发酵乳杆菌对LTA诱导的牛子宫内膜细胞炎性损伤干预作用的研究
|
摘要
旨在研究发酵乳杆菌能否通过抑制NF-kB信号通路的激活,调控下游炎性细胞因子,进而对脂磷壁酸(LTA)所诱导的牛子宫内膜细胞(BEND)炎性损伤发挥保护作用,并通过体外试验对其作用机制进一步研究,为解决牛子宫内膜炎的治疗难题提供方向。以牛子宫内膜细胞为研究对象,LTA和发酵乳杆菌分别作为毒药与解药来建立体外细胞试验模型,测定细胞因子TNF-α、IL-1β、IL-6、IL-8的分泌量和炎性相关基因TNF-α、IL-1β、IL-6、IL-8、TLR2、MyD88、NF-kB p65 mRNA的表达量。结果表明:LTA组、LTA+Lf组、Lf组炎性细胞因子TNF-α、IL-1β、IL-6、IL-8的蛋白表达水平均高于对照组。LTA组与对照组比较差异极显著(p<0.01);Lf组与对照组比较,TNF-α、IL-6细胞因子差异不显著(p>0.05),IL-1β、IL-8细胞因子差异显著(p<0.05);LTA+Lf组与LTA组比较,TNF-α、IL-1β、IL-6、IL-8的蛋白表达量显著降低(p<0.05)。LTA组、LTA+Lf组、Lf组的TNF-α、IL-1β、IL-6、IL-8、TLR2、MyD88、NF-kB p65基因mRNA的表达量均高于对照组。LTA组与对照组相比,7种基因m RNA的表达量极显著增加(p<0.01);Lf组与对照组相比,TNF-α、IL-6基因mRNA的表达量增加不显著(p>0.05),IL-8基因mRNA的表达量增加显著(p<0.05),IL-1β、TLR2、MyD88、NF-kB p65基因mRNA的表达量极显著增加(p<0.01);LTA+Lf组与LTA组相比,7种基因mRNA的表达量均极显著降低(p<0.01)。发酵乳杆菌能够抑制NF-kB信号传导通路,调控炎性细胞因子的表达与释放,抑制炎性反应的发生,缓解LTA的细胞毒性,对LTA诱导的牛子宫内膜细胞炎性损伤起干预作用。
This study is aim to investigate whether to play a protective effect of Lactobacillus fermentum on lipoteichoic acid(LTA) isolated from Staphylococcus aureus-induced inflammatory injury of endometrial cells in Bos taurus by inhibiting the activation of NF-kB signaling pathway and regulating the expression of downstream inflammatory cytokines. The mechanism of action through in vitro testing provided a reference for prevention and treatment of staphylococcus aureus-induced bovine endometritis. Establishing cell experiment model in vitro by taking endometrial cells in Bos taurus as the research object, LTA and Lactobacillus fermentum as poisons and antidotes and testing the secretion of inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-8) and the expression of inflammatory related genes mRNA(TNF-α,IL-1β,IL-6,IL-8,TLR2,MyD88,NF-kB p65). The results showed that the protein expression levels of inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in LTA group, LTA+Lf group and Lf group were higher than those in control group. There was a significant difference between the LTA group and the control group(p<0.01). Compared with the control group, the TNF-α and IL-6 cytokines were not significantly different(p>0.05), and the IL-1β and IL-8 cytokines were different significantly(p<0.05). The protein expression levels of TNF-α, IL-1β, IL-6 and IL-8 were significantly lower in the LTA+Lf group than those in the LTA group. The expression levels of TNF-α, IL-1β, IL-6,IL-8, TLR2, My D88 and NF-kB p65 mRNA in LTA group, LTA+Lf group and Lf group were higher than those in control group.LTA group compared with the control group, the mRNA expression level of the seven kinds increased significantly(p<0.01). The mRNA expression of IL-6 and TNF-α in Lf group does not increase significantly(p>0.05), compared with the control group. The expression of IL-8 gene mRNA increased significantly(p <0.05). The expression of IL-1β, TLR2, MyD88, NF-kB p65 gene mRNA increased significantly(p <0.01). The mRNA expression levels of the seven genes in LTA +Lf group were significantly decreased(p <0.01) compared with the LTA group. Lactobacillus fermentum can inhibit NF-kB signaling pathway, regulate the expression and release of inflammatory cytokines, inhibit the occurrence of inflammatory reaction, alleviate the cytotoxicity of LTA and play an intervention role in LTA-induced inflammatory damage of bovine endometrial cells.
