白藜芦醇抵御奶牛乳腺上皮细胞氧化应激的作用机制研究

发布时间：2018-07-04 15:53

本文选题：奶牛 + 乳腺上皮细胞　；参考：《浙江大学》2016年博士论文

【摘要】：乳腺上皮细胞是乳汁合成的主要场所,与乳产量和乳品质密切相关。乳腺上皮细胞在生理及病理阶段,易发生氧化应激。抵御乳腺上皮细胞的氧化应激和增强其抗氧化能力是维持乳腺健康和高效泌乳的有效手段。白藜芦醇是一种高效的抗氧化剂,大量研究发现了它的保健和治疗功效,然尚无研究将其用于健康的乳腺抗氧化研究。本研究首先采用H2O2刺激MAC-T细胞构建了奶牛乳腺上皮细胞氧化应激模型,评价了白藜芦醇抵御乳腺上皮细胞氧化应激的效果。然后利用分子生物学手段探索了白藜芦醇通过Nrf2-ARE信号通路、MAPK及PI3K/AKT等信号通路对乳腺上皮细胞抵御氧化应激的保护作用机理。最后通过向哺乳期ICR小鼠日粮中添加低剂量及高剂量的白藜芦醇检测白藜芦醇在哺乳期饲喂的安全性,并明确其对乳腺的氧化还原平衡及对Nrf2-ARE信号通路的调控作用。主要研究结果如下：1.白藜芦醇保护奶牛乳腺上皮细胞抵御H2O2诱导的氧化损伤1.1构建H2O2诱导的奶牛乳腺上皮细胞氧化应激模型。采用了不同浓度不同时间的H2O2刺激MAC-T检测细胞增殖、细胞死亡率、ROS蓄积情况、细胞的氧化平衡情况、细胞内线粒体损伤(BAX和BCL2)和内质网损伤指标(GPR78和CHOP)基因的表达变化,结果发现500μM H2O2处理MAC-T细胞24 h可以引起显著地细胞活力损失,导致细胞凋亡发生,处理MAC-T细胞12 h引起显著地ROS过量产生,并伴随着总抗氧能力和超氧化物歧化酶活性的下降,500μM H2O2处理导致GPR78和CHOP基因表达在4h达到最大,BAX/BCL2最大值则在24h出现。由此确定500μM以上H2O2处理MAC-T细胞引起细胞氧化损伤,主要表现为细胞活力损伤、细胞死亡加剧、氧化还原平衡被破坏,并伴随着线粒体及内质网损伤,并在后续研究中采用500μM H2O2处理MAC-T细胞构建氧化应激损伤模型。1.2明确白藜芦醇对H2O2诱导的奶牛乳腺上皮细胞氧化损伤的保护效果。采用不同浓度白藜芦醇处理MAC-T细胞确定MAC-T 24 h内能够耐受的白藜芦醇的最高浓度是S0μM。接着基于前期构建的氧化应激模型,采用50μM以下不同浓度的白藜芦醇或溶剂对照预处理2h而后加入H2O2或PBS的处理特定时间来检测细胞增殖、细胞死亡率、ROS蓄积情况、细胞的氧化平衡情况、细胞内线粒体损伤(BAX和BCL2)和内质网损伤指标(GPR78和CHOP)基因的表达变化。结果发现,较之H2O2处理,白藜芦醇预处理后再加入H2O2的细胞活力损失、细胞凋亡率、细胞内的ROS蓄积程度随白藜芦醇的浓度的增加而下降。而且白藜芦醇的添加增加了细胞的总抗氧化能力。50μM白藜芦醇削弱了H2O2诱导的线粒体损伤和内质网损伤。结果显示白藜芦醇可以保护乳腺上皮细胞抵御H2O2诱导的氧化损伤2.白藜芦醇保护乳腺上皮细胞抵御H2O2诱导的氧化损伤的作用机制2.1检测白藜芦醇及H2O2处理对奶牛乳腺上皮细胞中Nrf2-ARE-抗氧化酶链的作用。首先检测白藜芦醇和H2O2单独/组合处理MAC-T后细胞内的抗氧化关键基因XNRD1、 HO-1、XCT、NQO-1的表达丰度变化,发现白藜芦醇预处理后无论是否有H2O2刺激,细胞内的抗氧化关键基因表达在特定的时间内均被激活表达,且较之H2O2单独处理组,白藜芦醇+H2O2处理的抗氧化基因均在8h显著增强。采用不同浓度白藜芦醇处理细胞后加入H2O2刺激发现白藜芦醇对于抗氧化基因的表达具有剂量依赖性,有报道证实这些基因是Nrf2-ARE信号通路的靶基因。接着我们检测发现白藜芦醇促进了H202处理下Nrf2蛋白的核转移程度,并促进了细胞内的ARE原件的激活。由此证实白藜芦醇激活了细胞内的Nrf2-ARE-抗氧化链。2.2明确白藜芦醇依赖Nrf2发挥奶牛乳腺细胞保护作用。通过siRNA沉默Nrf2基因表达后重新评估白藜芦醇的保护效果,研究发现细胞内的Nrf2-ARE-抗氧化链的关键靶基因表达在H2O2和白藜芦醇+H2O2处理中均被抑制。而且siRNA沉默Nrf2基因表达后白藜芦醇预处理不能保护细胞免受H2O2诱导的细胞活力损失,甚至siRNA沉默Nrf2基因表达后H2O2处理导致的内质网应激加剧。由此得出白藜芦醇是通过Nrf2-ARE信号通路实现保护乳腺上皮细胞的作用的。2.3探索白藜芦醇通过MAPK、PI3K/AKT信号通路对Nrf2-ARE信号通路激活的作用。检测白藜芦醇及H2O2单独/组合处理MAC-T后细胞内AKT、ERK1/2、JNK1/2、p38的磷酸化水平的时间剂量作用,结果发现H202单独处理激活了所有上述信号转导关键蛋白的活化,而白藜芦醇预处理则延长了AKT及ERK1/2的磷酸化时间。后期采用抑制剂预处理抑制细胞内PI3K/AKT、ERK1/2、JNK1/2、p38信号通路的激活,再采用白藜芦醇及H2O2单独/组合处理发现白藜芦醇可能是通过EKR、PI3K/AKT信号通路激活Nrf2-ARE信号通路实现保护乳腺上皮细胞的作用,而p38信号通路则抑制了白藜芦醇对Nrf2-ARE信号通路的激活作用及细胞保护作用的发挥。3.日粮添加白藜芦醇对哺乳期小鼠乳腺氧化损伤及生产性能的影响该部分采用单因素多水平试验探索了日粮中添加白藜芦醇对哺乳期ICR小鼠乳腺的抗氧化作用,白藜芦醇的添加剂量为0、0.02%、0.2%。通过母鼠采食量、体重、器官分数、乳腺重量变化发现白藜芦醇的添加不会对母鼠这些指标造成负面影响,通过间接法评估乳产量也未发现白藜芦醇具有促进乳产量的作用。对仔鼠的窝重,日增重及器官发育分数检测发现白藜芦醇不会对仔鼠以上发育指标产生负面作用,提示哺乳期食入一定剂量白藜芦醇是安全的。通过采集母鼠的乳腺、肝脏、肾脏等器官检测器官总抗氧化能力、脂质过氧化物、超氧化物歧化酶酶活力,发现白藜芦醇的添加显著降低了泌乳期ICR小鼠乳腺中的脂质过氧化程度,减少氧化损伤,而对肝脏和肾脏则无上述效果。同时对乳腺的抗氧化基因mRNA表达的检测发现,0.2%白藜芦醇添加的小鼠乳腺中SOD2、GPX1、PRX1、TXNRD1的基因表达显著上调,而0.02%以上的白藜芦醇添加小鼠乳腺中TXNRD1、HO1、Nrf2蛋白表达均显著上调,提示日粮添加白藜芦醇可以减少哺乳期小鼠乳腺氧化损伤,促进Nrf2-ARE-抗氧化链信号通路。综上所述,本研究成功构建了奶牛乳腺上皮细胞氧化应激模型,利用此模型研究了白藜芦醇在乳腺上皮细胞中的抗氧化损伤作用及其机制,并采用在体实验证实了白藜芦醇的乳腺保护作用及其对Nrf2-ARE-抗氧化链的调节作用。其中Nrf2-ARE-抗氧化链在乳腺抗氧化应激和细胞存活中发挥重要作用,白藜芦醇可能通过PI3K/AKT、ERK通路影响Nrf2-ARE-抗氧化链从而调控乳腺中的氧化还原平衡。本研究也为白藜芦醇在奶牛乳腺保护中的应用提供研究基础及提示。
[Abstract]:Breast epithelial cells are the main sites for milk synthesis, closely related to milk production and milk quality. Breast epithelial cells are prone to oxidative stress at the physiological and pathological stages. It is an effective means to protect breast health and enhance milk secretion by resisting oxidative stress in breast epithelial cells and enhancing their antioxidant capacity. Resveratrol is a highly effective method. Antioxidants, a large number of studies have found its health and therapeutic efficacy, but there is no study on its use in healthy breast antioxidation. First, H2O2 stimulated MAC-T cells to construct a model of oxidative stress in mammary epithelial cells of dairy cows and evaluated the effect of resveratrol against oxidative stress in mammary epithelial cells. Biological means explored the protective mechanism of resveratrol through Nrf2-ARE signaling pathway, MAPK and PI3K/AKT signaling pathway to protect breast epithelial cells against oxidative stress. Finally, the safety of resveratrol in lactation period was detected by adding low dose and high dose of resveratrol into the diet of ICR mice, and the safety of resveratrol in lactation period was determined. Redox balance of mammary glands and regulation of Nrf2-ARE signaling pathway. The main results are as follows: 1. resveratrol protects mammary epithelial cells against oxidative damage induced by H2O2, and constructs a H2O2 induced oxidative stress model of mammary epithelial cells induced by dairy cows. H2O2 stimulates MAC-T detection with different concentrations and time. Cell proliferation, cell mortality, ROS accumulation, cell oxidation balance, intracellular mitochondrial damage (BAX and BCL2) and endoplasmic reticulum damage index (GPR78 and CHOP) gene expression changes. The results showed that 500 u M H2O2 treatment MAC-T cells 24 h can cause significant cell vitality loss, lead to cell apoptosis, and treat MAC-T cells 12 h lead. A significant excess of ROS was produced, accompanied by a decrease in the total oxygen resistance and the activity of superoxide dismutase. The 500 mu M H2O2 treatment resulted in the maximum expression of GPR78 and CHOP genes in 4H and the maximum of BAX/BCL2 in 24h. The cell death was aggravated, the redox balance was destroyed, and the mitochondria and endoplasmic reticulum were damaged. In the follow-up study, 500 M H2O2 was used to treat MAC-T cells to construct oxidative stress damage model.1.2 to clarify the protective effect of resveratrol on the oxidative damage of breast epithelial cells induced by H2O2 in dairy cows. Different concentrations of resveratrol were used to treat M. AC-T cells determined that the highest concentration of resveratrol that could be tolerated in MAC-T 24 h was S0 M. and then based on the pre constructed oxidative stress model, using the resveratrol or solvent control under 50 u M concentrations to pretreat 2H and then add H2O2 or PBS to detect cell proliferation, cell mortality, ROS accumulation, and cells. The changes in the oxidative balance, mitochondrial damage (BAX and BCL2) and the