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1.0 μg/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8.0 μg/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200 μmol/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乳腺上皮细胞炎症,并通过调节P53/mTOR通路缓解脂多糖诱导的水牛乳腺上皮细胞β-酪蛋白合成减少.
Abstract
[Objective]The objective of this study was to investigate the mitigating effect of abrine on induced inhibi-tion 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;morpho-logical observations of mammary tissues were conducted using HE staining;cell viability was assessed using the CCK-8 method,while the secretion levels of β-casein were determined using ELISA kits;real-time quantitative PCR was em-ployed 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 epithe-lial cell viability of the 1.0 μg/mL LPS treatment group was significant decreased(P<0.05,the same below),buffab mam-mary epithelial cell viability of 2.0,4.0,and 8.0 μg/mL LPS treatments extremely significantly(P<0.001).Abrine at 25,50,100,and 200 μmol/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,TNFα and P53 genes and proteins in buffalo mammary epithlial cells,while extremely significantly reduced β-casein expression.Abrine co-treatment with LPS group resulted in significant increase in β-casein levels in buffalo mammary epithlial cells and signifi-cant decrease in NF-κB,IL-1β,TNFα and P53 genes and proteins relative expression compared to the LPS group.The LPS+Pifithrin-μ group showed significant increase in β-casein expression in buffalo mammary epithlial cells compared to the LPS group.Conversely,abrine+Kevetrin hydrochloride group exhibited significant decrease in β-casein expression in buffalo mammary epithlial cells compared to abrine group.[Conclusion]LPS decreases β-casein synthesis and expression levels of genes and their encoded proteins involved in β-casein synthesis;abrine alleviates LPS-induced mammary epithe-lial cells of buffalo by inhibiting NF-κB pathway,and alleviates LPS-induced reduction of β-casein synthesis in buffalo mammary epithelial cells by modulating P53/mTOR pathway.
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