This study is aim to investigate whether to play a protective effect of Lactobacillus fermentum on lipoteichoic acid(LTA) isolated from Staphylococcus aureus-induced inflammatory injury of endometrial cells in Bos taurus by inhibiting the activation of NF-kB signaling pathway and regulating the expression of downstream inflammatory cytokines. The mechanism of action through in vitro testing provided a reference for prevention and treatment of staphylococcus aureus-induced bovine endometritis. Establishing cell experiment model in vitro by taking endometrial cells in Bos taurus as the research object, LTA and Lactobacillus fermentum as poisons and antidotes and testing the secretion of inflammatory cytokines(TNF-α,IL-1β,IL-6,IL-8) and the expression of inflammatory related genes mRNA(TNF-α,IL-1β,IL-6,IL-8,TLR2,MyD88,NF-kB p65). The results showed that the protein expression levels of inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in LTA group, LTA+Lf group and Lf group were higher than those in control group. There was a significant difference between the LTA group and the control group(p<0.01). Compared with the control group, the TNF-α and IL-6 cytokines were not significantly different(p>0.05), and the IL-1β and IL-8 cytokines were different significantly(p<0.05). The protein expression levels of TNF-α, IL-1β, IL-6 and IL-8 were significantly lower in the LTA+Lf group than those in the LTA group. The expression levels of TNF-α, IL-1β, IL-6,IL-8, TLR2, My D88 and NF-kB p65 mRNA in LTA group, LTA+Lf group and Lf group were higher than those in control group.LTA group compared with the control group, the mRNA expression level of the seven kinds increased significantly(p<0.01). The mRNA expression of IL-6 and TNF-α in Lf group does not increase significantly(p>0.05), compared with the control group. The expression of IL-8 gene mRNA increased significantly(p <0.05). The expression of IL-1β, TLR2, MyD88, NF-kB p65 gene mRNA increased significantly(p <0.01). The mRNA expression levels of the seven genes in LTA +Lf group were significantly decreased(p <0.01) compared with the LTA group. Lactobacillus fermentum can inhibit NF-kB signaling pathway, regulate the expression and release of inflammatory cytokines, inhibit the occurrence of inflammatory reaction, alleviate the cytotoxicity of LTA and play an intervention role in LTA-induced inflammatory damage of bovine endometrial cells.
引文
[1] LEDOUX D,PONSART C,GRIMARD B,et al.Sire effect on early and late embryonic death in French Holstein cattle[J].Animal,2015,9(5):766-774.
[2]傅晓雪.一例奶牛子宫内膜炎的细菌分离鉴定及药敏试验[J].畜牧兽医科技信息,2016(12):27-28.
[3]吴金鹏,范建新.牛子宫内膜炎细菌的分离、鉴定与治疗[J].黑龙江动物繁殖,2017,25(2):41-42.
[4]王京仁,李淑红,成钢,等.肉牛子宫内膜炎细菌的分离鉴定与药敏试验[J].黑龙江畜牧兽医,2013(2):96-98.
[5]刘福元,张云峰,王海萍,等.奶牛子宫内膜炎病料细菌的分离及药敏试验[J].草食家畜,2013(1):65-68.
[6]林晶晶,陶慧,候瑛倩,等.脂磷壁酸对小鼠子宫内膜组织和内膜细胞中功能受体TLR2表达的影响[J].畜牧兽医学报,2013,44(5):811-816.
[7]张安,赵佳琦,李晓婷,等.脂磷壁酸对小鼠乳腺组织TLR2表达的影响[J].黑龙江八一农垦大学学报,2017,29(4):24-27.
[8]孟润莎,莫泽欢,徐琼.脂磷壁酸诱导人成牙本质样细胞炎症微环境的机制研究[J].中华口腔医学研究杂志,2017,11(5):257-265.
[9] GENIS S,BACH A,FABREGAS F,et al.Potential of lactic acid bacteria at regulating Escherichia coli infection and inflammation of bovine endometrium[J].Theriogenology,2015,85(4):625-637.
[10] MARTIN R,OLIVARES M,PEREZ M,et al.Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk[J].Veterinary Journal,2010,185(2):193-198.
[11] JONAS D,ROSENBAUM A,WEYRICH S,et al.Enzyme linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoaveolar fluid[J].Clin Microbiol,1995,33(5):1247-1257.
[12] BAI H,SAKURAI T,BAI R,et al.Establishment and characterization of immortalized bovine endometrial epithelial cells[J].Anim Sci J,2014,85:799-804.
[13] SHELDON I M,ROBERTS M H.Toll-like recetor 4 mediates the resonse of epithelial and stromal cell to lipopolysaccharide in the endometrium[J].Plos One,2010,5(9):12906.
[14] ZHANG K K.MURRAY G L.SEEMANN T,et al.Leptospiral LruA is required for virulence and modullates an interaction with mammalian apolipoprotein AI[J].Infect Immun,2013,81(10):3872-3879.
[15] HAIDER S,KNOFLER M.Human tumour necrosis factor:physiological and pathological roles in placenta and endometrium[J].Placenta,2009,30(2):111-123.
[16] CHEN Y W,LU W T,JIA J L.Evaluation of serum concentrations of tumor necrosis factor(TNF)-α,interleukin(IL)-2,IL-10,and nitric oxide(NO)during the estrous cycle,early pregnancy and abortion in goats[J].Animal Reproduction Science,2016,174:73-79.
