The inflammatory response is a key

The inflammatory response is a key element in the innate immune response system and is primarily mediated by cytokines [54]. Very little is known about the immunotoxicology of heavy metals at the genetic level in fish. The cytokines IL-1β and TNF-α are primarily produced by monocytes and macrophages and regulate multiple aspects of the immune response. TNF-α affects tissue vasculature in inflammation and also induces acute phase proteins from the liver [55]. In the present study, TNF-α levels were found to be significantly lower in the Pb treated group than in the control. IL-1β levels were lower in the Pb-treated group, though this difference not statistically significant. The observed Ddown-regulation of TNF-α and IL-1β implicates NF-κB signaling pathway. However, L. reuteri P16 supplementation had a positive on TNF-α and IL-1β expression. In a previous study, Paul etal [8] recorded decreased TNF-α level in sera and cell lysates of freshwater fish Channa punctatus exposed to lead acetate. Based on the results of this study, it can be stated that Pb renders the fish in an immunocompromised and inflammatory state. Thus, TNF-α and IL-1β are down-regulated, probably by the action of the heavy metal [8], but probiotic supplementation reversed these effects on the expression of those genes. The synthesis of heat shock proteins (Hsps) increases in response to a variety of physical and chemical stressors, including temperature and metal stress, and consequently these proteins can be good environmental stress biomarkers [56]. Expression of HSPs which increased in Pb-exposure group, was further decreased in the L. reuteri P16 supplementation groupOur results indicated that Pb exposure significantly elevated the transcription level of HSP70 and HSP90 in the head-kidney of fish. However, L. reuteri P16 supplementation decreases the expression of HSPs and thus alleviated stress condition in fish. In agreement with the results of our study, Mohapatra et al [19] demonstrated that dietary supplementation of a probiotic mixture consisting of Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae decreased the expression of HSP70 in L. rohita. Stress-reducing factors produced by probiotics might have lowered the HSP levels in fish, reduced Pb-induced stress, and resulted in better growth and immunity. Interestingly, co-treatment with Pb and L. reuteri P16 reversed Pb-induced adverse effects on the carp species studied. The potential beneficial effects and protection by probiotics against Pb toxicity in carp has been shown in Figure 8. The hematological parameters indicated the ability of L. reuteri P16 to stimulate erythropoiesis, thereby increasing the oxygen transport, and a similar result had been reported earlier [13]. Previously, we have demonstrated the excellent Pb-binding ability of this strain [28]. Therefore, supplementing diets with this strain can reduce intestinal absorption of Pb and reduce Pb accumulation in other tissues. The strain L. reuteri P16 possess strong antioxidant abilities [28]. In the present study, co-treatment with Pb and L. reuteri P16 reduced Provisional production of MDA and increased the activities of antioxidant enzymes (SOD and GPx). Thus, L. reuteri P16 alleviates the Pb-induced oxidative stress in carps. However, the underlying role of antioxidative activity of this strain against Pb-induced oxidative stress and dysfunction of the gut barrier need to be investigated further. In addition, co-treatment with Pb and L. reuteri P16 reversed the expressions of proinflammatory cytokines (TNF-α and IL-1β), which are associated with the NF-κB signaling pathway and subsequent decreases in reactive oxygen species production and prevents Pb-induced cellular apoptosis. However, comprehensive Pb tolerance mechanisms in LAB strains have not been investigated. Therefore, Pb stress response network in LAB strains are yet to be elucidated. Further, key proteins and pathways involved in Pb tolerance of lactobacilli strains are unknown. Hence, isobaric tags for relative and absolute quantification (iTRAQ)-based comparative and functional proteomic approaches could be utilized to explore Pb-tolerance related to key proteins and pathways in L. reuteri strains. Moreover, at this early stage of research, we do not know how probiotics repair the metabolic damages occurring in fish exposed to heavy metals; therefore, efforts should be made in the future to incorporate multiple targets (genes, proteins, and metabolites) for exploring heavy metal defense mechanisms of probiotics in hosts. Notably, treatment with L. reuteri P16 alone did not exert any adverse effects on growth performance, hemato

