姜黄素干预AFB1致肉鸡亚慢性肝损伤的分子机制

发布时间：2018-07-12 18:29

本文选题：黄曲霉毒素B1 + 自噬　；参考：《东北农业大学》2017年博士论文

【摘要】：黄曲霉毒素B1(aflatoxin B1,AFB1)是畜禽饲料中最常见的致癌污染物。肉鸡AFB1中毒事件频频发生,除了能影响肉鸡生长效率、繁殖力等,还会因其免疫抑制作用导致中毒鸡只对多种传染病易感性增强,加大其患病率和死亡率;同时,饲料中的AFB1及其代谢物可以蓄积在肉鸡的肝脏、肾脏和肌肉组织中,人食入后可引起慢性中毒甚至癌症,对畜牧业、公共卫生和人体健康威胁极大。目前关于AFB1致肉鸡亚慢性肝损伤的机制尚不完全明确。姜黄素是从姜科和天南星科植物根茎中提取的二酮类化合物,具有抗肿瘤、抗氧化、抗炎等作用。近期研究发现姜黄素在脂肪肝、肝炎、肝硬化、肝癌等疾病中具有肝保护作用,然而其对AFB1诱导的肉鸡亚慢性肝损伤的保护效果及其分子机制尚不明确。AA肉鸡是中国市场占有量最大的肉鸡品种。本研究以1日龄AA肉雏鸡为实验动物,建立AFB1亚慢性肝损伤模型并应用不同剂量姜黄素干预,采用DNA/RNA染色、透射电镜和Western Blot、Real Time-PCR等先进分子生物学技术,探讨AFB1亚慢性暴露对肉鸡肝脏细胞自噬、凋亡、炎症和增殖相关基因表达的影响以及TLR4/NF-κB通路在此过程中扮演的角色,探明姜黄素能否通过影响自噬、凋亡、炎症和增殖相关基因表达干预AFB1致肉鸡亚慢性肝损伤及其干预效果的可持续性。形态学和超微形态学检查结果:AFB1连续暴露28天,与空白对照组(C组)相比,透射电镜观察肝细胞超微结构发现,AFB1染毒组(A组)肝细胞自噬小体罕见,线粒体结构紊乱、嵴断裂,细胞核染色质边集化;采用姜黄素连续干预28天,低剂量姜黄素(L组)对上述病理改变干预效果不明显,中(M组)、高(H组)剂量姜黄素干预明显改善上述病理变化,H组肝细胞自噬小体重新出现,细胞核染色质分布均匀,线粒体结构清晰;姜黄素对照组(CC组)肝细胞超微结构未见异常。肝组织切片DNA/RNA染色及凋亡和坏死细胞计数结果显示,A组凋亡和坏死细胞数量显著增加(P0.01),姜黄素干预能剂量依赖性减少凋亡、坏死细胞数(L、M、H组,P0.01),CC组未见明显异常。停止暴露AFB1和姜黄素干预7天后,透射电镜观察发现,A组肝细胞自噬小体罕见,细胞核染色质聚集或边集,线粒体嵴结构不清,细胞间隙出现纤维样物质。其它各组中除L组肝细胞核仍有轻微的染色质聚集以外,各组肝细胞溶酶体活动性强,自噬现象明显,细胞核染色质分布均匀,线粒体结构清晰。DNA/RNA染色及凋亡和坏死细胞计数结果显示,A组凋亡和坏死细胞数明显多于C组(P0.01),姜黄素干预组凋亡和坏死细胞数较A组剂量依赖性减少(P0.01),CC组较C组无明显差异。说明姜黄素能有效干预AFB1亚慢性暴露诱发的肝细胞自噬抑制和凋亡、坏死增加,而且姜黄素的干预效果在停药7天内具有持续性。自噬相关蛋白、基因检测结果:AFB1连续暴露28天,与C组比较,A组肝脏LC3a蛋白表达、LC3b-II/LC3b-I比值和beclin-1 mRNA表达显著减少(P0.01),m TOR蛋白、m RNA以及肝细胞质P53蛋白表达显著增加(P0.01)。姜黄素连续干预28天,与A组比较,姜黄素干预组LC3a蛋白表达、LC3b-II/LC3b-I比值、beclin-1 m RNA表达明显增加(P0.05或P0.01),m TOR蛋白、m RNA以及细胞质P53蛋白表达量明显减少(P0.05或P0.01)。CC组与C组比较,LC3b-II/LC3b-I比值和beclin-1 m RNA表达水平升高(P0.01)。停毒停药7天后,各组肝脏上述各指标变化趋势与28天相似,仅CC组各项指标与C组无统计学差异。