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α-单月桂酸甘油酯(希特力)的抗炎特性

时间:2019-04-08 11:02来源:未知 作者:admin 点击:



 
introduction
简介
 

Researchers are continuously investigating what the limiting factors are for our production animals to reach 100% of their genetic growth potential. Farm management, climate conditions and nutrition are all important factors. However, it became clear that the immunological processes, and more particularly inflammation, can be a high cost for animal growth as well. Hence, if we are able to reduce this negative impact of pro-inflammatory processes by supplying anti-inflammatory compounds, we are one step closer to achieve 100% of the genetic growth potential of livestock.

研究人员正在不断研究生产动物达到其遗传生长潜力最大化的限制因素。农场管理,气候条件和营养都是重要因素。然而,很明显,免疫过程,尤其是炎症,对动物生长也是很高的一个成本。因此,如果我们能够通过提供抗炎化合物来减少促炎过程的负面影响,我们距离实现100%的家畜遗传生长潜力更近了一步。

 

Inflammation is defined as a reaction of tissue to any harmful stimulus. This stimulus can either be of physical, chemical or immunological origin, but can also be caused by microorganisms like bacteria, parasites, and viruses. As the gastrointestinal tract (GIT) is the largest immune organ of the body and the place where 70% of the immune cells are located, these harmful stimuli will often result in the activation of the gastrointestinal immune (GI) system. Hence, specialized cells start to do their job, especially by producing pro-inflammatory cytokines (e.g. interleukin-6 (IL-6)). Cytokines are polypeptides produced mainly by monocytes, macrophages, and lymphocytes and regulate the intensity and duration of an immune response.

炎症定义为组织对任何有害刺激的反应。这种刺激可以是物理的,化学的或免疫的,但也可以由细菌,寄生虫和病毒等微生物引起。由于胃肠道(GIT)是身体的最大免疫器官和70%免疫细胞所在的位置,这些有害刺激通常会导致胃肠道免疫(GI)系统的激活。因此,特化细胞开始发挥作用,特别是通过产生促炎细胞因子(例如白细胞介素-6(IL-6))。细胞因子是主要由单核细胞,巨噬细胞和淋巴细胞产生的多肽,并调节免疫应答的强度和持续时间。

 

It is known that even mild immune challenges already induce a reduction in feed intake and consequently can have a negative impact on growth. In addition, the immune cells require nutrients and energy to perform their function. There is also an increased protein demand during inflammation, because the production of acute-phase proteins and antibodies requires protein. Processes as lipolysis, proteolysis and glycolysis (degradation of fat, protein and glucose respectively) will provide the required nutrients and energy for the induced immune response. However, due to the reduced feed intake, body reserves may be used to release the required nutrients and energy. In case of a severe or prolonged infection this will result in a clear loss of body weight. In humans, for instance, a severe infection can lead to a loss in body weight of 15-30%.

众所周知,即使轻微的免疫挑战诱导了采食量的减少,因此可能对生长产生负面影响。此外,免疫细胞需要营养和能量来发挥其功能。炎症期间蛋白质需求也增加,因为急性期蛋白质和抗体的产生需要蛋白质。脂解,蛋白水解和糖酵解(分别降解脂肪,蛋白质和葡萄糖)的过程将为诱导的免疫应答提供所需的营养和能量。然而,由于采食量减少,身体储备可用于释放所需的营养和能量。在严重或长期感染的情况下,这将导致明显的体重减轻。 例如,在人类中,严重感染可导致体重减轻15-30%。

 

It is important to remember that the GI immune system should be activated in case of high pathogenic pressure, but we should avoid that the immune system over-reacts when small challenges occur. If we can manage to reduce the negative impact of pro-inflammatory processes by supplying anti-inflammatory compounds, we are one step closer to achieve 100% of the genetic growth potential of an animal. Therefore, the aim of this in vitro trial was to study whether a-monoglycerides have anti-inflammatory properties.

重要的是要记住,在高致病性压力的情况下应该激活GI免疫系统,但我们应该避免免疫系统在发生小的挑战时过度反应。如果我们能够通过提供抗炎化合物来减少促炎过程的负面影响,我们距离实现100%的动物遗传生长潜力更近了一步。因此,该体外试验的目的是研究FraC12否具有抗炎特性。

 
Materials and Methods
(材料和方法)
 
 

At the laboratory of Prof. Niewold at KU Leuven in Belgium, an in vitro model was used to study the potential anti-inflammatory effects of several compounds. In this model, macrophage-like cells were used and were challenged with lipopolysaccharides (LPS) from E. coli. Upon this challenge, the macrophage-like cells produced Nitric Oxide (NO). When the selected compound can reduce NO production, it means that the compound has anti-inflammatory properties.

