CN113440505A - N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用 - Google Patents
N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用 Download PDFInfo
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Abstract
本发明公开了N‑乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用，能够有效减轻或治疗由草甘膦诱导肠道细胞损伤药物的问题。所述的应用包括减轻因草甘膦引起的猪肠上皮细胞中活性抑制，下调由草甘膦诱导的猪肠道上皮细胞中活性氧含量和白细胞介素IL‑6的mRNA相对表达量。下调由草甘膦诱导的猪肠道上皮细胞微管相关蛋白轻链3(LC3)由LC3‑I到LC3‑II转化比率的表达，上调草甘膦引起的猪肠上皮细胞中自噬相关底物P62的蛋白消耗，具有保护动物肠道的完整性和健康的优点。
Description
技术领域
本发明涉及兽医领域，具体涉及一种N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用。
背景技术
N-乙酰半胱氨酸，以下简称NAC，结构式如下所示：
NAC是含巯基的氨基酸衍生物，其作为活性氧(ROS)清除剂和谷胱甘肽的底物，可对抗不同原因所致的组织氧化损伤，是一种具有研究价值的用途广泛的药物。主要作为一种抗氧化剂，对体内乙酰氨基酚的亲电子代谢物活性氧(ROS)，如羟基(OH)、二氧化氮(NO2)和过氧化氢(H2O2)快速反应中和它们。另外，NAC调节细胞的代谢活性,调整基因的表达和信号转导***,在临床和实验中得到了广泛的应用。
草甘膦(Glyphosate，GLP)是一种内吸、高效的有机磷性质的广谱除草剂，是全世界范围内使用量最大、应用最广泛、年销量最高的除草剂，且使用量仍在持续上升。草甘膦作为污染物广泛存在于土壤和地表水中，动物饲料中也都含有不同浓度的草甘膦残留。人类不可避免的通过水、食物、空气等多种方式接触草甘膦。草甘膦会对动物多个器官造成损害，当动物体长期接触草甘膦，会产生内分泌***、免疫***、神经***、消化***、细胞与基因损伤。
猪肠道是集消化、吸收、内分泌、免疫和防御功能于一体的器官，易受到农药、毒素等有害外源性物质的损伤。猪肠道上皮细胞(IPEC-J2)是防御外部刺激的第一道物理屏障。IPEC-J2来源于未哺乳仔猪空肠上皮细胞。它是一种非转化、非致瘤性肠上皮细胞系，在体内活性相当。适用于体外模型，可以代表正常生理条件下的猪肠上皮细胞。综上所述，如何研发一种可减轻或治疗由于草甘膦诱导的猪肠道损伤的药物，是亟待要解决的技术问题。
发明内容
基于以上不足之处，本发提供一种N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用，以解决现有技术中缺乏有效减轻或治疗由草甘膦诱导肠道细胞损伤药物的问题。
本发明的技术方案如下：一种N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用。
进一步的，以上所述的应用，用于减轻因草甘膦引起的猪肠上皮细胞中活性抑制。
进一步的，以上所述的应用，用于下调因草甘膦引起的猪肠上皮细胞中活性氧含量。
进一步的，以上所述的应用，用于下调因草甘膦引起的猪肠上皮细胞中促炎性因子IL-6的mRNA表达量。
进一步的，以上所述的应用，用于下调因草甘膦引起的猪肠上皮细胞中自噬相关蛋白LC3-II/LC3-I比率的表达。
进一步的，以上所述的应用，用于上调因草甘膦抑制的猪肠上皮细胞中自噬相关底物P62的蛋白表达。
所述N-乙酰半胱氨酸制备的药物是片剂、胶囊剂、颗粒剂、滴丸剂、混悬剂、糖浆剂、各种肠溶制剂或注射剂。各种剂型可以按照药学领域的常规生产方法制备，例如使活性成分与一种或多种载体混合，然后将其制成所需的剂型。
本发明具有如下优点及有益效果：N-乙酰半胱氨酸可以减轻草甘膦诱导IPEC-J2细胞的增殖毒性、氧化、炎症和自噬，具有保护动物肠道的完整性和健康的优点。
附图说明
图1为本发明实施例提供的NAC保护GLP造成的IPEC-J2细胞活性损伤的作用图，标注不同小写字母表示差异显著，相同字母表示差异不显著。
图2为本发明实施例提供的NAC抑制GLP诱导的细胞活性氧荧光图。
图3为本发明实施例提供的NAC抑制GLP诱导的细胞活性氧含量作用图，标注不同小写字母表示差异显著，相同字母表示差异不显著。
图4为本发明实施例提供的NAC抑制GLP诱导的细胞炎症因子IL-6mRNA相对表达量的作用图，标注不同小写字母表示差异显著，相同字母表示差异不显著。
图5为本发明实施例提供的NAC对GLP诱导的细胞自噬相关蛋白影响的蛋白质印记图。
图6为本发明实施例提供的NAC缓解GLP引起的自噬相关蛋白P62消耗的蛋白质印记量化图。标注不同小写字母表示差异显著，相同字母表示差异不显著。
图7为本发明实施例提供的NAC抑制GLP引起的自噬相关蛋白LC3脂化的蛋白质印记量化图。
标注不同小写字母表示差异显著，相同字母表示差异不显著。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
