Chebulagic acid regulates LPS-induced inflammatory response in dairy cow endom-etrial tissue through MAPK/NF-κB pathway
杨英 1刘鑫煜 1赵非凡 1郭文瑞 1高瑞峰1
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作者信息
1. 内蒙古农业大学兽医学院,内蒙古呼和浩特 010018
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摘要
分离并体外培养奶牛子宫内膜组织,用1.0 mg/L脂多糖(lipopolysaccharide,LPS)刺激1 h后与诃子鞣酸(Che-bulagic acid,CA)(12.5、25.0、50.0、100.0 mg/L)共培养,然后收集不同处理组的奶牛子宫内膜组织进行试验.应用ELISA和实时荧光定量PCR法检测TNF-α、IL-6、IL-1β的蛋白分泌水平和基因表达水平;HE染色观察奶牛子宫内膜组织损伤程度;应用免疫荧光技术鉴定高迁移率蛋白-1(high mobility grope box protein 1,HMGB-1)、透明质酸酶结合蛋白-2(hyaluronidase binding protein 2,HABP-2)的表达;Western blot 检测 ERK、p65 和 IκBα 的磷酸化表达水平.结果显示,经CA处理LPS诱导的奶牛子宫内膜组织炎症反应模型后,TNF-α在6、24 h和IL-6在6、12、24 h的蛋白分泌水平显著下降,IL-1β和IL-6在6、9、12 h和TNF-α在6 h的基因表达水平均显著下调;HE染色切片发现,LPS+CA组相比LPS组均有所改善,上皮细胞脱落程度减轻,腺体和血管结构较为完整,炎性细胞浸润程度降低,无明显的坏死或出血;LPS+CA组也显著下调了 HMGB-1和HABP-2的表达;LPS+CA组的ERK、IκBα和p65的磷酸化水平显著降低.结果表明,CA能够通过抑制LPS诱导的奶牛子宫内膜组织炎症反应中MAPK和NF-κB通路的激活,下调其奶牛子宫组织中炎症因子的表达水平从而减轻LPS诱导的奶牛子宫内膜组织炎症反应.由此推断,CA可能是一种潜在的奶牛子宫内膜炎的治疗药物,值得进一步的研究和开发.
Abstract
To investigate the role of chebulagic acid(CA)in regulating lipopolysaccharide(LPS)-in-duced inflammatory response in endometrial tissue of dairy cows,and to provide new ideas for the treatment and new drug development of endometritis in dairy cows.The endometrial tissues of dairy cows were isolated and cultured in vitro,stimulated with 1 mg/L LPS for 1 h and then co-cultured with CA(12.5,25.0,50.0,100.0 mg/L).Then the endometrial tissues of different treat-ment groups were collected for experiments.The protein secretion and gene expression levels of TNF-α,IL-6 and IL-1β were detected by ELISA and real-time fluorescence quantitative PCR.HE staining was used to observe the degree of endometrial tissue damage.The expressions of high mobility protein 1(HMGB-1)and hyaluronidase binding protein 2(HABP-2)were detected by immunofluorescence.The phosphorylation levels of ERK,p65 and IκBα were detected by Western blot.The results showed that the protein secretion levels of TNF-α at 6,24 h and IL-6 at 6,12 and 24 h,and the gene expression levels of IL-1β and IL-6 at 6,9,and 12 h and TNF-α at 6 h were sig-nificantly down-regulated after CA treatment with the LPS-induced endometrial tissue inflammation response model of dairy cows.HE staining showed that compared with the LPS group,the LPS+CA group had some improvements,the degree of epithelial cell exfoliation was reduced,the struc-ture of glands and blood vessels was relatively complete,the degree of inflammatory cell infiltra-tion was reduced,and there was no obvious necrosis or hemorrhage.The expression of HMGB-1 and HABP-2 in LPS+CA group was also significantly down-regulated.The phosphorylation levels of ERK,IκBα and p65 in the LPS+CA group were significantly decreased.In conclusion,CA can reduce LPS-induced inflammation in the endometrial tissue of dairy cows by inhibiting the activa-tion of MAPK and NF-κB pathways and down-regulating the expression of inflammatory factors in the uterine tissue.It is concluded that CA may be a potential therapeutic agent for endometritis in dairy cows and deserves further research and development.
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