几种功能性物质抑制产肠毒性大肠杆菌的效果及作用机制研究

Inhibitory effect and mechanism of several functional substances on enterotoxic Escherichia coli

不同浓度葛根素(PR)、丙酮酸乙酯(EP)、酪醇(TS)、氧化锌(ZnO)处理IPEC-1细胞,CCK-8法检测细胞活力;乳酸脱氢酶(lactate dehydrogena,sLeDH)试剂盒检测几种功能性物质对ETEC K88感染IPEC-1细胞损伤程度的影响;实时荧光定量PCR法检测不同功能性物质对ETEC K88感染IPEC-1细胞相关基因相对表达水平的影响。结果:PR、EP和ZnO均可促进IPEC-1细胞增殖,其最适添加浓度分别为5μmol/L、1 mmol/L和1μmol/L;TS无促进IPEC-1细胞增殖作用。ETEC K88感染IPEC-1细胞后导致LDH活性显著升高(P<0.05);与K88组相比,添加5μmol/L PR、1 mmol/L EP和1μmol/L ZnO均能显著降低LDH活性(P<0.05)。ETEC K88感染后引起白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)、趋化因子配体2(CXCL2)、热休克蛋白70(HSP70)和核因子E2相关因子(Nrf2)的相对表达量显著升高(P<0.05),细胞周期蛋白D(cyclin D)和绒毛蛋白(Villi)n基因的相对表达量显著降低(P<0.05)。PR显著提高了细胞IL-8和Villin基因的相对表达量(P<0.05);EP显著降低了(P<0.05)细胞TNF-α和CXCL2基因的相对表达量;ZnO显著提高了Villin和Cyclin D基因的相对表达量(P<0.05)。此外,PR、EP和ZnO均能降低大肠杆菌16S rRNA基因的相对表达量(P<0.05)。综上所述,PR、EP和ZnO均能抑制ETEC K88的增殖,具有抑菌效果,EP可以降低K88造成的炎性基因的表达,PR可以增加肠绒毛基因表达,ZnO可以增加细胞周期基因的表达。

IPEC-1 cells were treated with different concentrations of puerarin(PR),ethyl pyruvate(EP),tyrosol(TS)or zinc oxide(ZnO),and then cell viability was detected by CCK-8 method.The lactate dehydrogenase(LDH)kit was used to detect the effect of functional substances on the cytotoxic effects of ETEC K88.Real-time PCR was used to detect the effects of different functional substances on the relative expression levels of genes related to IPEC-1 cells infected with ETEC K88.Results∶PR,EP and ZnO could promote the proliferation of IPEC-1 cells,and the optimal concentrations were 5μM,1 mM and 1μM,respectively.TS had no obvious effect on the proliferation of IPEC-1 cells.After being infected with ETEC K88,the LDH activity of IPEC-1 cells significantly increased(P<0.05).Compared with the K88 infected group,the addition of 5μM PR,1 mM EP and 1μM ZnO significantly reduced LDH activity(P<0.05).Infection with ETEC K88 caused significant increases in the relative expression of interleukin-6(IL-6),interleukin-8(IL-8),tumor necrosis factor-α(TNF-α),chemokine ligand 2(CXCL2),heat shock protein 70(HSP70)and nuclear factor E2-related factor(Nrf2)genes(P<0.05).The relative expression of cyclin protein D(cyclin D)and villin protein(villin)genes was significantly decreased(P<0.05).PR significantly increased the relative expression of IL-8 and villin genes in IPEC-1 cells(P<0.05).EP significantly down-regulated the relative expression of TNF-αand CXCL2 genes in cells(P<0.05).ZnO significantly increased the relative expression of villin and cyclin D genes(P<0.05).In addition,PR,EP and ZnO significantly reduced the relative expression of 16S rRNA gene in E.coli genes(P<0.05).In conclusion,PR,EP and ZnO can inhibit the proliferation of ETEC K88 and have antibacterial effects,EP can reduce the expression of inflammatory genes caused by K88,PR can increase the expression of intestinal villi genes,and ZnO can increase the expression of cell cycle genes.