本文選題：黃曲霉毒素B1 + 自噬��； 參考：《東北農業(yè)大學》2017年博士論文

【摘要】：黃曲霉毒素B1(aflatoxin B1,AFB1)是畜禽飼料中最常見的致癌污染物。肉雞AFB1中毒事件頻頻發(fā)生,除了能影響肉雞生長效率、繁殖力等,還會因其免疫抑制作用導致中毒雞只對多種傳染病易感性增強,加大其患病率和死亡率;同時,飼料中的AFB1及其代謝物可以蓄積在肉雞的肝臟、腎臟和肌肉組織中,人食入后可引起慢性中毒甚至癌癥,對畜牧業(yè)、公共衛(wèi)生和人體健康威脅極大。目前關于AFB1致肉雞亞慢性肝損傷的機制尚不完全明確。姜黃素是從姜科和天南星科植物根莖中提取的二酮類化合物,具有抗腫瘤、抗氧化、抗炎等作用。近期研究發(fā)現(xiàn)姜黃素在脂肪肝、肝炎、肝硬化、肝癌等疾病中具有肝保護作用,然而其對AFB1誘導的肉雞亞慢性肝損傷的保護效果及其分子機制尚不明確。AA肉雞是中國市場占有量最大的肉雞品種。本研究以1日齡AA肉雛雞為實驗動物,建立AFB1亞慢性肝損傷模型并應用不同劑量姜黃素干預,采用DNA/RNA染色、透射電鏡和Western Blot、Real Time-PCR等先進分子生物學技術,探討AFB1亞慢性暴露對肉雞肝臟細胞自噬、凋亡、炎癥和增殖相關基因表達的影響以及TLR4/NF-κB通路在此過程中扮演的角色,探明姜黃素能否通過影響自噬、凋亡、炎癥和增殖相關基因表達干預AFB1致肉雞亞慢性肝損傷及其干預效果的可持續(xù)性。形態(tài)學和超微形態(tài)學檢查結果:AFB1連續(xù)暴露28天,與空白對照組(C組)相比,透射電鏡觀察肝細胞超微結構發(fā)現(xiàn),AFB1染毒組(A組)肝細胞自噬小體罕見,線粒體結構紊亂、嵴斷裂,細胞核染色質邊集化;采用姜黃素連續(xù)干預28天,低劑量姜黃素(L組)對上述病理改變干預效果不明顯,中(M組)、高(H組)劑量姜黃素干預明顯改善上述病理變化,H組肝細胞自噬小體重新出現(xiàn),細胞核染色質分布均勻,線粒體結構清晰;姜黃素對照組(CC組)肝細胞超微結構未見異常。肝組織切片DNA/RNA染色及凋亡和壞死細胞計數(shù)結果顯示,A組凋亡和壞死細胞數(shù)量顯著增加(P0.01),姜黃素干預能劑量依賴性減少凋亡、壞死細胞數(shù)(L、M、H組,P0.01),CC組未見明顯異常。停止暴露AFB1和姜黃素干預7天后,透射電鏡觀察發(fā)現(xiàn),A組肝細胞自噬小體罕見,細胞核染色質聚集或邊集,線粒體嵴結構不清,細胞間隙出現(xiàn)纖維樣物質。其它各組中除L組肝細胞核仍有輕微的染色質聚集以外,各組肝細胞溶酶體活動性強,自噬現(xiàn)象明顯,細胞核染色質分布均勻,線粒體結構清晰。DNA/RNA染色及凋亡和壞死細胞計數(shù)結果顯示,A組凋亡和壞死細胞數(shù)明顯多于C組(P0.01),姜黃素干預組凋亡和壞死細胞數(shù)較A組劑量依賴性減少(P0.01),CC組較C組無明顯差異。說明姜黃素能有效干預AFB1亞慢性暴露誘發(fā)的肝細胞自噬抑制和凋亡、壞死增加,而且姜黃素的干預效果在停藥7天內具有持續(xù)性。自噬相關蛋白、基因檢測結果:AFB1連續(xù)暴露28天,與C組比較,A組肝臟LC3a蛋白表達、LC3b-II/LC3b-I比值和beclin-1 mRNA表達顯著減少(P0.01),m TOR蛋白、m RNA以及肝細胞質P53蛋白表達顯著增加(P0.01)。姜黃素連續(xù)干預28天,與A組比較,姜黃素干預組LC3a蛋白表達、LC3b-II/LC3b-I比值、beclin-1 m RNA表達明顯增加(P0.05或P0.01),m TOR蛋白、m RNA以及細胞質P53蛋白表達量明顯減少(P0.05或P0.01)。CC組與C組比較,LC3b-II/LC3b-I比值和beclin-1 m RNA表達水平升高(P0.01)。停毒停藥7天后,各組肝臟上述各指標變化趨勢與28天相似,僅CC組各項指標與C組無統(tǒng)計學差異。說明姜黃素可能通過促進beclin-1表達、抑制mTOR和細胞質P53表達誘導LC3a和LC3b-II/LC3b-I水平增加進而干預AFB1亞慢性暴露引起的肝細胞自噬抑制,而且其干預作用在停藥7天內具持續(xù)性。凋亡相關蛋白、基因檢測結果:AFB1連續(xù)暴露28天,與C組比較,A組肝臟Caspase-3、Bax、細胞核P53蛋白、p53 m RNA、bcl-2 m RNA表達量顯著增加(P0.01)。姜黃素連續(xù)干預28天,與A組相比,姜黃素干預組bcl-2 m RNA表達劑量依賴性增加(P0.05或P0.01),其它各指標均呈劑量依賴性降低(P0.05或P0.05或P0.01)。CC組較C組bcl-2 m RNA表達增加(P0.01)。停毒停藥7天后,與C組比較,A組除bcl-2 mRNA表達下降(P0.01)以外,其它各指標變化趨勢與28天相同(P0.05或P0.01)。與A組比較,姜黃素干預組各指標變化趨勢與28天相同(P0.05或P0.05或P0.01)。CC組與C組比較其細胞核P53蛋白、p53 mRNA、caspase-3 m RNA、Bax蛋白表達水平明顯下降(P0.01),bcl-2 m RNA表達顯著增加(P0.01)。說明抑制促凋亡基因、蛋白表達并促進抗凋亡基因的表達可能是姜黃素干預AFB1亞慢性暴露誘導的肉雞肝細胞凋亡的重要分子機制,而且姜黃素的干預效果在停藥7天內具有持續(xù)性。炎癥和細胞增殖相關基因表達及TLR4/NF-κB通路活性檢測結果:AFB1連續(xù)暴露28天,與C組比較,A組肝臟TNF-α、cyclin D1、IL-10、HO-1 m RNA表達及細胞質NF-κB p65蛋白表達顯著減少(P0.05或P0.01),TLR4蛋白、細胞核NF-κB p65蛋白以及TGF-β、IL-1β、IL-6、IL-8、TLR4、NF-κB p65 m RNA表達增加(P0.01)。姜黃素連續(xù)干預28天,相比于A組,姜黃素干預組TNF-α、cyclin D1、TGF-β、IL-10、HO-1 mRNA表達以及細胞質NF-κB p65蛋白表達增加(P0.05或P0.05或P0.01),TLR4蛋白、細胞核NF-κB p65蛋白以及IL-1β、IL-6、IL-8、TLR4、NF-κB p65 m RNA表達量減少(P0.05或P0.01)。CC組較C組TNF-α、cyclin D1、TGF-β、IL-10、HO-1 mRNA表達水平顯著升高(P0.01),TLR4蛋白和NF-κB p65 mRNA表達水平顯著降低(P0.01)。停毒停藥后第7天,與C組比較,A組除cyclin D1和TGF-βmRNA表達無變化以外,其它各項指標變化趨勢均與28天相同(P0.01)。與A組相比,姜黃素干預組各指標變化趨勢與28天相似(P0.05或P0.05或P0.01),僅TGF-βm RNA表達無明顯變化。CC組與C組比較,TNF-α、cyclin D1、IL-10 m RNA表達增加(P0.01),TLR4蛋白和基因以及細胞核NF-κB p65蛋白表達減少(P0.01)。說明姜黃素可能通過促進抗炎基因和細胞增殖基因表達,并通過抑制TLR4/NF-κB通路抑制促炎基因表達,進而干預AFB1亞慢性暴露誘導的肝臟炎癥和細胞增殖能力抑制,且其干預作用在停藥7天內具有持續(xù)性。本研究結果表明,促進肝細胞自噬活動和細胞增殖相關基因的表達,抑制肝細胞凋亡、壞死及炎癥,是姜黃素干預AFB1致肉雞亞慢性肝損傷的重要機制,而且姜黃素的干預效果在停藥7天內具有持續(xù)性。
[Abstract]:Aflatoxin B1 (aflatoxin B1, AFB1) is the most common carcinogenic pollutant in livestock and poultry feed. The occurrence of AFB1 poisoning occurs frequently in broiler chickens. Besides it can affect the growth efficiency and fecundity of broilers, the susceptibility to a variety of infectious diseases can be increased by the immunosuppressive effect, and the morbidity and mortality of the chickens are increased. At the same time, the AFB in the feed is AFB. 1 and its metabolites can be accumulated in the liver, kidneys and muscle tissues of broilers, which can cause chronic poisoning and even cancer after