expression of endoplasmic reticulum damage index (GPR78 and CHOP) were found. The results showed that compared with the H2O2 treatment, the cell viability loss, the rate of cell apoptosis and the degree of ROS accumulation in the cells decreased with the increase of the concentration of resveratrol after the pretreatment of resveratrol. The addition of resveratrol increased the total antioxidant capacity of the cells.50 mu M resveratrol weakened the mitochondrial damage induced by H2O2 and the endoplasmic reticulum damage. The results showed that resveratrol protects against H2O2 induced oxidative damage in breast epithelial cells and 2. resveratrol protects breast epithelial cells against H2O2 induced oxidative damage. The effect of resveratrol and H2O2 on the antioxidant enzyme chain of Nrf2-ARE- in mammary epithelial cells of dairy cows was detected by 2.1. First, we detected the changes in the expression of XNRD1, HO-1, XCT, NQO-1 in the cells of resveratrol and H2O2 alone / combined with MAC-T, and found whether the resveratrol was pretreated with H2O2, whether or not there was a H2O2 stimulation. The expression of key antioxidative key genes was activated in a specific time, and the antioxidant genes of resveratrol +H2O2 were significantly enhanced in 8h compared with the H2O2 alone treatment group. The expression of resveratrol was dose-dependent on the expression of antioxidation genes after the use of resveratrol treated cells with different concentrations, and the expression of resveratrol was dose-dependent. The reports confirmed that these genes are the target genes of the Nrf2-ARE signaling pathway. Then we found that resveratrol promoted the degree of nuclear transfer of Nrf2 protein under H202 treatment and promoted the activation of the ARE original in the cells. It was confirmed that resveratrol activated the Nrf2-ARE- antioxidant chain.2.2 in cells to make clear that resveratrol relies on Nrf2 to play milk. The protective effect of bovine mammary gland cells was reevaluated by siRNA silencing Nrf2 gene expression. The study found that the key target gene expression of Nrf2-ARE- antioxidant chain in the cells was suppressed in H2O2 and resveratrol +H2O2 treatment. Moreover, the siRNA silent Nrf2 gene was pretreated with resveratrol and the cells could not protect the cells. Free from H2O2 induced cell vitality loss, even after the siRNA silencing of Nrf2 gene expression, the endoplasmic reticulum stress caused by H2O2 treatment is aggravated. Thus, resveratrol is a.2.3 to protect mammary epithelial cells through the Nrf2-ARE signaling pathway to explore the activation of resveratrol via the MAPK, PI3K/AKT signaling pathway to the Nrf2-ARE signaling pathway. Use. Test the time dose effect of resveratrol and H2O2 alone / combination on the phosphorylation level of AKT, ERK1/2, JNK1/2, p38 in cells after MAC-T. The results showed that H202 activated all the activation of all the key signal transduction proteins, while resveratrol pretreated the phosphorylation