The inflammatory response is a key element in the innate immune response system and is primarily mediated by cytokines [54]. Very little is known about the immunotoxicology of heavy metals at the genetic level in fish. The cytokines IL-1β and TNF-α are primarily produced by monocytes and macrophages and regulate multiple aspects of the immune response. TNF-α affects tissue vasculature in inflammation and also induces acute phase proteins from the liver [55]. In the present study, TNF-α levels were found to be significantly lower in the Pb treated group than in the control. IL-1β levels were lower in the Pb-treated group, though this difference not statistically significant. The observed Ddown-regulation of TNF-α and IL-1β implicates NF-κB signaling pathway. However, L. reuteri P16 supplementation had a positive on TNF-α and IL-1β expression. In a previous study, Paul etal [8] recorded decreased TNF-α level in sera and cell lysates of freshwater fish Channa punctatus exposed to lead acetate. Based on the results of this study, it can be stated that Pb renders the fish in an immunocompromised and inflammatory state. Thus, TNF-α and IL-1β are down-regulated, probably by the action of the heavy metal [8], but probiotic supplementation reversed these effects on the expression of those genes.  The synthesis of heat shock proteins (Hsps) increases in response to a variety of physical and chemical stressors, including temperature and metal stress, and consequently these proteins can be good environmental stress biomarkers [56]. Expression of HSPs which increased in Pb-exposure group, was further decreased in the L. reuteri P16 supplementation groupOur results indicated that Pb exposure significantly elevated the transcription level of HSP70 and HSP90 in the head-kidney of fish. However, L. reuteri P16 supplementation decreases the expression of HSPs and thus alleviated stress condition in fish. In agreement with the results of our study, Mohapatra et al [19] demonstrated that dietary supplementation of a probiotic mixture consisting of Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae decreased the expression of HSP70 in L. rohita. Stress-reducing factors produced by probiotics might have lowered the HSP levels in fish, reduced Pb-induced stress, and resulted in better growth and immunity.  Interestingly, co-treatment with Pb and L. reuteri P16 reversed Pb-induced adverse effects on the carp species studied. The potential beneficial effects and protection by probiotics against Pb toxicity in carp has been shown in Figure 8. The hematological parameters indicated the ability of L. reuteri P16 to stimulate erythropoiesis, thereby increasing the oxygen transport, and a similar result had been reported earlier [13]. Previously, we have demonstrated the excellent Pb-binding ability of this strain [28]. Therefore, supplementing diets with this strain can reduce intestinal absorption of Pb and reduce Pb accumulation in other tissues. The strain L. reuteri P16 possess strong antioxidant abilities [28]. In the present study, co-treatment with Pb and L. reuteri P16 reduced Provisional production of MDA and increased the activities of antioxidant enzymes (SOD and GPx). Thus, L. reuteri P16 alleviates the Pb-induced oxidative stress in carps. However, the underlying role of antioxidative activity of this strain against Pb-induced oxidative stress and dysfunction of the gut barrier need to be investigated further. In addition, co-treatment with Pb and L. reuteri P16 reversed the expressions of proinflammatory cytokines (TNF-α and IL-1β), which are associated with the NF-κB signaling pathway and subsequent decreases in reactive oxygen species production and prevents Pb-induced cellular apoptosis. However, comprehensive Pb tolerance mechanisms in LAB strains have not been investigated. Therefore, Pb stress response network in LAB strains are yet to be elucidated. Further, key proteins and pathways involved in Pb tolerance of lactobacilli strains are unknown. Hence, isobaric tags for relative and absolute quantification (iTRAQ)-based comparative and functional proteomic approaches could be utilized to explore Pb-tolerance related to key proteins and pathways in L. reuteri strains. Moreover, at this early stage of research, we do not know how probiotics repair the metabolic damages occurring in fish exposed to heavy metals; therefore, efforts should be made in the future to incorporate multiple targets (genes, proteins, and metabolites) for exploring heavy metal defense mechanisms of probiotics in hosts. Notably, treatment with L. reuteri P16 alone did not exert any adverse effects on growth performance, hemato