feeding into the animal, public health and human health. The mechanism of AFB1 induced subchronic liver injury in broilers is not yet completely clear. Two ketones have the effects of anti-tumor, antioxidation and anti-inflammatory. Recent studies have found that curcumin has a protective effect on fatty liver, hepatitis, liver cirrhosis, liver cancer and other diseases. However, the protective effect of AFB1 induced subchronic liver injury in Broilers and its molecular mechanism are not yet clear and.AA broilers are the largest in the Chinese market. In this study, 1 days old AA chicks were used as experimental animals to establish a AFB1 subchronic liver damage model and to use different doses of curcumin intervention. DNA/RNA staining, transmission electron microscopy and Western Blot, Real Time-PCR and other advanced molecular biology techniques were used to investigate the autophagy, apoptosis, inflammation and proliferation of subchronic AFB1 in chicken liver cells. The effects of related gene expression and the role played by the TLR4/NF- kappa B pathway in this process, and to explore whether curcumin can interfere with the sustainability of subchronic liver injury and intervention effects in Broilers by interfering with autophagy, apoptosis, inflammation and proliferation related gene expression. Morphological and ultrastructural examination results: 28 days of continuous exposure to AFB1, and 28 days. Compared with the blank control group (group C), the ultrastructure of liver cells was observed by transmission electron microscopy. The autophagic corpuscles of hepatocytes in the AFB1 group (group A) were rare, the mitochondrial structure was disorganized, the crista broke and the chromatin chromatin of the nucleus was collected; the intervention of the low dose curcumin (group L) with curcumin (group L) for 28 days was not obvious in the middle (group M) and high (group H). The effects of curcumin on the pathological changes were obviously improved. The autophagic corpuscles of the hepatocytes in the H group reappeared, the chromatin of the nucleus was evenly distributed, the structure of the mitochondria was clear, and the ultrastructure of the hepatocytes in the curcumin control group (group CC) was not abnormal. The DNA/RNA staining and the apoptosis and necrotic cells of the liver tissue section showed that the apoptosis and necrotic cells in the A group were apoptotic and necrotic cells. There was a significant increase in quantity (P0.01), and curcumin intervention could reduce apoptosis in a dose-dependent manner, the number of necrotic cells (L, M, H, P0.01), and no obvious abnormality in group CC. 7 days after the intervention of AFB1 and curcumin exposure, the transmission electron microscopy showed that the autophagic corpuscles of the hepatocytes in A group were rare, the nuclei chromatin aggregation or edge set, the mitochondrial crista structure indistinct, and the cell gap. In the other groups, in other groups, the lysosome activity was strong, autophagy was strong, autophagy was obvious, and the chromatin of the nucleus was evenly distributed. The mitochondria structure clear.DNA/RNA staining and apoptosis and necrotic cell count showed that the number of apoptotic and necrotic cells in the A group was obviously more than that of C in the L group. Group (P0.01), the number of apoptotic and necrotic cells in the curcumin intervention group decreased in a dose-dependent manner than that in the A group (P0.01), and there was no significant difference between the CC group and the C group. It indicated that curcumin could effectively interfere with the inhibition and apoptosis of autophagy induced by AFB1 subchronic exposure and the increase of necrosis, and the effect of curcumin on the autophagy related protein in 7 days. Gene detection results: AFB1 was continuously