说明姜黄素可能通过促进beclin-1表达、抑制mTOR和细胞质P53表达诱导LC3a和LC3b-II/LC3b-I水平增加进而干预AFB1亚慢性暴露引起的肝细胞自噬抑制,而且其干预作用在停药7天内具持续性。凋亡相关蛋白、基因检测结果:AFB1连续暴露28天,与C组比较,A组肝脏Caspase-3、Bax、细胞核P53蛋白、p53 m RNA、bcl-2 m RNA表达量显著增加(P0.01)。姜黄素连续干预28天,与A组相比,姜黄素干预组bcl-2 m RNA表达剂量依赖性增加(P0.05或P0.01),其它各指标均呈剂量依赖性降低(P0.05或P0.05或P0.01)。CC组较C组bcl-2 m RNA表达增加(P0.01)。停毒停药7天后,与C组比较,A组除bcl-2 mRNA表达下降(P0.01)以外,其它各指标变化趋势与28天相同(P0.05或P0.01)。与A组比较,姜黄素干预组各指标变化趋势与28天相同(P0.05或P0.05或P0.01)。CC组与C组比较其细胞核P53蛋白、p53 mRNA、caspase-3 m RNA、Bax蛋白表达水平明显下降(P0.01),bcl-2 m RNA表达显著增加(P0.01)。说明抑制促凋亡基因、蛋白表达并促进抗凋亡基因的表达可能是姜黄素干预AFB1亚慢性暴露诱导的肉鸡肝细胞凋亡的重要分子机制,而且姜黄素的干预效果在停药7天内具有持续性。炎症和细胞增殖相关基因表达及TLR4/NF-κB通路活性检测结果:AFB1连续暴露28天,与C组比较,A组肝脏TNF-α、cyclin D1、IL-10、HO-1 m RNA表达及细胞质NF-κB p65蛋白表达显著减少(P0.05或P0.01),TLR4蛋白、细胞核NF-κB p65蛋白以及TGF-β、IL-1β、IL-6、IL-8、TLR4、NF-κB p65 m RNA表达增加(P0.01)。姜黄素连续干预28天,相比于A组,姜黄素干预组TNF-α、cyclin D1、TGF-β、IL-10、HO-1 mRNA表达以及细胞质NF-κB p65蛋白表达增加(P0.05或P0.05或P0.01),TLR4蛋白、细胞核NF-κB p65蛋白以及IL-1β、IL-6、IL-8、TLR4、NF-κB p65 m RNA表达量减少(P0.05或P0.01)。CC组较C组TNF-α、cyclin D1、TGF-β、IL-10、HO-1 mRNA表达水平显著升高(P0.01),TLR4蛋白和NF-κB p65 mRNA表达水平显著降低(P0.01)。停毒停药后第7天,与C组比较,A组除cyclin D1和TGF-βmRNA表达无变化以外,其它各项指标变化趋势均与28天相同(P0.01)。与A组相比,姜黄素干预组各指标变化趋势与28天相似(P0.05或P0.05或P0.01),仅TGF-βm RNA表达无明显变化。CC组与C组比较,TNF-α、cyclin D1、IL-10 m RNA表达增加(P0.01),TLR4蛋白和基因以及细胞核NF-κB p65蛋白表达减少(P0.01)。说明姜黄素可能通过促进抗炎基因和细胞增殖基因表达,并通过抑制TLR4/NF-κB通路抑制促炎基因表达,进而干预AFB1亚慢性暴露诱导的肝脏炎症和细胞增殖能力抑制,且其干预作用在停药7天内具有持续性。本研究结果表明,促进肝细胞自噬活动和细胞增殖相关基因的表达,抑制肝细胞凋亡、坏死及炎症,是姜黄素干预AFB1致肉鸡亚慢性肝损伤的重要机制,而且姜黄素的干预效果在停药7天内具有持续性。
[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.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.31

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