time of AKT and ERK1/2. Later, the inhibitors were used as inhibitors. Pretreatment inhibited the activation of intracellular PI3K/AKT, ERK1/2, JNK1/2, p38 signaling pathway, and resveratrol and H2O2 alone / combined treatment found that resveratrol may be activated by EKR, PI3K/AKT signaling pathway to activate Nrf2-ARE signaling pathway to protect mammary epithelial cells, while p38 signaling inhibits resveratrol on Nrf2-ARE. The effect of the activation of the number pathway and the effect of cell protection on the oxidative damage and production performance of.3. diet supplemented with resveratrol on mammary gland of breast-feeding mice. This part uses a single factor and multi level test to explore the antioxidant effect of resveratrol on mammary gland of mammary ICR mice in the diet. The amount of resveratrol is 0,0.02% 0.2%., through the feed intake, body weight, organ fraction, and breast weight change, found that resveratrol did not have a negative effect on the female mice. The effect of resveratrol on milk yield was not detected by indirect method. The litter weight, daily weight gain and organ development score of the offspring were found to be found in resveratrol. It would not have a negative effect on the development index of the offspring, suggesting that a certain dose of resveratrol was safe during lactation. The total antioxidant capacity, lipid peroxide and superoxide dismutase activity were detected by collecting the mammary gland, liver, kidney and other organs of the mice, and the addition of resveratrol significantly reduced the ICR in lactation period. The degree of lipid peroxidation in the rat breast reduced oxidative damage and had no effect on the liver and kidney. At the same time, the expression of antioxidant gene mRNA in the mammary gland was detected. 0.2% the gene expression of SOD2, GPX1, PRX1, TXNRD1 in the mammary gland of mice added with resveratrol was significantly up-regulated, while more than 0.02% of resveratrol added TXNRD in the mammary gland of mice. 1, the expression of HO1 and Nrf2 protein was significantly up-regulated, suggesting that the diet supplemented with resveratrol could reduce the oxidative damage of mammary glands in breast-feeding mice and promote the signaling pathway of Nrf2-ARE- antioxidant chain. The effect of antioxidant injury and its mechanism, and the effects of resveratrol on the protection of resveratrol and its regulation on Nrf2-ARE- antioxidant chain are verified in vivo. The antioxidant chain of Nrf2-ARE- plays an important role in the antioxidant stress and cell survival of the breast. The resveratrol may affect the antioxidant activity of Nrf2-ARE- through the PI3K/AKT and ERK pathway. The chain also regulates the redox balance in the mammary gland. This study also provides a research basis and hint for the application of resveratrol in the protection of mammary gland in dairy cows.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823

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