摘要目的 研究双歧杆菌乳杆菌三联活菌治疗急性肝功能衰竭(ALF)的作用机制.方法 BALB/c小鼠10只腹腔注射3.0 g/kg D-GalN建立ALF模型,双歧杆菌乳杆菌三联活菌片灌胃治疗,Western印迹法和实时荧光定量-PCR检测Jagged1、Notch1、Hes5 mRNA和蛋白以及Notch受体胞内段(NICD)蛋白的表达,免疫组织化学染色法检测小鼠肝组织CD68的表达,同时测定血清ALT、AST、IL-10、高迁移率族蛋白B1(HMGB1)和血浆脂多糖水平.同时设模型组和正常对照小鼠各10只.体外培养小鼠巨噬细胞株RAW264.7细胞,分别用正常小鼠、ALF小鼠、双歧杆菌乳杆菌三联活菌治疗小鼠血浆处理RAW264.7细胞.实时荧光定量-PCR和Western印迹法检测细胞Jagged1、Notch1、Hes5mRNA和蛋白以及NICD蛋白的变化,同时检测细胞上清液IL-10、HMGB1和脂多糖水平.多组间比较用单因素方差分析,两组间比较用q检验.结果 模型组小鼠血ALT、AST、HMGB1、IL-10、脂多糖,肝组织Jagged1、Notch1、NICD、Hes5、CD68水平均较正常对照组明显升高(均P＜0.01).模型组与双歧杆菌乳杆菌三联活菌治疗组比较,血清HMGB1[(101.9±12.4) μg/L比(82.6±9.7) μg/L,q=6.36,P＜0.01]、血清IL-10[(4 628±842) pg/mL比(3 183±769) pg/mL,q=6.79,P＜0.01]、血浆脂多糖[(11.80±0.89) EU/mL比(7.40±0.92) EU/mL,q=18.81,P＜0.01]、肝组织Jagged1 mRNA(7.63±1.41比5.55±0.71,q=7.22,P＜0.01)、Jagged1蛋白(0.71±0.07比0.56±0.07,q=7.20,P＜0.01)、Notch1 mRNA(7.10±0.66比3.66±0.67,q=20.06,P＜0.01)、Notch1蛋白(0.66±0.11比0.38±0.08,q=9.57,P＜0.01)、NICD蛋白(0.76±0.07比0.47±0.05,q=12.68,P＜0.01)、Hes5mRNA(7.95±0.71比3.94±0.68,q=22.40,P＜0.01)、Hes5蛋白(1.20±0.07比1.04±0.12,q=5.61,P＜0.01)和CD68蛋白(7 685±417比5 180±610,q=16.38,P＜0.01)差异均有统计学意义.ALF小鼠血浆可显著提高RAW264.7细胞上清液HMGB1、IL-10、脂多糖和细胞Jagged1、Notch1、NICD、Hes5水平.双歧杆菌乳杆菌三联活菌治疗小鼠血浆干预RAW264.7细胞后,细胞上清液HMGB1、IL-10、脂多糖和细胞Jagged1、Notch1、NICD、Hes5水平均较ALF小鼠血浆干预组明显降低(均P＜0.01).结论 双歧杆菌乳杆菌三联活菌可通过降低血浆脂多糖水平,抑制肝脏巨噬细胞内Notch信号通路活化,减少HMGB1、IL-10的分泌,延缓ALF的发生发展.

abstractsObjective To investigate the mechanism of live combined Bifidobacterium and Lactobacillus tablets in acute liver failure (ALF) treatment.Methods Ten mice were injected intraperitoneally with 3.0 g/kg D-galactosamine to establish the model of ALF and treated with live combined Bifidobacterium and Lactobacillus tablets.Protein levels of Jagged1,Notch1,Notch intracellular domain (NICD),Hes5 and the mRNA expressions of Jagged1,Notch1,Hes5 were measured via Western blot and real time-polymerase chain reaction (PCR),respectively.The protein level of CD68 was detected by immunohistochemical staining method.Meanwhile,serum alanine aminotransferase (ALT),aspartate aminotransferase (AST),interleukin (IL)-10,high mobility group protein B1 (HMGB1) and plasma lipopolysaccharide (LPS) were measured.Moreover,model group and control group were also established with 10 mice each.In vitro,RAW264.7 cells were cultured with normal mice plasma,plasma of ALF mice and plasma of treated mice,respectively.Real time-PCR and Western blot were used to determine the mRNA expressions of Jagged1,Notch1,Hes5 and proteins levels of Jagged1,Notch1,NICD,Hes5.The levels of IL-10,HMGB1 and LPS in the supernatant of RAW264.7 cells were detected as well.The total significant differences among groups were compared by one way ANOVA,and q test was used to evaluate the significance of subgroup differences.Results The levels of serum ALT,AST,HMGB1,IL10,plasma LPS,and the expressions of Jagged1,Notch1,NICD,Hes5,CD68 were higher in ALF model group than control group (all P＜0.01).Compared with the ALF model group,all of these indexes could be improved in mice with live combined Bifidobacterium and Lactobacillus tablets (HMGB1:[82.6±9.7] μg/L vs [101.9±12.4] μg/L,q=6.36,P＜0.01; IL-10.:[3 183±769] pg/mL vs [4 628±842] pg/mL,q=6.79,P＜0.01; plasma LPS:[7.40±0.92] EU/mL vs [11.80±0.89] EU/mL,q=18.81,P＜0.01; Jagged1 mRNA:5.55±0.71 vs 7.63±1.41,q=7.22,P＜0.01;Jagged1 protein:0.56±0.07 vs 0.71±0.07,q=7.20,P＜0.01; Notch1 mRNA:3.66±0.67 vs 7.10±0.66,q=20.06,P＜0.01; Notch1 protein:0.38±0.08 vs 0.66±0.11,q=9.57,P＜0.01;NICD protein:0.47±0.05 vs 0.76±0.07,q=12.68,P＜0.01; Hes5 mRNA:3.94±0.68 vs 7.95± 0.71,q=22.40,P＜0.01; Hes5 protein:1.04±0.12 vs 1.20±0.07,q=5.61,P＜0.01; CD68 protein:5 180±610 vs 7 685 ±417,q=16.38,P＜0.01).And the differences were statistically significant.After RAW264.7 cells cultured with the plasma of ALF model mice,the levels of HMGB1,IL-10 and LPS in the supernatant and the expressions of Jagged1,Notch1,NICD and Hes5 in cells were increased,whereas if RAW264.7 cells were cultured with the plasma of treated mice,indexes mentioned above were significantly decreased (all P＜0.01).Conclusions Live combined Bifidobacterium and Lactobacillus tablet could prevent the occurrence and development of ALF by decreasing the plasma level of LPS,inhibiting the activation of Notch signaling pathway in macrophages and reducing the secretion of HMGB1 and IL-10.