硒对S.aureus诱导的奶牛乳腺上皮细胞Nod2/MAPK/mTORs信号通路关键蛋白表达的影响

Effects of Selenium on the Key Factors in Nod2/MAPK/mTORs Signaling Pathways in the bMECs Infected S. aureus

【目的】硒(Se)能否通过Nod2/MAPK/mTOR途径调控金黄色葡萄球菌诱导的奶牛乳腺上皮细胞炎性损伤,有待于进一步研究。因此本研究将探究硒对金黄色葡萄球菌(S. aureus)感染的奶牛乳腺上皮细胞(bMECs)Nod2/MAPK/mTORs信号通路中关键蛋白表达的影响,从而为阐明硒的免疫调控机制提供理论依据。【方法】首先将bMECs以10^6细胞/孔接种于6孔板中,当细胞超过80%的汇合度时,用含2、4和8μmol·L^-1浓度硒的培养基替换原来的培养基,继续孵育12 h,然后用PBS洗涤每孔3次,将S. aureus按MOI=1:1的比例加入6孔板中,继续培养0.5 h,然后收集bMECs细胞进行相关蛋白的检测。本试验共分3大组,即对照(Con)组(bMECs)、模型(Mod)组(bMECs+S. aureus)和试验组。其中试验组又分3个亚剂量组,即Low组(bMECs+2μmol·L^-1 Se+S. aureus)、Mid组(bMECs+4μmol·L^-1 Se+S. aureus)和Hig组(bMECs+8μmol·L^-1 Se+S. aureus),每组设3个重复。利用BCA蛋白测定试剂盒对收集的bMECs细胞进行总蛋白提取。应用Western blotting技术检测bMECs中Nod2和RIP2蛋白表达水平及JNK,AKT和mTOR蛋白磷酸化水平。将蛋白样品加到10%的SDS聚丙烯酰胺凝胶电泳中,上样量为20μg/孔,之后将蛋白转移到聚偏氟乙烯(PVDF)膜上。将PVDF膜用5 mL 5%脱脂乳阻断2 h,脱脂乳脱脂后用TBST清洗后,分别用5 mL的Nod2、RIP2、JNK、AKT、mTOR和β-actin的一抗孵育过夜,回收一抗。之后在PVDF膜中分别加入5 mL上述蛋白的二抗,室温孵育2 h,回收二抗。PVDF用TBST洗涤5次,最后在暗室条件下进行化学显影。【结果】S. aureus能显著提高bMECs中Nod2和RIP2蛋白表达水平及JNK,AKT和mTOR蛋白磷酸化水平(P<0.01)。S. aureus感染0.5 h后,Nod2蛋白水平显著升高(P<0.01)。在培养基里添加2μmol·L^-1的硒可极显著抑制Nod2蛋白的表达(P<0.01),在培养基里添加8μmol·L^-1的硒可显著抑制Nod2的表达(P<0.05);S. aureus感染0.5 h后,RIP2蛋白水平显著升高(P<0.05),而在培养基里添加8μmol·L^-1硒可显著抑制RIP2蛋白的表达(P<0.05);S. aureus感染0.5 h后,与对照组相比,模型组JNK蛋白磷酸化水平显著升高(P<0.01)。在培养基里添加4μmol·L^-1的硒能显著抑制JNK蛋白的磷酸化水平(P<0.05),在培养基里添加8μmol·L^-1的硒能显著抑制JNK蛋白的磷酸化水平(P<0.01);S. aureus感染0.5 h后,与对照组相比,模型组AKT蛋白磷酸化水平显著升高(P<0.01)。在培养基里添加4μmol·L^-1硒可极显著抑制JNK蛋白的磷酸化水平(P<0.01),在培养基里添加8μmol·L^-1硒可显著抑制AKT蛋白的磷酸化水平(P<0.05);S. aureus感染0.5 h后,模型组mTOR蛋白磷酸化水平显著升高(P<0.01)。在培养基里分别添加4μmol·L^-1和8μmol·L^-1硒均能显著抑制mTOR蛋白磷酸化水平(P<0.05)。【结论】硒可通过抑制bMECs Nod2/MAPK/mTORs信号通路中关键因子蛋白的表达而减轻S. aureus诱导的bMECs炎症反应。

