CN113476430A - 白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能损伤的药物中的应用 - Google Patents
白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能损伤的药物中的应用 Download PDFInfo
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Abstract
本发明公开一种白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能损伤的药物中的应用，利用白藜芦醇对草甘膦诱导的猪乳腺上皮细胞损伤进行修复的问题。白藜芦醇可以显著缓解由草甘膦抑制的猪乳腺上皮细胞活力。上调由草甘膦诱导的猪乳腺上皮细胞抗凋亡基因BCL2和BCL‑XL的表达，上调草甘膦引起的猪乳腺上皮细胞中解毒酶GST和GSH基因的表达。本发明公开了白藜芦醇可以减轻草甘膦诱导PMECs细胞的增殖毒性、氧化，具有保护动物乳腺上皮细胞的完整性和健康的优点。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能损伤的药物中的应用。
背景技术
草甘膦(Glyphosate，GLP)，是目前世界上使用最广泛的广谱型出苗后除草剂。用于各种作物，特别是水稻、玉米和大豆。现在已经有大量研究表明GLP会对动物产生毒性作用，包括器官毒性、细胞毒性、遗传毒性、生殖毒性以及免疫系统、抗氧化系统和内分泌系统等方面。更重要的是有报道证明人的血清和母乳中也会残留GLP和及其主要代谢物氨甲基膦酸AMPA，而乳腺是合成乳液的主要场所，它能够从血液中吸收所需要的蛋白质、脂类、糖类等营养物质，并转化成乳液中的营养成分。它不仅可以把母亲的营养提供给后代，而且还可以提高后代的免疫力，这与动物繁殖息息相关。但是目前缺乏GLP对猪乳腺上皮细胞(PMECs)的系统研究。因此，选择GLP对PMECs进行体外研究以探求GLP刺激是否会对PMECs产生损伤。
白藜芦醇(Resveratrol，Res)是一种天然多酚化合物，存在于葡萄、花生和桑葚等植物中。它具有多种生物活性，包括抗氧化、抗炎和抗肿瘤的发生发展血小板聚集、动脉粥样硬化与衰老等。白藜芦醇的一个主要靶点是抗氧化转录因子Nrf2。饲料中添加白藜芦醇可以清除清除各种氧化剂的活性并抑制其生成的能力，可以中和多种氧化剂，减少ROS的产生，并增强了SOD，GSH和GPx的活性，从而提高乳腺上皮细胞的抗氧化损伤能力，减少氧自由基对乳腺的损害，保护细胞、组织和器官免受自由基造成的损伤，稳定机体细胞膜和蛋白质的结构，保护动物机体的各项生理功能。
发明内容
基于以上不足之处，本发明提供一种白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能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猪乳腺细胞损伤药物的问题。
为了实现上述目的，本发明技术方案如下：一种白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能损伤的药物中的应用。
进一步，所述的应用，用于减轻草甘膦引起的猪乳腺上皮细胞中活性抑制。
进一步，所述的应用，用于上调草甘膦引起的猪肠乳腺上皮细胞中抗凋亡基因BCL2和BCL-XL的mRNA表达量。
进一步，所述的应用，用于下调草甘膦引起的猪乳腺上皮细胞中解毒酶GSH和GST的mRNA表达量。
本发明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：本发明公开了白藜芦醇(Res)对草甘膦诱导猪乳腺上皮细胞氧化保护作用。随着草甘膦浓度的增加，细胞活力显著降低，白藜芦醇可以显著增加细胞活力。此外，白藜芦醇还可以显著上调由草甘膦诱导的抗凋亡基因BCL2和BCL-XL的mRNA表达量，显著上调由草甘膦诱导的GSH和GST的mRNA表达量。本发明公开了白藜芦醇可以减轻草甘膦诱导PMECs细胞的增殖毒性、氧化，具有保护动物乳腺上皮细胞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Res对GLP造成的PMECs细胞活性损伤的保护作用图，标注不同小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
图2为发明实施例提供的Res对GLP细胞抗凋亡基因BCL2和BCL-XL的mRNA表达量对比图；
图3为发明实施例提供的Res对GLP细胞解毒酶GSH和GST的mRNA相对表达量的影响对比图，标注不同小写字母表示差异显著(P<0.05)，相同字母表示差异不显著(P>0.05)。
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。
本发明实施例中，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)均购自中国碧云天生物公司；Res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)。
本发明实施例中，PMECs细胞系为由美国德州农工大学伍国耀教授馈赠。PMECs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。Res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。试验数据先用MicrosoftExcel 2016整理，之后用SPSS 19.0(SPSS Inc，Chicago，IL，USA)统计软件分析所有试验数据，并将三个独立试验的数据表示为平均值±标准误差，然后通过单因素分析(One-WayANOVA)进行数据分析。各组之间的差异通过Duncan测试进行评估。P<0.05表示该分析结果差异显著
实施例1、Res缓解GLP诱导PMECs细胞增殖毒性
将IPEC-J2细胞与50μmol/L的Res和180mg/L的GLP共培养4h，结果如图1所示，50μmol/L的Res减轻了GLP造成的细胞损伤，细胞活力显著提高(P<0.05)。
实施例2、Res缓解GLP诱导的PMECs细胞凋亡
1.Res减轻GLP诱导PMECs细胞抗凋亡因子BCL2和Bax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检测系统进行实时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，
BCL2引物序列如下，
F:ACTTCTGCGAAAGCGAATTGCC，
R:AGCCTCCGTTTTGCCTTATCC；
BCL-XL引物序列如下，
F:GCGTGGAGAGCGTAGACAAG，
R:CCGCCGTTCTCCTGGATC。
由图2可以看出，Res的添加使PMECs细胞BCL2和Bax的mRNA表达量显著升高(P<0.05),由此初步推断NAC可以缓解GLP诱导的细胞凋亡。
2.Res提高GLP诱导PMECs细胞解毒酶基因GSH和GST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检测系统进行实时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，
GSH引物序列如下，
F:TGCACGAATTCTCAGCCAAGGAC
R:GGTGACGATGCACACGTAGCC；
GST引物序列如下，
F:GGACTCTCCTGGTCCTGAATGCC,
R:AGAACTGGCACCAGACCTGAGG。
由图3可以看出，Res的添加使PMECs细胞GSH和GST的mRNA表达量显著升高(P<0.05),由此初步推断NAC可以缓解GLP诱导的细胞氧化应激。
Claims (4)
1.一种白藜芦醇在制备治疗由草甘膦引起的猪乳腺上皮细胞抗氧化功能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，用于减轻草甘膦引起的猪乳腺上皮细胞中活性抑制。
3.根据权利要求1所述的应用，其特征在于，用于上调草甘膦引起的猪肠乳腺上皮细胞中抗凋亡基因BCL2和BCL-XL的mRNA表达量。
4.根据权利要求1所述的应用，其特征在于，用于下调草甘膦引起的猪乳腺上皮细胞中解毒酶GSH和GST的mRNA表达量。
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