在比利时鲁汶大学的Niewold教授的实验室中,使用体外模型研究了几种化合物的潜在抗炎作用。在该模型中,使用来自大肠杆菌的脂多糖(LPS)攻击巨噬细胞样细胞。在这次攻击后,巨噬细胞样细胞产生一氧化氮(NO)。当所选化合物可以减少NO产生时,这意味着该化合物具有抗炎特性。

 

The Raw 264.7 macrophage-like cells were incubated for 24 hours at 37°C. Then 50 μl/well of serially diluted oxytetracycline dihydrate (OTC) (control group) and serially diluted extracts (a-mono-, di- and triglycerides of lauric acid; a-monolaurin; a-mono-, di- and triglycerides of caprylic acid (C8)/ capric acid (C10); and a-mono-, di- and triglycerides of pelargonic acid (C9)) were added and incubated for 4 hours at 37°C. Subsequently, 50 μl medium containing LPS from E. coli (50 ng LPS/ml) was added, giving a final concentration of 12.5 ng LPS/ml, and incubated for 24 hours at 37°C. Then, 100 μl of medium was taken, and pipetted into another 96 wells plate. Quantification of NO production was performed with Griess reagent using a serial dilution of NaNO2 as a standard. The inhibitory concentration 50 (IC50) was calculated using the sigmoidal dose response (variable slope) method, using GraphPad Prism 5 for Windows. The inhibitory concentration 50 indicates from which dose level onwards an anti-inflammatory effect was observed. All samples were tested in duplicate.

将Raw 264.7巨噬细胞样细胞在37℃温育24小时。然后50μl/孔来连续稀释的土霉素二水合物(OTC)(对照组)和连续稀释的提取物(月桂酸的a-单,二和甘油三酯;α-单月桂酸甘油酯;辛酸(C8)/癸酸(C10)的a-单,二和甘油三酯; 壬酸(C9)的a-单,二和甘油三酯)并在37℃下温育4小时。随后,加入含有来自大肠杆菌的LPS(50ng LPS / ml)的50μl培养基,得到12.5ng LPS / ml的终浓度,并在37℃温育24小时。然后,取出100μl培养基,并移液到另一个96孔板中。使用连续稀释的NaNO2作为标准,用Griess试剂进行NO产生的定量。抑制浓度50 (IC50)采用s形剂量响应(变斜率)法,用GraphPad Prism 5来分析。抑制浓度50表明从哪个剂量水平开始观察到抗炎作用。所有样品一式两份进行测试。

 

Results
结果 
 

 

The model is based on the reduction of production of NO by macrophages. Nitric Oxide is an intercellular messenger that has been recognized as one of the most versatile players in the immune system. Cells of the innate immune system, like macrophages, use pattern recognition receptors to recognize the molecular patterns associated with pathogens. Activated macrophages then inhibit pathogen replication by releasing a variety of effector molecules, including NO. In addition to macrophages, many other immune-system cells produce and respond to NO. Thus, NO is important as a toxic defense molecule against infectious organisms. (Tripathi et al., 2007) 

该模型基于巨噬细胞减少NO的产生。NO是一种细胞间信使,已被公认为免疫系统中最通用的参与者之一。先天免疫系统的细胞,如巨噬细胞,使用模式识别受体来识别与病原体相关的分子模式。然后,活化的巨噬细胞通过释放多种效应分子(包括NO)来抑制病原体复制。除了巨噬细胞,许多其他免疫系统细胞产生并响应NO。因此,NO作为针对传染性生物的毒性防御分子是重要的。(Tripathi等,2007)

 

Even though NO has some anti-bacterial activity, it can have a negative effect on the intestinal bacterial diversity as well. Nitric oxide is rapidly transformed to nitrate in the lumen. A nitrate rich environment is beneficial for strains like E. coli, but non-favorable for the obligate anaerobic bacteria like Clostridia and Bacteriodia. Hence, the balance between the good (Clostridia and Bacteriodia species) and the bad bacteria (E. coli) is disturbed. In addition, inflammation increases the oxygen level in the lumen, which also results in a reduction of the obligate anaerobic bacteria and consequently an increased loss of bacterial diversity. (Gresse et al., 2017)

