相思子碱通过P53/mTOR通路缓解脂多糖对水牛乳腺上皮细胞β-酪蛋白合成的影响  

Abrine mitigates the effects of lipopolysaccharide onβ-casein synthesis in buffalo mammary epithelial cells by modulating the P53/mTOR pathway

作　　者：周金陈 肖鹏[1,2] 李孟琪 张博 郑海英 杨春艳 田新如 黄依林 杨小淦 尚江华[2] ZHOU Jin-chen;XIAO Peng;LI Meng-qi;ZHANG Bo;ZHENG Hai-ying;YANG Chun-yan;TIAN Xin-ru;HUANG Yi-lin;YANG Xiao-gan;SHANG Jiang-hua(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;Guangxi Buffalo Research Institute/Guangxi Key Laboratory of Buffalo Genetics,Reproduction and Breeding,Nanning,Guangxi 530001,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]广西水牛研究所/广西水牛遗传繁育重点实验室,广西南宁530001

出　　处：《南方农业学报》2024年第7期1887-1896,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31872350);广西科技重大专项(桂科AA22068099);广西自然科学基金项目(2021GXNSFAA196021);国家现代农业产业技术体系广西奶水牛产业创新团队项目(nycytxgxcxtd-2021-21-01)。

摘　　要：【目的】探究相思子碱(Abrine)通过调节P53/mTOR通路缓解脂多糖(Lipopolysaccharide,LPS)对水牛乳腺上皮细胞酪蛋白合成的影响,为缓解乳房炎引起的水牛乳品质下降提供理论依据。【方法】使用脂多糖构建水牛乳腺上皮细胞炎症模型,同时使用相思子碱、P53抑制剂(Pifithrin-μ)、P53激活剂(Kevetrin hydrochloride)与水牛乳腺上皮细胞共同孵育12 h;通过HE染色观察水牛乳腺组织形态,采用CCK-8法检测细胞活性,使用ELISA试剂盒检测水牛乳腺上皮细胞β-酪蛋白分泌水平,利用实时荧光定量PCR测定NF-κB、IL-1β、TNFα、β-casein、mTOR、P53、JAK2、STAT5和AKT1基因相对表达量,并以Western blotting检测NF-κB、IL-1β、TNFα、β-casein、mTOR和P53蛋白相对表达量。【结果】临床型乳房炎乳腺组织间隙及腺泡腔被大量中性粒细胞浸润,部分腺泡结构呈现一定程度的萎缩。经1.0μg/mL脂多糖处理,水牛乳腺上皮细胞活力显著降低(P<0.05,下同),2.0、4.0和8.0μg/mL脂多糖处理,水牛乳腺上皮细胞活力极显著降低(P<0.001),25、50、100和200μmol/L相思子碱处理能缓解脂多糖的影响,水牛乳腺上皮细胞活力明显升高。与空白对照组相比,脂多糖处理组水牛乳腺上皮细胞NF-κB、IL-1β、TNFα和P53基因和蛋白相对表达量显著升高,β-酪蛋白表达水平显著降低;与脂多糖组相比,相思子碱+脂多糖处理组水牛乳腺上皮细胞β-酪蛋白水平显著升高,NF-κB、IL-1β、TNFα、P53基因和蛋白相对表达量显著降低;与脂多糖组相比,脂多糖+Pifithrin-μ组水牛乳腺上皮细胞β-酪蛋白表达水平显著升高,与相思子碱组相比,相思子碱+Kevetrin hydrochloride组水牛乳腺上皮细胞β-酪蛋白表达水平显著降低。【结论】脂多糖降低了水牛乳腺上皮细胞β-酪蛋白合成水平与β-酪蛋白合成相关基因及其编码蛋白的表达水平;相思子碱能通过抑制NF-κB通路缓解脂多糖诱导的水牛�【Objective】The objective of this study was to investigate the mitigating effect of abrine on induced inhibition by lipopolysaccharide(LPS)of casein synthesis in buffalo mammary epithelial cells by modulating the P53/mTOR pathway,to provide a theoretical basis for mitigating the decline in milk quality in buffaloes caused by mastitis.【Method】The LPS-induced inflammation model was established in buffalo mammary epithelial cells,co-incubated with abrine,P53 inhibitor(Pifithrin-μ),P53 activator(Kevetrin hydrochloride)and buffab mammary epithelial cells for 12 h;morphological observations of mammary tissues were conducted using HE staining;cell viability was assessed using the CCK-8 method,while the secretion levels ofβ-casein were determined using ELISA kits;real-time quantitative PCR was employed to measure the gene relative expression levels of NF-κB,IL-1β,TNFα,β-casein,mTOR,P53,JAK2,STAT5,and AKT1;additionally,Western blotting was used to assess the protein relative expression levels of NF-κB,β-casein,mTOR,and P53.【Result】Clinical mastitis(CMS)mammary tissues showed infiltration of neutrophils in the interstitium and alveolar cavities,accompanied by atrophy at certain degree in some alveolar structures.The buffalo mammary epithelial cell viability of the 1.0μg/mL LPS treatment group was significant decreased(P<0.05,the same below),buffab mammary epithelial cell viability of 2.0,4.0,and 8.0μg/mL LPS treatments extremely significantly(P<0.001).Abrine at 25,50,100,and 200μmol/L alleviated the impact of LPS,resulting in obvious increase in cell viability.Compared to the blank control group,LPS treatment group significantly elevated relative expression NF-κB,IL-1β,TNFαand P53 genes and proteins in buffalo mammary epithlial cells,while extremely significantly reducedβ-casein expression.Abrine cotreatment with LPS group resulted in significant increase inβ-casein levels in buffalo mammary epithlial cells and significant decrease in NF-κB,IL-1β,TNFαand P53 genes and proteins relative express

关 键 词：水牛乳腺上皮细胞 相思子碱 脂多糖 Β-酪蛋白 P53/mTOR通路 

分 类 号：S853.74[农业科学—临床兽医学]