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炎症反应是在先天免疫应答系统的关键要素，主要是由细胞因子[54]介导的。很少有人知道关于重金属在鱼类基因水平的免疫毒理学。细胞因子IL-1β和TNF-α主要由单核细胞和巨噬细胞产生和调节免疫应答的多个方面。TNF-α会影响炎症组织脉管系统和也诱导急性期蛋白由肝脏[55]。在本研究中，发现TNF-α水平为显著下在铅治疗组比对照。IL-1β水平铅处理组中降低，尽管这种差异不是统计学显著。TNF-α和IL-1β牵连NF-κB信号传导途径的观察Ddown调节。然而，罗伊氏乳杆菌P16补充对TNF-α和IL-1β表达的正。在先前的研究中，保罗等人[8]记录在暴露于醋酸铅淡水鱼鳢毛虫血清和细胞裂解物降低TNF-α水平。在此基础上研究的结果，可以说，铅呈现鱼的免疫功能低下和炎症状态。因此，TNF-α和IL-1β是下调，可能是由重金属[8]的动作，但补充益生菌逆转这些基因的表达这些效果。 热休克蛋白的合成（的Hsp）增加响应于各种物理和化学应激，包括温度和金属应力，因此这些蛋白质可以是良好的环境应力的生物标记[56]。杆菌热休克蛋白在其中的Pb-暴露组增加，在L.进一步降低的表达P16补充groupOur结果表明，铅曝光显著升高HSP70和HSP90的鱼的头肾转录水平。然而，罗伊氏乳杆菌P16补充减少的HSPs的鱼中的表达，从而缓和应力的条件。在我们的研究结果一致，Mohapatra等人[19]证实了由枯草芽孢杆菌的益生菌混合物的饮食补充，乳酸乳球菌，和酿酒酵母中L.鲮降低HSP70的表达。益生菌产生的应力降低因素可能已经降低在鱼的HSP的水平，减少铅引起的应力，并导致更好的生长和免疫力。 有趣的是，铅，罗伊氏乳杆菌P16联合治疗后逆转所研究的鲤科鱼类铅引起的不良影响。通过针对在鲤鱼铅毒性益生菌的潜在有益效果和保护已被示于图8的血液学参数表明L的能力罗伊氏P16到刺激红细胞生成，从而提高了氧的运输，和一个类似的结果已被先前报道[ 13。以前，我们已经证明此菌株[28]的优良铅结合能力。因此，在该菌株补充饮食可以减少铅的肠道吸收，并降低在其它组织中的Pb的积累。该菌株罗伊氏乳杆菌P16具有很强的抗氧化能力[28]。与Pb和L.本研究中，共治疗罗伊氏P16减少临时生产MDA的和增加的抗氧化酶（SOD和GPX）的活动。因此，罗伊氏乳杆菌P16减轻了鲤鱼铅诱导的氧化应激。然而，针对铅诱导的氧化应激和的肠屏障功能障碍的需要这种菌株的抗氧化活性的潜在作用，进一步调查。另外，Pb和罗伊氏乳杆菌P16共治疗逆转促炎细胞因子（TNF-α和IL-1β），其与NF-κB信号传导途径和活性氧的产生和随后的防止铅降低相关联的表达式诱导细胞凋亡。然而，在LAB菌株全面铅耐受机制都没有受到调查。因此，在LAB菌株Pb胁迫响应网络尚待阐明。进一步，关键蛋白和参与乳酸杆菌菌株的铅耐受性途径不明。因此，用于相对和绝对定量（iTRAQ的）同量异位标记为基础的比较和功能蛋白质组学方法可以被利用来探索罗伊氏乳杆菌菌株有关的关键蛋白质和途径的Pb-公差。此外，在研究的早期阶段，我们不知道如何益生菌修复鱼类暴露于重金属发生的代谢损伤; 因此，应努力在未来作出合并多重目标（基因，蛋白质和代谢物）探索在主机益生菌的重金属防御机制。值得注意的是，与L.治疗杆菌P16本身并不会对业绩增长的任何不利影响，hemato 用于相对和绝对定量（iTRAQ的）同量异位标记为基础的比较和功能蛋白质组学方法可以被利用来探索罗伊氏乳杆菌菌株有关的关键蛋白质和途径的Pb-公差。此外，在研究的早期阶段，我们不知道如何益生菌修复鱼类暴露于重金属发生的代谢损伤; 因此，应努力在未来作出合并多重目标（基因，蛋白质和代谢物）探索在主机益生菌的重金属防御机制。值得注意的是，与L.治疗杆菌P16本身并不会对业绩增长的任何不利影响，hemato 用于相对和绝对定量（iTRAQ的）同量异位标记为基础的比较和功能蛋白质组学方法可以被利用来探索罗伊氏乳杆菌菌株有关的关键蛋白质和途径的Pb-公差。此外，在研究的早期阶段，我们不知道如何益生菌修复鱼类暴露于重金属发生的代谢损伤; 因此，应努力在未来作出合并多重目标（基因，蛋白质和代谢物）探索在主机益生菌的重金属防御机制。值得注意的是，与L.治疗杆菌P16本身并不会对业绩增长的任何不利影响，hemato 在研究的初期阶段，我们不知道如何益生菌修复鱼类暴露于重金属发生的代谢损伤; 因此，应努力在未来作出合并多重目标（基因，蛋白质和代谢物）探索在主机益生菌的重金属防御机制。值得注意的是，与L.治疗杆菌P16本身并不会对业绩增长的任何不利影响，hemato 在研究的初期阶段，我们不知道如何益生菌修复鱼类暴露于重金属发生的代谢损伤; 因此，应努力在未来作出合并多重目标（基因，蛋白质和代谢物）探索在主机益生菌的重金属防御机制。值得注意的是，与L.治疗杆菌P16本身并不会对业绩增长的任何不利影响，hemato