即使NO具有一些抗菌活性,它也会对肠道细菌多样性产生负面影响。一氧化氮在腔中迅速转化为硝酸盐。富含硝酸盐的环境对大肠杆菌等菌株有益,但对梭状芽孢杆菌和Bacteriodia等专性厌氧菌不利。因此,良好(梭状芽孢杆菌和细菌)和坏细菌(大肠杆菌)之间的平衡受到干扰。此外,炎症增加了腔中的氧水平,这也导致专性厌氧细菌的减少,并因此导致细菌多样性的损失增加。(Gresse等,2017)

 

Increasing evidence indicates that NO also plays a part in acute and chronic inflammation. Inhibiting NO synthase (NOS), the enzymes that convert L-arginine to NO, reduced the degree of inflammation in rats with acute inflammation. This indicates that it is important to modulate NO production. Hence, the aim of this in vitro study was to study whether different types of glycerides were able to reduce NO production. If NO production is reduced, we can assume that the molecule has anti-inflammatory properties.

越来越多的证据表明NO也在急性和慢性炎症中起作用。抑制NO合成酶(NOS),即将L-精氨酸转化为NO的酶,降低了急性炎症大鼠的炎症程度。 这表明调节NO产生很重要。因此,该体外研究的目的是研究不同类型的甘油酯是否能够减少NO产生。如果NO产量减少,我们可以假设该分子具有抗炎特性。

 

Figure 1 shows the effect of pure a-monolaurin, a-mono-, di- and triglycerides of C12 and OTC on NO production by LPS challenged macrophage-like cells.

图1显示了纯的a-单月桂酸甘油酯,C12的a-单,二和甘油三酯和OTC对脂多糖(LPS)激发的的巨噬细胞样细胞产生NO的影响。


 

 

Figure 1. Effect of α-monolaurin, a-mono-, di- and triglycerides of C12, and OTC on Nitric Oxide (NO) production induced by LPS challenge.

图1. a-单月桂酸甘油酯,月桂酸的a-单,二和甘油三酯和OTC对脂多糖LPS激发诱导的一氧化氮(NO)产生的影响。

 

In this study OTC was used as a control group and clearly showed the expected reduction in NO production at an IC50 of 400 ppm. Alpha-monolaurin also showed a clear reduction in NO production, but already at IC50 of 200 ppm. Surprisingly, there was no difference in IC50 level between the combination of mono-, di-and triglycerides of lauric acid containing approximately 40% α-monolaurin and the sample containing 90% a-monolaurin.

在该研究中,OTC作为对照组,并清楚地显示预期的NO产生减少在400ppm的IC50减少。α-月桂酸甘油酯也显示出NO产生的明显减少,但已经达到200ppm 的IC50。令人惊讶的是,在a-单,二和甘油三酯组合的样品含量约为40%的α-单月桂酸甘油酯和含有90%单月桂酸甘油酯的之间的IC50水平没有差异。

 

The combination of a-mono-, di- and triglycerides of C8/C10 showed intermediate results with an IC50 of 800 ppm. Alpha-mono-, di- and triglycerides of C9 also proved to have anti-inflammatory properties at an IC50 of 1000 ppm (Figure 2).

C8 / C10的a-单,二和甘油三酯组合显示中间结果,IC50为800ppm。C9的a-单,二和甘油三酯组合在IC50为1000ppm也被证明具有抗炎特性,(图2)


 

 
Conclusions
结论
 

The GI immune system should be activated in case of high pathogenic pressure, but over-reaction of the immune system should be avoided when only small challenges occur. If we are able to reduce negative impact of pro-inflammatory processes by supplying anti-inflammatory compounds, we are one step closer to achieve 100% of the genetic growth potential of an animal. From this in vitro trial, it became clear that glycerides of C8/C10, C9 and C12 have anti-inflammatory properties next to their anti-microbial effects. This gives us more insight in the mode of action of a-monoglycerides. Moreover, it gives us extra information why a-monoglycerides stimulate animal growth and performance in case of low pathogenic pressure.

在高致病性压力的情况下应该激活GI免疫系统,但是当仅发生小的挑战时应该避免免疫系统的过度反应。如果我们能够通过提供抗炎化合物来减少促炎过程的负面影响,我们距离实现100%的动物遗传生长潜力更近一步。从该体外试验中可以清楚地看出,C8 / C10,C9和C12的甘油酯除了具有抗微生物作用外,还具有抗炎特性。这使我们更加了解FraC12的作用方式。 此外,它为我们提供了额外的信息,为什么FraC12会在低致病压力的情况下刺激动物的生长和表现。

 

来源:荷兰FRAmelco/佰高威盛公司

 

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