【Objective】Whether selenium(Se) could regulate the inflammatory damage of bovine mammary epithelial cells(bMECs) induced by S. aureus through Nod2/MAPK/mTOR pathway remains to be further studied. So in the study, the effects of Se on the key proteins in the Nod2/MAPK/mTORs signaling pathway in the bovine mammary epithelial cells(bMECs) infected by S.aureus was studied in order to provide a theoretical basis for elucidating the immune regulation mechanism of Se.【Method】 Firstly,the bMECs were inoculated into the 6 well plates with 10^6 cells/well. When more than 80% of the cells were confluent, the medium was replaced with the one containing different concentrations of Se(2, 4 and 8 μmol·L^-1) and continued to culture for 12 h. Then after washing each well for 3 times with PBS, S.aureus was added into 6-well plates at a ratio of MOI=1:1 and continued to culture for 0.5 h. The bMECs were collected for further detection of related proteins expression. The experiment was divided into three groups: control(Con) group(bMECs), model(Mod) group(bMECs+S. aureus) and experimental group. The experimental group was divided into three sub-dose groups, namely Low group(bMECs+2 μmol·L^-1 Se+S. aureus), Mid group(bMECs+4 μmol·L^-1 Se+S.aureus) and Hig group(bMECs+8 μmol·L^-1 Se+S. aureus), with three replicates each group. Total protein was extracted from the above bMECs using a bicinchoninic acid(BCA) protein assay kit. The expressions level of Nod2 and RIP2 and the phosphorylation level of JNK, AKT and mTOR proteins in bMECs were detected by Western blotting. The protein samples were loaded into 10% SDS polyacrylamide gel for electrophoresis, and the uniform volume of protein was 20 μg/hole. Then the protein was transferred to polyvinylidene fluoride(PVDF) membranes. The PVDF membranes were blocked with 5 mL 5% nonfat milk for 2 h, then skimmed the nonfat milk and washed the membranes with TBST, subsequently the membranes were incubated overnight with5 mL primary antibodies including Nod2, RIP2, JNK, AKT, mTOR and β-actin. The primary antibodies were recovered, and 5 mL second antibodies were added to the membranes and incubated for 2 h at room temperature. Subsequently the second antibodies were recovered the membranes were washed with TBST for 5 times. Finally the membranes were developed with chemiluminescent substrate under darkroom conditions.【Result】S. aureus could significantly increase the expression of Nod2 and RIP2 proteins and the phosphorylation of JNK, AKT and mTOR proteins in bMECs(P<0.01). At 0.5 h after S. aureus infection, the level of Nod2 protein increased significantly(P<0.01). The expression of Nod2 protein was significantly inhibited by adding 2 μmol·L^-1 Se to the medium(P<0.01), and the expression of Nod2 was significantly inhibited by adding 8 μmol·L^-1 Se to the medium(P<0.05);at 0.5 h after S.aureus infection, RIP2 protein level was significantly increased(P<0.05), while the expression of RIP2 protein was significantly inhibited by adding 8 μmol·L^-1 Se to the medium(P<0.05);at 0.5 h after S. aureus infection, the phosphorylation level of JNK protein in model group was significantly higher than that in control group(P<0.01). The phosphorylation of JNK protein was significantly inhibited by adding 4 μmol·L^-1 Se to the medium(P<0.05), and the phosphorylation of JNK protein was significantly inhibited by adding 8 μmol·L^-1 Se to the medium(P<0.01);at 0.5 h after S. aureus infection, the phosphorylation level of AKT protein in the model group was significantly higher than that in the control group(P<0.01). The phosphorylation level of JNK protein was significantly inhibited by adding 4 μmol·L^-1 Se to the medium(P<0.01). The phosphorylation level of AKT protein was significantly inhibited by adding 8 μmol·L^-1 Se to the medium(P<0.05). After 0.5 h of S. aureus infection, the phosphorylation level of mTOR protein was significantly increased in the model group(P<0.01). The phosphorylation of mTOR protein was significantly inhibited by adding 4 and8 μmol·L^-1 Se to the medium(P<0.05).【Conclusion】Se could alleviate the inflammatory response of bMECs induced by S. aureus by inhibiting the protein expression of key factors in the bMECs Nod2/MAPK/mTORs signaling pathway.