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结果 (简体中文) 2:[复制]

复制成功！

炎症反应是先天免疫反应系统的关键元素，主要由细胞因子介导[54]。在鱼类的遗传水平上，对重金属的免疫毒理学知之甚少。细胞因子IL-1+和TNF-α主要由单核细胞和巨噬细胞产生，并调节免疫反应的多个方面。TNF-α 影响炎症中的组织血管，也诱导肝脏的急性相蛋白 [55]。在本研究中，发现Pb处理组中的TNF-α水平明显低于对照组。在Pb处理的组中，IL-1+水平较低，尽管这种差异在统计学上没有显著性。观察到的TNF-α和IL-1+的降调涉及NF-βB信号通路。然而，L. reuteri P16 补充对 TNF-α 和 IL-1+ 表达呈阳性。在先前的研究中，Paul etal [8] 记录到暴露于铅醋酸盐的血清和细胞里结的酸酯和细胞莱酸盐中TNF-α水平下降。根据这项研究的结果，可以说明Pb使鱼处于免疫功能低下和炎症状态。因此，TNF-α和IL-1+是低调节的，可能是由重金属[8]的作用，但益生菌补充逆转这些影响对这些基因的表达。 热休克蛋白（Hsps）的合成会随着各种物理和化学应力（包括温度和金属应力）而增加，因此这些蛋白质可能是良好的环境应激生物标志物[56]。在P.reuteri P16补充组中，在Pb暴露组中增加的HPS表达进一步下降。结果显示，Pb暴露显著提升了鱼头肾中HSP70和HSP90的转录水平。然而，L. reuteri P16 补充减少 HPS 的表达，从而减轻鱼类的压力状况。与研究结果一致，Mohapatra等人[19]证明，膳食补充由亚氏杆菌、乳酸乳酸杆菌和糖霉菌组成的益生菌混合物在L.Rohita中降低HSP70的表达。益生菌产生的减压因子可能降低鱼类的HSP水平，降低Pb引起的压力，并产生更好的生长和免疫力。 有趣的是，与Pb和L.reuteri P16的共治疗逆转了Pb对所研究的鲤鱼物种的不利影响。图8显示了益生菌对鲤鱼中Pb毒性的潜在有益影响和保护。血液学参数表明L.reuteri P16刺激红细胞生成的能力，从而增加氧气输送，类似的结果也早先报告[13]。此前，我们已经演示了该菌株的优秀Pb结合能力[28]。因此，补充这种菌株的饮食可以减少肠道对Pb的吸收，减少其他组织的Pb积累。菌株 L. reuteri P16 具有很强的抗氧化能力 [28]。在本研究中，与Pb和L.reuteri P16联合治疗减少了MDA的临时生产，并增加了抗氧化酶（SOD和GPx）的活性。因此，L. reuteri P16 缓解了鲤鱼中由 Pb 引起的氧化应激。然而，这种菌株对Pb引起的氧化应激和肠道屏障功能障碍的抗氧化活性的基础性作用需要进一步研究。此外，与Pb和L.reuteri P16的共治疗逆转了原炎细胞因子（TNF-α和IL-1+）的表达，这些细胞因子与NF-βB信号通路和随后减少活性氧物种生产有关，并防止Pb诱导细胞凋亡。然而，LAB菌株中的综合Pb公差机制尚未研究。因此，LAB菌株中的Pb应力响应网络还有待阐明。此外，乳酸杆菌菌株的Pb耐受性所涉及的关键蛋白质和途径是未知的。因此，可用于相对和绝对定量（iTRAQ）的左右重标记，用于探索与L.reuteri菌株中的关键蛋白质和通路相关的Pb耐受性。此外，在研究的早期阶段，我们不知道益生菌如何修复暴露于重金属的鱼类的代谢损伤;因此，今后应努力纳入多种靶点（基因、蛋白质和代谢物），以探索宿主中益生菌的重金属防御机制。值得注意的是，仅使用L.reuteri P16治疗对生长性能没有任何不利影响，造异体