[17] SAYAKA S,NOE N,MASAKATSU K,et al.A phytoestrogen supplement prevented the altered gene expression associated with pregnant implantation induced by IL-1βin endometrial epithelial cells[J].Reproductive Biology,2017,17(3):259-267.
[18] WU CJ,CONZE D B,LI T,et al.Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kB activation[J].Nature Cell Biology,2006,8:398-406.
[19] RUTH P,SUSANNE H,LIDIJA C,et al.Inhibition of influenza virus-induced NF-kappaB and Raf/MEK/ERK activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo[J].Antiviral Research,2011,92(1):45-56.
[20] CHEN Z J.Ubiquitin signalling in the NF-kB pathway[J].Nature Cell Biology,2005,7:758-765.
[21] BEINKE S,LEY S.Functions of NF-kappa B1 and NF-kappa B2 in immune cell biology[J].Biochem J,2004,382:393-409.
[2]傅晓雪.一例奶牛子宫内膜炎的细菌分离鉴定及药敏试验[J].畜牧兽医科技信息,2016(12):27-28.
[3]吴金鹏,范建新.牛子宫内膜炎细菌的分离、鉴定与治疗[J].黑龙江动物繁殖,2017,25(2):41-42.
[4]王京仁,李淑红,成钢,等.肉牛子宫内膜炎细菌的分离鉴定与药敏试验[J].黑龙江畜牧兽医,2013(2):96-98.
[5]刘福元,张云峰,王海萍,等.奶牛子宫内膜炎病料细菌的分离及药敏试验[J].草食家畜,2013(1):65-68.
[6]林晶晶,陶慧,候瑛倩,等.脂磷壁酸对小鼠子宫内膜组织和内膜细胞中功能受体TLR2表达的影响[J].畜牧兽医学报,2013,44(5):811-816.
[7]张安,赵佳琦,李晓婷,等.脂磷壁酸对小鼠乳腺组织TLR2表达的影响[J].黑龙江八一农垦大学学报,2017,29(4):24-27.
[8]孟润莎,莫泽欢,徐琼.脂磷壁酸诱导人成牙本质样细胞炎症微环境的机制研究[J].中华口腔医学研究杂志,2017,11(5):257-265.
[9] GENIS S,BACH A,FABREGAS F,et al.Potential of lactic acid bacteria at regulating Escherichia coli infection and inflammation of bovine endometrium[J].Theriogenology,2015,85(4):625-637.
[10] MARTIN R,OLIVARES M,PEREZ M,et al.Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk[J].Veterinary Journal,2010,185(2):193-198.
[11] JONAS D,ROSENBAUM A,WEYRICH S,et al.Enzyme linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoaveolar fluid[J].Clin Microbiol,1995,33(5):1247-1257.
[12] BAI H,SAKURAI T,BAI R,et al.Establishment and characterization of immortalized bovine endometrial epithelial cells[J].Anim Sci J,2014,85:799-804.
[13] SHELDON I M,ROBERTS M H.Toll-like recetor 4 mediates the resonse of epithelial and stromal cell to lipopolysaccharide in the endometrium[J].Plos One,2010,5(9):12906.
[14] ZHANG K K.MURRAY G L.SEEMANN T,et al.Leptospiral LruA is required for virulence and modullates an interaction with mammalian apolipoprotein AI[J].Infect Immun,2013,81(10):3872-3879.
[15] HAIDER S,KNOFLER M.Human tumour necrosis factor:physiological and pathological roles in placenta and endometrium[J].Placenta,2009,30(2):111-123.
[16] CHEN Y W,LU W T,JIA J L.Evaluation of serum concentrations of tumor necrosis factor(TNF)-α,interleukin(IL)-2,IL-10,and nitric oxide(NO)during the estrous cycle,early pregnancy and abortion in goats[J].Animal Reproduction Science,2016,174:73-79.
[17] SAYAKA S,NOE N,MASAKATSU K,et al.A phytoestrogen supplement prevented the altered gene expression associated with pregnant implantation induced by IL-1βin endometrial epithelial cells[J].Reproductive Biology,2017,17(3):259-267.
[18] WU CJ,CONZE D B,LI T,et al.Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kB activation[J].Nature Cell Biology,2006,8:398-406.
[19] RUTH P,SUSANNE H,LIDIJA C,et al.Inhibition of influenza virus-induced NF-kappaB and Raf/MEK/ERK activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo[J].Antiviral Research,2011,92(1):45-56.
[20] CHEN Z J.Ubiquitin signalling in the NF-kB pathway[J].Nature Cell Biology,2005,7:758-765.
[21] BEINKE S,LEY S.Functions of NF-kappa B1 and NF-kappa B2 in immune cell biology[J].Biochem J,2004,382:393-409.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |