硒对奶牛乳腺炎抗炎作用和炎症信号转导通路调节机制的研究
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摘要
奶牛乳腺炎引起世界范围内乳业巨大的经济损失，如产奶量下降，牛奶质量降低，疾病治疗费用的增加等。奶牛乳腺炎是以乳腺组织发生炎症反应和病理变化为特征的疾病。硒是机体必须微量元素，以硒蛋白的形式存在于机体中，在细胞的抗氧化防御体系起到至关重要的作用，是感染性疾病和自体免疫性疾病炎症反应的调节剂。奶牛硒缺乏和临床型乳腺炎发病率升高密切相关。因此我们研究了微量元素硒对奶牛乳腺炎的抗炎作用和相关信号转导通路的调节机制，为防治乳腺炎提供理论依据和基础实验数据。
大肠杆菌是临床型奶牛乳腺炎最重要的致病菌，LPS是大肠杆菌的主要毒力因子，LPS致乳腺炎模型常用来模拟革兰氏阴性菌的感染而进行乳腺炎的基础性研究。首先，通过乳池灌注LPS建立小鼠乳腺炎模型。饲喂小鼠含不同硒水平的日粮，研究硒对LPS诱导的乳腺炎症反应的影响。通过乳腺组织病理学观察、相关酶活性检测和细胞因子表达分析，研究不同日粮硒水平对LPS诱导的小鼠乳腺炎炎症反应的影响。结果显示，摄入硒的水平会影响乳腺组织谷胱甘肽过氧化物酶的活性和硒蛋白GPX1、GPX4的表达水平，呈剂量依赖效应。乳腺组织病理学切片显示缺硒小鼠乳腺出现较多炎性细胞浸润。缺硒小鼠乳腺中组织中促炎因子表达水平高于正常硒含量小鼠。这些结果说明，低硒日粮促进LPS诱导的小鼠乳腺炎症反应，硒可能通过抑制NF-κB磷酸化降低促炎因子的表达。
然后，我们建立了LPS诱导的原代小鼠乳腺上皮细胞炎症模型。在培养液中添加不同浓度的亚硒酸钠，检测LPS刺激下细胞因子和炎性介质的分泌以及常见的两条炎症信号转导通路NF-κB和MAPK的变化。实验数据表明，亚硒酸钠能够抑制LPS诱导的TNF-α分泌，并呈现剂量依赖性，并下调了炎性分子iNOS和COX-2的蛋白表达水平，但对IL-6、IL-1β无明显抑制作用。亚硒酸钠显著抑制了LPS诱导的小鼠乳腺上皮细胞NF-κB的活化，同时显著抑制了p38、JNK和ERK MAPKs磷酸化.
最后，我们构建了原代奶牛乳腺上皮细胞炎症模型。通过细胞培养液中添加不同浓度的亚硒酸钠，检测LPS刺激下的TLR4和下游信号分子和细胞因子表达。结果表明，LPS刺激下，TRL4、NF-κB和下游细胞因子表达升高。亚硒酸钠影响下游细胞因子基因的表达，但这种改变可能是通过抑制NF-κB完成的。
Bovine mastitis is characterized by inflammation and pathological changes in theudder tissues caused by mammary gland inflammation; as a result, milk yield isreduced, milk quality is compromised, and veterinary costs are increased, therebycausing worldwide economic losses in the dairy industry. The essential trace elementselenium (Se), in the form of selenoproteins, plays a pivotal role in the antioxidantdefense system of the cell，acts as modulator of inflammatory response in infectiousand autoimmune disease. A known consequence that Se-deficiency has beenassociated with increased clinical mastitis cases. So we explored theanti-inflammatory effects and associated mechanisms of selenium on bovine mastitis.These results provided theoretical foundation and the basis of experimental data forprophylaxis and treatment of bovine mastitis.
The main pathogen of clinical bovine mastitis is E.coli. Lipopolysaccharide (LPS)is a powerful bacterial virulence factor of E.coli. The model of LPS-induced mousemastitis is recognized as a valuable tool to study the effects of bovine mastitis becausethey mimic the responses observed during natural mastitis. First, effects of diaryselenium levels on LPS-induced models of mouse mastitis were investigated. Theanimals were fed with diaries that contain the different selenium level. At the end ofexperiment, an intramammary infusion of LPS was used to establish mouse mastitismodel.
Histopathological analysis, enzymatic activity test and pro-inflammatory genesanalysis were conducted to study effects of diary selenium levels on LPS-inducedmouse mammary mastitis. Results showed that activity of glutathione peroxidase andmRNA expression of GPX1、GPX4of mammary gland was affected by diaryselenium levels in a dose depended way. Histopathological observation of mammarygland in LPS-induced Se-deficient mouse mammary gland showed more infiltrationof inflammatory cells. Pro-inflammatory genes expression levels in mammary glandof Se-deficient group were higher than Se-adequate group. These results showed that LPS-induced inflammatory responses of mammary gland were affected by diary Selevels. NF-κB activity might be involved in these processes.
Second, in vitro inflammatory model was performed by LPS-stimulated primarymouse mammary gland epithelial cells. We investigated the effect of differentconcentrations of Na2SeO3on LPS-induced cytokines, inflammatory mediator,mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). Resultsshowed that Na2SeO3inhibited LPS-stimulated TNF-α secretion but not IL-6or IL-1β.Na2SeO3inhibited LPS-stimulated NF-κB activity, p38, ERK and JNK MAPKsphosphorylation.
Finally, in vitro inflammatory model was performed by LPS-induced primarybovine epithelial cells. Different concentration of Na2SeO3was added in cell culturemedia. TLR4expression and regarding down stream signal molecules and cytokinesin LPS-stimulated bovine epithelial cell were investigated. Results showed that TLR4,NF-κB and down stream cytokines expression were up regulated after LPSstimulation. Na2SeO3inhibited LPS-stimulated downstream cytokines expression byinhibit NF-κB expression.
大肠杆菌是临床型奶牛乳腺炎最重要的致病菌，LPS是大肠杆菌的主要毒力因子，LPS致乳腺炎模型常用来模拟革兰氏阴性菌的感染而进行乳腺炎的基础性研究。首先，通过乳池灌注LPS建立小鼠乳腺炎模型。饲喂小鼠含不同硒水平的日粮，研究硒对LPS诱导的乳腺炎症反应的影响。通过乳腺组织病理学观察、相关酶活性检测和细胞因子表达分析，研究不同日粮硒水平对LPS诱导的小鼠乳腺炎炎症反应的影响。结果显示，摄入硒的水平会影响乳腺组织谷胱甘肽过氧化物酶的活性和硒蛋白GPX1、GPX4的表达水平，呈剂量依赖效应。乳腺组织病理学切片显示缺硒小鼠乳腺出现较多炎性细胞浸润。缺硒小鼠乳腺中组织中促炎因子表达水平高于正常硒含量小鼠。这些结果说明，低硒日粮促进LPS诱导的小鼠乳腺炎症反应，硒可能通过抑制NF-κB磷酸化降低促炎因子的表达。
然后，我们建立了LPS诱导的原代小鼠乳腺上皮细胞炎症模型。在培养液中添加不同浓度的亚硒酸钠，检测LPS刺激下细胞因子和炎性介质的分泌以及常见的两条炎症信号转导通路NF-κB和MAPK的变化。实验数据表明，亚硒酸钠能够抑制LPS诱导的TNF-α分泌，并呈现剂量依赖性，并下调了炎性分子iNOS和COX-2的蛋白表达水平，但对IL-6、IL-1β无明显抑制作用。亚硒酸钠显著抑制了LPS诱导的小鼠乳腺上皮细胞NF-κB的活化，同时显著抑制了p38、JNK和ERK MAPKs磷酸化.
最后，我们构建了原代奶牛乳腺上皮细胞炎症模型。通过细胞培养液中添加不同浓度的亚硒酸钠，检测LPS刺激下的TLR4和下游信号分子和细胞因子表达。结果表明，LPS刺激下，TRL4、NF-κB和下游细胞因子表达升高。亚硒酸钠影响下游细胞因子基因的表达，但这种改变可能是通过抑制NF-κB完成的。
Bovine mastitis is characterized by inflammation and pathological changes in theudder tissues caused by mammary gland inflammation; as a result, milk yield isreduced, milk quality is compromised, and veterinary costs are increased, therebycausing worldwide economic losses in the dairy industry. The essential trace elementselenium (Se), in the form of selenoproteins, plays a pivotal role in the antioxidantdefense system of the cell，acts as modulator of inflammatory response in infectiousand autoimmune disease. A known consequence that Se-deficiency has beenassociated with increased clinical mastitis cases. So we explored theanti-inflammatory effects and associated mechanisms of selenium on bovine mastitis.These results provided theoretical foundation and the basis of experimental data forprophylaxis and treatment of bovine mastitis.
The main pathogen of clinical bovine mastitis is E.coli. Lipopolysaccharide (LPS)is a powerful bacterial virulence factor of E.coli. The model of LPS-induced mousemastitis is recognized as a valuable tool to study the effects of bovine mastitis becausethey mimic the responses observed during natural mastitis. First, effects of diaryselenium levels on LPS-induced models of mouse mastitis were investigated. Theanimals were fed with diaries that contain the different selenium level. At the end ofexperiment, an intramammary infusion of LPS was used to establish mouse mastitismodel.
Histopathological analysis, enzymatic activity test and pro-inflammatory genesanalysis were conducted to study effects of diary selenium levels on LPS-inducedmouse mammary mastitis. Results showed that activity of glutathione peroxidase andmRNA expression of GPX1、GPX4of mammary gland was affected by diaryselenium levels in a dose depended way. Histopathological observation of mammarygland in LPS-induced Se-deficient mouse mammary gland showed more infiltrationof inflammatory cells. Pro-inflammatory genes expression levels in mammary glandof Se-deficient group were higher than Se-adequate group. These results showed that LPS-induced inflammatory responses of mammary gland were affected by diary Selevels. NF-κB activity might be involved in these processes.
Second, in vitro inflammatory model was performed by LPS-stimulated primarymouse mammary gland epithelial cells. We investigated the effect of differentconcentrations of Na2SeO3on LPS-induced cytokines, inflammatory mediator,mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). Resultsshowed that Na2SeO3inhibited LPS-stimulated TNF-α secretion but not IL-6or IL-1β.Na2SeO3inhibited LPS-stimulated NF-κB activity, p38, ERK and JNK MAPKsphosphorylation.
Finally, in vitro inflammatory model was performed by LPS-induced primarybovine epithelial cells. Different concentration of Na2SeO3was added in cell culturemedia. TLR4expression and regarding down stream signal molecules and cytokinesin LPS-stimulated bovine epithelial cell were investigated. Results showed that TLR4,NF-κB and down stream cytokines expression were up regulated after LPSstimulation. Na2SeO3inhibited LPS-stimulated downstream cytokines expression byinhibit NF-κB expression.
引文
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