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结果 (简体中文) 3:[复制]

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炎症反应是先天免疫反应系统中的一个关键因素，主要由细胞因子介导[54]。在鱼类的遗传水平上，重金属的免疫毒理学知之甚少。细胞因子IL-1β和TNF-α主要由单核细胞和巨噬细胞产生，调节免疫反应的多个方面。TNF-α在炎症中影响组织血管系统，并诱导来自肝脏的急性期蛋白[55]。在本研究中，发现铅处理组的TNF-α水平显著低于对照组。铅治疗组IL-1β水平较低，但差异无统计学意义。观察到的TNF-α和IL-1β的Ddown调节与NF-κB信号通路有关。然而，补充鲁氏乳杆菌P16对TNF-α和IL-1β的表达呈阳性。在先前的一项研究中，Paul etal[8]记录了暴露于醋酸铅的淡水鱼斑点香肠的血清和细胞裂解物中TNF-α水平的降低。根据这项研究的结果，可以说铅使鱼处于免疫受损和炎症状态。因此，TNF-α和IL-1β下调，可能是重金属的作用[8]，但益生菌的补充逆转了这些对这些基因表达的影响。 热休克蛋白（heat shock proteins，hsp）的合成随着各种物理和化学应激源（包括温度和金属应激）的反应而增加，因此这些蛋白可以成为良好的环境应激生物标记物[56]。铅暴露组HSPs的表达增加，而鲁氏疟原虫P16组HSPs的表达进一步降低。结果表明，铅暴露显著提高了鱼头肾HSP70和HSP90的转录水平。然而，补充鲁氏乳杆菌P16可降低鱼类HSPs的表达，从而缓解应激状态。与我们的研究结果一致，Mohapatra等人[19]证明，饮食中添加由枯草芽孢杆菌、乳酸乳球菌和酿酒酵母组成的益生菌混合物降低了罗氏乳杆菌中HSP70的表达。益生菌产生的减压因子可能降低了鱼类的热休克蛋白水平，降低了铅引起的应激，并导致更好的生长和免疫。 有趣的是，铅和鲁氏衣原体P16联合处理对鲤鱼的不利影响进行了研究。图8显示了益生菌对鲤鱼Pb毒性的潜在有益作用和保护作用。血液学参数表明，罗伊氏菌P16有刺激红细胞生成的能力，从而增加了氧转运，早期也有类似的结果[13]。在此之前，我们已经证明了该菌株具有优异的铅结合能力[28]。因此，用该菌株补充日粮可以减少肠道对铅的吸收，减少铅在其他组织中的积累。菌株p16具有较强的抗氧化能力[ 28 ]。在本研究中，与Pb和R.Tuvii P16的协同处理降低了MDA的暂时生成，并增加了抗氧化酶（SOD和GPX）的活性。因此，L.Rewiti p16减轻了Pb诱导的鲤鱼氧化应激。然而，该菌株的抗氧化活性对铅诱导的氧化应激和肠屏障功能障碍的潜在作用需要进一步研究。此外，铅和鲁氏衣原体P16联合治疗可逆转促炎性细胞因子（TNF-α和IL-1β）的表达，这些细胞因子与NF-κB信号通路相关，并随后降低活性氧的产生，防止铅诱导的细胞凋亡。然而，实验室菌株对铅的综合耐受机制尚未得到研究。因此，实验室菌株的铅胁迫响应网络尚待阐明。此外，与乳酸杆菌耐铅性相关的关键蛋白和途径尚不清楚。因此，基于相对和绝对定量（iTRAQ）的比较和功能蛋白质组学方法的等压标签可用于探索与reuteri菌株关键蛋白和途径相关的铅耐受性。此外，在这一研究的早期阶段，我们还不知道益生菌是如何修复重金属对鱼类的代谢损伤的，因此，今后应努力将多种靶点（基因、蛋白质和代谢物）结合起来，探索益生菌在宿主体内的重金属防御机制。值得注意的是，单独使用罗伊氏菌P16对生长性能、血液

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