exposed for 28 days. Compared with group C, the expression of LC3a protein in liver of A group, LC3b-II/LC3b-I ratio and beclin-1 mRNA expression were significantly decreased (P0.01), m TOR protein, m RNA and liver cytoplasmic protein expression were significantly increased. The expression of LC3b-I ratio and beclin-1 m RNA increased significantly (P0.05 or P0.01), m TOR protein, m RNA and cytoplasmic P53 protein were significantly decreased (P0.05 of and RNA). After 7 days of stopping drug withdrawal, the changes in the indexes of liver indexes in each group were similar to those of the 28 days. There is no statistical difference between group C and group C. It is suggested that curcumin may inhibit the inhibition of autophagy in liver cells induced by AFB1 subchronic exposure by promoting the expression of beclin-1 and inhibiting the increase of LC3a and LC3b-II/LC3b-I levels induced by mTOR and cytoplasmic P53 expression, and the intervention effect is persistent within 7 days of drug withdrawal. Test results: AFB1 was continuously exposed for 28 days. Compared with group C, the expression of Caspase-3, Bax, nuclear P53 protein, p53 m RNA, bcl-2 m RNA was significantly increased in group A. The dose dependence of curcumin was increased for 28 days. The expression of Bcl-2 m RNA in group.CC was increased (P0.01) in group.CC than in group C. After 7 days of stopping poison and stopping drug, the trend of other indexes in A group was the same as that of 28 days except Bcl-2 mRNA (Bcl-2 mRNA). Compared with those in the group of 28 days, the trend of each index in the curcumin intervention group was the same as that in the 28 day. Group P53 protein, p53 mRNA, caspase-3 m RNA, Bax protein expression level decreased significantly (P0.01), bcl-2 m RNA expression increased significantly (P0.01). It indicated that inhibiting apoptosis gene, protein expression and promoting the expression of anti apoptotic gene may be the important molecular mechanism of curcumin to interfere with the apoptosis of chicken liver cells induced by chronic exposure. And the effect of curcumin was sustained within 7 days. The expression of inflammation and cell proliferation related gene expression and TLR4/NF- kappa B pathway activity detection results: AFB1 was continuously exposed for 28 days. Compared with group C, the expression of TNF- alpha, cyclin D1, IL-10, HO-1 m RNA, and cytoplasm of cytoplasmic kappa protein expression were significantly reduced. NF- kappa B p65 protein and TGF- beta, IL-1 beta, IL-6, IL-8, TLR4, NF- kappa B p65 m. 65 protein and IL-1 beta, IL-6, IL-8, TLR4, NF- kappa B p65 m RNA expression decreased (P0.05 or P0.01). The change trend of all the other indexes was the same as that of the 28 day (P0.01). Compared with the A group, the change trend of the indexes of the curcumin intervention group was similar to that of the 28 days (P0.05 or P0.05 or P0.01), but only TGF- beta m RNA had no obvious changes in the expression of.CC group and C group. The expression of NF- kappa B p65 protein decreased (P0.01). It indicates that curcumin may inhibit the expression of anti-inflammatory genes and cell proliferation genes and inhibit the expression of proinflammatory genes by inhibiting the TLR4/NF- kappa B pathway, and then interferes with the liver inflammation and proliferation inhibition induced by subchronic exposure to AFB1, and the intervention effect is persistent within 7 days of drug withdrawal. The results show that promoting the expression of autophagy and cell proliferation related genes and inhibiting the apoptosis, necrosis and inflammation of liver cells is an important mechanism for the intervention of curcumin to AFB1 induced subchronic liver injury in broilers, and the effect of curcumin is sustained within 7 days.
【學位授予單位】：東北農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.31

