。方法:基于宁波地区人群因水产品膳食摄入而暴露的多种重金属污染物实际比例,配制重金属混合物染毒液。首先采用MTT实验,研究重金属混合物对HL7702细胞生存活力的影响。在此基础上,选取重金属混合物LC75毒性浓度进行细胞染毒,分别加入不同浓度的待筛药物(表没食子儿茶素没食子酸酯(Epigallocatechin-3-Gallate,EGCG)、毛地黄黄酮、L-半胱氨酸、牛磺酸、姜黄素、二巯基丙醇、二巯基丁二酸钠、二巯基丙磺酸钠、二氢杨梅素、大蒜素),通过MTT比色法筛选出有效的拮抗药物;再通过线粒体跨膜电位检测、ATP检测、MDA检测、ROS检测、细胞凋亡分析、蛋白免疫印迹和细胞免疫荧光染色等分子生物学实验手段,探讨多种重金属联合毒性的分子机制和有效药物的拮抗机制。结果:MTT实验结果显示,重金属混合物可诱导HL7702细胞生存活力降低,形态皱缩,其细胞毒性的LC25、LC50、LC75浓度分别为20.58、23.16、25.73mg/L;EGCG和毛地黄黄酮可改善重金属混合物诱导的细胞形态改变和生存活力降低程度;ROS检测结果显示,EGCG和毛地黄黄酮均可降低重金属混合物诱导的ROS水平;线粒体膜电位检测结果显示,毛地黄黄酮可抑制重金属混合物对线粒体功能的损伤,维持染毒后细胞的ATP水平;细胞凋亡检测结果显示,EGCG和毛地黄黄酮均可降低重金属混合物诱导的细胞凋亡水平;蛋白免疫印迹和细胞免疫荧光实验结果表明,重金属混合物可增大Bax/Bcl-2比例和线粒体外Cytochrome c水平,上调HO-1、MT、Cleaved Caspase-9、Cleavd Caspase-3、Cleaved PARP、p-JNK、p-ERK和p-p38蛋白水平,EGCG和毛地黄黄酮均可抑制重金属混合物诱导的JNK、ERK、p38的磷酸化,降低HO-1蛋白的应激性表达。另外,毛地黄黄酮可阻止线粒体释放Cytochrome c,抑制Capase-9、Caspase-3、PARP等蛋白的剪切活化。结论:重金属混合物可诱导HL7702细胞氧化应激、脂质氧化、线粒体功能损伤,活化MAPK蛋白信号通路,进而应激性上调细胞氧化应激相关蛋白HO-1和MT的水平,同时激活细胞色素C凋亡信号通路,剪切活化Capase-9、Caspase-3、PARP等蛋白,从而诱导细胞凋亡;EGCG和毛地黄黄酮均可保护HL7702细胞免受重金属混合物诱导的氧化应激损伤,下调MAPK通路磷酸化水平;毛地黄黄酮维持线粒体跨膜势能,抑制Cytochrome c进入细胞内液,阻断Cytochrome c/Apaf1/Caspase-9凋亡复合体的形成,降低Caspase-3的活化,从而抑制重金属混合物诱导的HL7702细胞凋亡。
[Abstract]:Objective: to study the combined toxicity of nickel, zinc, lead, chromium, cadmium, copper, mercury and manganese under the actual exposure ratio, to screen out effective antagonists and to further study the antagonistic mechanism of the antagonists. To provide scientific basis for preventing chronic harm of heavy metals caused by dietary exposure pathway. Methods: based on the actual proportion of heavy metal contaminants exposed to aquatic products in Ningbo area, a mixture of heavy metals was prepared. The effects of heavy metal mixtures on the viability of HL7702 cells were studied by MTT assay. On this basis, the toxic concentration of heavy metal mixture LC75 was selected for cell exposure, and different concentrations of drugs to be screened (epigallocatechin Gallic acid ester (Epigallocatechin-3-Gallate,EGCG), flavone of Rehmannia solani, L-cysteine) were added, respectively. Taurine, curcumin, dimercapto propanol, sodium dimercaptosuccinate, sodium dimercaptopropanesulfonate, dihydromyricetin, allicin), the effective antagonists were screened by MTT colorimetry. Then through the mitochondrial transmembrane potential detection, ATP detection, MDA detection, ROS detection, apoptosis analysis, Western blot and cell immunofluorescence staining and other molecular biological experimental means, To explore the molecular mechanism of combined toxicity of many heavy metals and the antagonistic mechanism of effective drugs. Results: the results of MTT test showed that heavy metal mixture could induce the viability of HL7702 cells to decrease and the morphology to shrink. The cytotoxic LC25,LC50,LC75 concentration of HL7702 cells was 20.58 ~ 23.16 ~ 25.73 mg 路L ~ (-1) 路L ~ (-1), respectively. EGCG and flavone could improve the changes of cell morphology and decrease of survival activity induced by heavy metal mixture, the results of ROS showed that both EGCG and flavone could decrease the ROS level induced by heavy metal mixture. The results of mitochondrial membrane potential test showed that flavone could inhibit the damage of heavy metal mixture to mitochondrial function and maintain the ATP level of exposed cells. The results of apoptosis detection showed that both EGCG and flavone could decrease the level of apoptosis induced by heavy metal mixture. The results of Western blot and cellular immunofluorescence assay showed that heavy metal mixture could increase the ratio of Bax/Bcl-2 and the level of Cytochrome c outside mitochondria, and up-regulate HO-1,MT,Cleaved Caspase-9,Cleavd Caspase-3,Cleaved PARP,p-JNK,. P-ERK and p-p38 protein levels, EGCG and flavone could inhibit the phosphorylation of JNK,ERK,p38 induced by heavy metal mixture, and decrease the stress expression of HO-1 protein. In addition, flavone could inhibit the release of Cytochrome c from mitochondria and inhibit the shearing activation of Capase-9,Caspase-3,PARP and other proteins. Conclusion: heavy metal mixtures can induce oxidative stress, lipid oxidation, mitochondrial functional damage, activation of MAPK signal pathway, and up-regulate the levels of HO-1 and MT in HL7702 cells. At the same time, activated cytochrome C apoptosis signaling pathway, shearing activation of Capase-9,Caspase-3,PARP and other proteins, thereby inducing apoptosis; Both EGCG and flavone could protect HL7702 cells from oxidative stress induced by heavy metal mixture and down-regulate the phosphorylation level of MAPK pathway. Flavonoids maintained mitochondrial transmembrane potential energy, inhibited Cytochrome c into the cell fluid, blocked the formation of Cytochrome c/Apaf1/Caspase-9 apoptotic complex, and reduced the activation of Caspase-3, thus inhibiting the apoptosis of HL7702 cells induced by heavy metal mixture.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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