双歧杆菌对脂多糖刺激的大鼠肠上皮细胞肿瘤坏死因子受体相关因子6、糖原合成激酶-3β和miRNA-146a mRNA表达的影响

Effects of bifidobacterium on mRNA expression of tumor necrosis factor receptor - associated factor 6, glyco- gen synthase kinase -3β and miRNA -146a in intestinal epithelial cells induced by lipopolysaccharide in rats

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5mg／L的LPS刺激细胞5h后，分别做下列处理：LPS组培养液中加入1mL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1mL1×1010 CFU／L灭活婴儿双歧杆菌继续培养24h。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（0．724±0．05）、GSK-3β（0．464±0．14）的mRNA相对表达量均低于LPS组（1．014±0．14，1．024±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（t=4．837，P=0．000），而TRAF-6、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。

Objective 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 supematant was added in cul- tured supematant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 ± 1010CFU/L added in inactivated bacteria group and continued culturing for 24 hours. The mRNA expressions of TRAF6, GSK - 3β and miRNA - 146a were detected by reverse transcription - polymerase chain reaction （ RT - PCR）. Results The level of TRAF6,GSK -3β 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 - 3β 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 diffe- rences with LPS group （ t = O. 732,1. 463, P 〉 0.05 ）. The level of TRAF6 of cultured snpernatant 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 supematant and inactivated bacteria both have certain protective effect on the IEC -6 induced by LPS. One of the protective mechanisms of bifidobaeterium cultured supernatant may be achieved by elevating the expression ofmiRNA - 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.