试验材料：磷酸盐缓冲液(PBS)，0.25％胰酶-乙二胺四乙酸(EDTA)，青霉素-链霉素，二甲基亚砜(DMSO)和噻唑基溴化四唑鎓(MTT)，活性氧测定试剂盒均购自中国碧云天生物公司；NAC纯度≥99％购自Sigma-Aldrich公司(美国)；DMEM/F12培养基和胎牛血清(FBS)购自Hyclone公司(美国)。草甘膦(农达)购自Monsanto公司(美国)。
本发明的实施例中，IPEC-J2细胞系为中国农业大学动物科学技术学院馈赠。IPEC-J2细胞培养液为含10％FBS和1％青霉素-链霉素的DMEM/F12培养基，接种于25cm2培养瓶中，置于37℃，5％CO2浓度的培养箱内培养。NAC和草甘膦均采用不含血清和抗生素的培养液稀释成不同浓度，现配现用。
以下实施例中，试验中每种处理均进行三次独立实验。试验数据使用MicrosoftExcel2016进行整理和计算，再使用SPSS 19.0统计软件(芝加哥，美国)进行单因素方差分析(One-Way ANOVA)，采用Duncan法进行多重比较。试验结果以平均值(Mean)和标准误(SEM)的形式表示。以P<0.05作为差异显著性判断标准。
实施例1、NAC缓解GLP诱导IPEC-J2细胞增殖毒性
将IPEC-J2细胞与不同浓度的NAC(0、0.5、1mM)和不同浓度GLP(0、30、60mg/L)共培养12h，结果如图1所示，不同浓度的NAC减轻了GLP造成的细胞损伤。当采用1mM NAC处理细胞时，细胞活力显著提高(P<0.05)。根据上述结果，选择1mM NAC孵育细胞12h用于后续实验。
实施例2、NAC缓解GLP诱导的IPEC-J2细胞损伤
1.NAC抑制GLP诱导的IPEC-J2细胞活性氧含量
取对数生长期的IPEC-J2细胞，用胰酶消化后以2.5×105cells/孔的密度在6孔板中培养细胞，直到细胞融合率达80％，然后在四种不同的方法(对照，GLP，NAC，GLP+NAC)处理细胞，之后按照1：1000用无血清培养液稀释DCFH-DA，使终浓度10μmol/L。处理时间结束后，去除细胞培养液，用PBS冲洗两遍。加入稀释好的DCFH-DA。37℃细胞培养箱内孵育20min。用PBS洗涤细胞三次，以充分去除未进入细胞内的DCFH-DA。用荧光酶标仪使用488nm激发波长，525nm发射波长，实时检测刺激前后荧光的强弱。并在荧光显微镜下观察。
由图2-3可以看出，NAC的添加使IPEC-J2细胞活性氧含量显著降低(P<0.05),由此初步推断NAC可以缓解GLP诱导的氧化损伤。
2.NAC减轻GLP诱导IPEC-J2细胞炎性因子IL-6的mRNA相对表达量
采用TRIZOL总RNA提取法，用紫外光吸收检测RNA浓度，A260/A280比值在1.8-2.0之间。并通过琼脂糖凝胶验证RNA的完整性。根据制造商的说明，用SYBR Premix Ex TaqTM试剂盒将总RNA反转录成cDNA，并用于实时聚合酶链反应。使用SYBR Real-Time PCR试剂盒实时PCR检测***进行实时PCR。PCR条件为预变性：95℃，30s，循环1次，RT-PCR反应：95℃，5s，61℃，34s循环40次。β-actin作为内参基因，使用2-ΔΔCT方法分析各个基因的相对表达量。荧光定量PCR所用引物是根据Genbank中已知基因序列，由生工生物工程有限公司(上海)设计并合成，IL-6引物序列如下：
F:AATGTCGAGGCCGTGCAGATTAG，
R:TTCATCCACTCGTTCTGTGACTGC。
由图4可以看出，NAC的添加使IPEC-J2细胞IL-6的mRNA表达量显著降低(P<0.05),由此初步推断NAC可以缓解GLP诱导的炎症损伤。
3.NAC减轻GLP诱导IPEC-J2细胞自噬水平
样品裂解液在12000rPm，4℃，离心10min后，对蛋白质定量处理。40μg的总蛋白通过SDS聚丙烯酰胺凝胶进行电泳，之后通过湿电转移法将蛋白转移到PVDF膜上。分别将LC3、p62抗体工作液放入封闭好的PVDF膜，进行四度孵育过夜，之后，用羊抗兔IgG-HRP的二抗孵育。蛋白质信号使用增强化学发光检测***进行检测。将胶片进行扫描，用凝胶图象处理***(Gel-Pro-Analyzer软件)分析目标条带的光密度值，如图5所示，NAC对GLP诱导的细胞自噬相关蛋白影响的蛋白质印记图。
由图6可以看出，NAC的添加使得P62蛋白表达显著升高(P<0.05)，由图7可以看出，NAC的添加使得IPEC-J2细胞LC3-II/LC3-I蛋白比率显著降低(P<0.05)。由此初步推断NAC可以缓解GLP诱导的自噬。
Claims (6)
1.一种N-乙酰半胱氨酸在制备治疗由草甘膦引起的猪肠道损伤的药物中的应用。
2.根据权利要求1所述的应用，其特征在于：用于减轻因草甘膦引起的猪肠上皮细胞中活性抑制的应用。
3.根据权利要求1所述的应用，其特征在于：用于下调因草甘膦引起的猪肠上皮细胞中活性氧含量。
4.根据权利要求1所述的应用，其特征在于：用于下调因草甘膦引起的猪肠上皮细胞中促炎性因子IL-6的mRNA表达量。
5.根据权利要求1所述的应用，其特征在于：用于下调因草甘膦引起的猪肠上皮细胞中自噬相关蛋白LC3-II/LC3-I比率的表达。
6.根据权利要求1所述的应用，其特征在于：用于上调因草甘膦抑制的猪肠上皮细胞中自噬相关底物P62的蛋白表达。
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