多種重金屬聯(lián)合毒性的拮抗藥物篩選及拮抗分子機(jī)制研究
發(fā)布時間：2018-11-27 12:13
【摘要】：目的:探討實際暴露比例下,鎳、鋅、鉛、鉻、鎘、銅、汞、錳八種重金屬的聯(lián)合毒性,篩選出有效的拮抗藥物,并進(jìn)一步研究拮抗藥物的拮抗機(jī)制,為預(yù)防膳食暴露途徑所致的重金屬慢性危害提供科學(xué)依據(jù)。方法:基于寧波地區(qū)人群因水產(chǎn)品膳食攝入而暴露的多種重金屬污染物實際比例,配制重金屬混合物染毒液。首先采用MTT實驗,研究重金屬混合物對HL7702細(xì)胞生存活力的影響。在此基礎(chǔ)上,選取重金屬混合物L(fēng)C75毒性濃度進(jìn)行細(xì)胞染毒,分別加入不同濃度的待篩藥物(表沒食子兒茶素沒食子酸酯(Epigallocatechin-3-Gallate,EGCG)、毛地黃黃酮、L-半胱氨酸、�；撬�、姜黃素、二巰基丙醇、二巰基丁二酸鈉、二巰基丙磺酸鈉、二氫楊梅素、大蒜素),通過MTT比色法篩選出有效的拮抗藥物;再通過線粒體跨膜電位檢測、ATP檢測、MDA檢測、ROS檢測、細(xì)胞凋亡分析、蛋白免疫印跡和細(xì)胞免疫熒光染色等分子生物學(xué)實驗手段,探討多種重金屬聯(lián)合毒性的分子機(jī)制和有效藥物的拮抗機(jī)制。結(jié)果:MTT實驗結(jié)果顯示,重金屬混合物可誘導(dǎo)HL7702細(xì)胞生存活力降低,形態(tài)皺縮,其細(xì)胞毒性的LC25、LC50、LC75濃度分別為20.58、23.16、25.73mg/L;EGCG和毛地黃黃酮可改善重金屬混合物誘導(dǎo)的細(xì)胞形態(tài)改變和生存活力降低程度;ROS檢測結(jié)果顯示,EGCG和毛地黃黃酮均可降低重金屬混合物誘導(dǎo)的ROS水平;線粒體膜電位檢測結(jié)果顯示,毛地黃黃酮可抑制重金屬混合物對線粒體功能的損傷,維持染毒后細(xì)胞的ATP水平;細(xì)胞凋亡檢測結(jié)果顯示,EGCG和毛地黃黃酮均可降低重金屬混合物誘導(dǎo)的細(xì)胞凋亡水平;蛋白免疫印跡和細(xì)胞免疫熒光實驗結(jié)果表明,重金屬混合物可增大Bax/Bcl-2比例和線粒體外Cytochrome c水平,上調(diào)HO-1、MT、Cleaved Caspase-9、Cleavd Caspase-3、Cleaved PARP、p-JNK、p-ERK和p-p38蛋白水平,EGCG和毛地黃黃酮均可抑制重金屬混合物誘導(dǎo)的JNK、ERK、p38的磷酸化,降低HO-1蛋白的應(yīng)激性表達(dá)。另外,毛地黃黃酮可阻止線粒體釋放Cytochrome c,抑制Capase-9、Caspase-3、PARP等蛋白的剪切活化。結(jié)論:重金屬混合物可誘導(dǎo)HL7702細(xì)胞氧化應(yīng)激、脂質(zhì)氧化、線粒體功能損傷,活化MAPK蛋白信號通路,進(jìn)而應(yīng)激性上調(diào)細(xì)胞氧化應(yīng)激相關(guān)蛋白HO-1和MT的水平,同時激活細(xì)胞色素C凋亡信號通路,剪切活化Capase-9、Caspase-3、PARP等蛋白,從而誘導(dǎo)細(xì)胞凋亡;EGCG和毛地黃黃酮均可保護(hù)HL7702細(xì)胞免受重金屬混合物誘導(dǎo)的氧化應(yīng)激損傷,下調(diào)MAPK通路磷酸化水平;毛地黃黃酮維持線粒體跨膜勢能,抑制Cytochrome c進(jìn)入細(xì)胞內(nèi)液,阻斷Cytochrome c/Apaf1/Caspase-9凋亡復(fù)合體的形成,降低Caspase-3的活化,從而抑制重金屬混合物誘導(dǎo)的HL7702細(xì)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
本文編號：2360715
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114
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