β-谷甾醇对脂多糖诱导的小鼠乳腺上皮细胞炎症反应及乳蛋白合成的影响

Effects ofβ⁃Sitosterol on Lipopolysaccharide⁃Induced Inflammatory Response and Milk Protein Synthesis in Mouse Mammary Epithelial Cells

本试验旨在探讨β-谷甾醇(BSS)对脂多糖(LPS)诱导的小鼠乳腺上皮细胞(HC11细胞)炎症反应及乳蛋白合成的影响。以体外培养的HC11细胞为模型,分为5组,对照组(生长培养基处理)、乳腺炎症模型组(LPS组,1μg/mL LPS处理)以及炎症模型药物组(BSS+LPS组,分别用5、10、20μmol/L的β-谷甾醇预处理1 h后加入1μg/mL LPS),每组设置5个重复。检测炎性细胞因子和乳蛋白mRNA表达以及乳蛋白合成信号通路相关基因mRNA和蛋白表达。结果表明:1)与对照组相比,LPS组的HC11细胞肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、环氧合酶-2(COX-2)和诱导型一氧化氮合酶(iNOS)的mRNA相对表达量显著升高(P<0.05)。与LPS组相比,5、10、20μmol/L BSS+LPS组的HC11细胞TNF-α、IL-6、IL-1β、COX-2和iNOS的mRNA相对表达量显著降低(P<0.05)。2)与对照组相比,LPS组的HC11细胞β酪蛋白(CSN2)、κ酪蛋白(CSN3)和乳清蛋白(LALBA)的mRNA相对表达量显著降低(P<0.05)。与LPS组相比,5、10、20μmol/L BSS+LPS组的HC11细胞CSN2、CSN3和LALBA的mRNA相对表达量显著升高(P<0.05)。3)与对照组相比,LPS组的HC11细胞信号转导和转录激活因子5(STAT5)、酪氨酸激酶2(JAK2)、哺乳动物雷帕霉素靶蛋白(mTOR)、真核细胞翻译启动因子4E结合蛋白1(4EBP1)和核糖体S6蛋白激酶1(S6K1)的mRNA相对表达量显著降低(P<0.05),同时磷酸化信号转导和转录激活因子5(p-STAT5)、磷酸化酪氨酸激酶2(p-JAK2)和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的蛋白相对表达量显著降低(P<0.05)。与LPS组相比,5、10、20μmol/L BSS+LPS组的HC11细胞STAT5、JAK2、mTOR、4EBP1和S6K1的mRNA相对表达量显著升高(P<0.05),同时p-STAT5、p-JAK2和p-mTOR的蛋白相对表达量显著升高(P<0.05)。由此可见,β-谷甾醇能够抑制LPS诱导的HC11细胞炎症反应和促进乳蛋白的合成。

The purpose of this experiment was to explore the effect ofβ-sitosterol(BSS)on lipopolysaccha-ride(LPS)-induced inflammatory response and milk protein synthesis in mouse mammary epithelial cells(HC11 cells).The experiment used HC11 cells cultured in vitro as the model,and divided into 5 groups:con-trol group(growth medium treated),mammary inflammatory model group(LPS group,1μg/mL LPS trea-ted)and inflammatory model medicine groups(BSS+LPS groups,after 5,10 and 20μmol/Lβ-sitosterol treated 1 h,respectively,added 1μg/mL LPS),and set 5 replicates per group.The mRNA expression of in-flammatory cytokines and milk protein,as well as the mRNA and protein expression of genes related to milk protein synthesis signaling pathway were detected.The results showed as follows:1)compared with the control group,the mRNA relative expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),inter-leukin-1β(IL-1β),cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS)in HC11 cells of LPS group were significantly increased(P<0.05).Compared with the LPS group,the mRNA relative expres-sion levels of TNF-α,IL-6,IL-1β,COX-2 and iNOS in HC11 cells of 5,10 and 20μmol/L BSS+LPS groups were significantly decreased(P<0.05).2)Compared with the control group,the mRNA relative ex-pression levels ofβcasein(CSN2),κcasein(CSN3)and whey protein(LALBA)in HC11 cells of LPS group were significantly decreased(P<0.05).Compared with the LPS group,the mRNA relative expression levels of CSN2,CSN3 and LALBA in HC11 cells of 5,10 and 20μmol/L BSS+LPS groups were significantly increased(P<0.05).3)Compared with the control group,the mRNA relative expression levels of signal transducer and activator of transcription 5(STAT5),Janus kinase 2(JAK2),mammalian target of rapamycin(mTOR),eukaryotic translation initiation factor 4E binding protein 1(4EBP1)and ribosomaiprotein S6 ki-nase 1(S6K1)in HC11 cells of LPS group were significantly decreased(P<0.05),meanwhile,the protein relative expression levels of phosphorylation signal transducer and activator of transcription 5(p-STAT5),phosphorylation Janus kinase 2(p-JAK2)and phosphorylation mammalian target of rapamycin(p-mTOR)were significantly decreased(P<0.05).Compared with the LPS group,the mRNA relative expression levels of STAT5,JAK2,mTOR,4EBP1 and S6K1 in HC11 cells of 5,10 and 20μmol/L BSS+LPS groups were significantly increased(P<0.05),meanwhile,the protein relative expression levels of p-STAT5,p-JAK2 and p-mTOR were significantly increased(P<0.05).In conclusion,β-sitosterol can inhibit LPS-induced inflam-matory response in HC11 cells and promote milk protein synthesis.