摘要目的 探讨灭活双歧杆菌或双歧杆菌培养上清对脂多糖(LPS)刺激的大鼠肠上皮细胞株(IEC-6)肿瘤坏死因子受体相关因子6(TRAF6)、糖原合成激酶-3β(GSK-3β)和miRNA-146a mRNA表达的影响.方法 取对数生长期的IEC-6采用随机数字表法分为LPS组、培养上清组和灭活菌组.3组细胞均用5 mg/L的LPS刺激细胞5h后,分别做下列处理:LPS组培养液中加入1 mL无菌9g/L盐水继续培养24h;培养上清组培养液中加入婴儿双歧杆菌培养上清1mL继续培养24h;灭活菌组培养液中加入1mL 1×1010 CFU/L灭活婴儿双歧杆菌继续培养24 h.反转录(RT)-PCR法检测各组细胞中TRAF6、GSK-3β及miRNA-146a mRNA的表达.结果 培养上清组细胞TRAF-6(0.72±0.05)、GSK-3β(0.46±0.14)的mRNA相对表达量均低于LPS组(1.01±0.14,1.02±0.25),miRNA-146a的mRNA相对表达量(3.05±0.40)高于LPS组(1.01 ±0.12),差异均有统计学意义(t=5.278、6.316、13.218,P=0.000);灭活菌组细胞GSK-3β的mRNA相对表达量(0.59±0.13)低于LPS组,差异有统计学意义(=4.837,P =0.000),而TRAF6、miRNA-146a的mRNA相对表达量(1.05±0.11,0.78 ±0.22)与LPS组比较,差异均无统计学意义(t=0.732、1.463,P＞0.05).培养上清组细胞TRAF6的mRNA相对表达量低于灭活菌组,miRNA-146a的mRNA相表达量高于灭活菌组,差异均有统计学意义(t=6.009、14.687,P=0.000).结论 双歧杆菌培养上清和灭活菌对LPS刺激的IEC-6均有一定的保护作用;双歧杆菌培养上清的保护作用机制之一可能是通过升高miRNA-146a,降低炎症相关因子TRAF6和损伤因子GSK-3β水平实现的;灭活双歧杆菌的保护作用可能是通过降低损伤因子GSK-3β水平实现的.

abstractsObjective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.