【參考文獻】

相關期刊論文 前7條

1 王敏;馮彩霞;郭立杰;田偉;;姜黃素應用于肝臟疾病的研究進展[J];解放軍醫(yī)藥雜志;2016年05期

2 夏斌;廖昆;張玉靜;邱楊;駱冰;莊群瑛;殷花;何承勇;林育純;林忠寧;;自噬參與促進AFB1致肝細胞DNA損傷的修復進程[J];熱帶醫(yī)學雜志;2014年10期

3 何聰;陳清勇;王劍;唐夏莉;焦德敏;;姜黃素下調mTOR誘導人肺癌A549細胞自噬的研究[J];腫瘤學雜志;2014年05期

4 黃新立;王學浩;;調控因子與肝臟再生[J];中國普外基礎與臨床雜志;2010年06期

5 ;The Roles of Innate Immune Cells in Liver Injury and Regeneration[J];Cellular & Molecular Immunology;2007年04期

6 彭海峰,劉能銀,段君英,閆玉礦,周兆棠,楊鎮(zhèn),裘法祖;兔血吸蟲病肝細胞的凋亡及基因調控[J];中華肝膽外科雜志;2000年03期

7 徐正婕,范建高;酒精誘發(fā)肝細胞凋亡的發(fā)病機制[J];國外醫(yī)學(消化系疾病分冊);1999年04期

相關會議論文 前2條

1 施曉晨;安雅男;申鳳鴿;于錄;;黃曲霉毒素誘導巨噬細胞自噬機制的研究[A];中國畜牧獸醫(yī)學會獸醫(yī)藥理毒理學分會第十一屆會員代表大會暨第十三次學術討論會與中國毒理學會獸醫(yī)毒理專業(yè)委員會第五次學術研討會論文集[C];2015年

2 張妮婭;朱明坤;孫鋁輝;徐珉娟;劉婕;齊德生;;姜黃素緩解AFB_1對肉雞肝臟損傷的效果研究[A];中國畜牧獸醫(yī)學會動物營養(yǎng)學分會第七屆中國飼料營養(yǎng)學術研討會論文集[C];2014年

相關博士學位論文 前1條

1 鄭海平;銀杏葉提取物對AFB1致大鼠肝癌抑制作用的研究[D];廣西醫(yī)科大學;2012年

相關碩士學位論文 前1條

1 趙君賢;活性炭納米粒子對5Fu藥理作用的影響及部分作用機制研究[D];中國人民解放軍軍事醫(yī)學科學院;2008年

，

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