| IEC-6 cells were used as a model to study effects of different sources and levels of zinc (nano-ZnO, ZnO and ZnSO4) on growth and bacterial adhesion of enterotoxigenic Escherichia coli (ETEC, strain K88), and related genes about immunity induced by ETEC in IEC-6 cells. This experiment included a total of two tests:Test 1:Effects of different sources and levels of zinc on growth and bacterial adhesion of ETEC1:Establishment of infection model induced by ETEC in IEC-6 cellsIEC-6 Cells (104cells/well) were treated with different concentrations (each wall containing 0, 1×102, 1×104, 1×106, 1×108 CFU bacteria) of ETEC for 1.5,3,4.5 hours to test cell viability. The results indicated that:all the ETEC treatments decreased CCK-8 OD of IEC-6 cells significantly (P<0.05) with time and dose effects. Cells infected with 1×106 CFU bacteria, to achieve a multiplicity of infection of 100 (bacteria/cells ratio), was used in the later experiment. This based on the results that cell viability significant lower than that of control and 1.5h group with multiplicity of infection of 100 and time of 3h, and large number of bacterials adhered around cells without too many dead cells. Thus, infection models of ETEC on small intestinal epithelial cells were established.2:Effects of different sources and levels of zinc on growth and bacterial adhesion of ETEC in IEC-6 cellsCells were treated with different sources (nano-ZnO, ZnO and ZnSO4) and concentrations (0,6,12,24,48,96μmol/L) of zinc or ETEC for every 1.5h in 24 hours, and ETEC group without zinc addition was set as control. The results showed that in contrast with control,6,12,24,48μmol/L nano-ZnO, all levels of ZnO and ZnSO4 treatments significant reduced OD value at 1.5h (P<0.05). The OD value of 48,96μmol/L Nano-ZnO groups and 96μmol ZnSO4 group significant lower than that of control group (P<0.05). All the ZnO treatments showed no significant differences at 24h (P>0.05). It suggested that all of the three zinc sources can inhibit the growth of ETEC, and the bacterio static actions of zinc may mainly based on its action on the different growth phase of ETEC. Furthermore, the resuts showed that there are no inhibition zone for 24h with addition of three zinc sources and ETEC, suggested that the growth of ETEC was not affected, and further support the former results.In contrast with control, nano-ZnO with the concentration of 6μmol/L, ZnO and ZnSO4 with the levels of 6,12,24μmol/L promoted ETEC adhesion in IEC-6 cells to some extent. Nano-ZnO with the concentration of 12,24,48,96μmol/L reduced bacterial adhesion, and 24,48,96μmol/L group reached significant level (P<0.05).Test 2:Effects of different sources and levels of zinc on ETEC-induced gene expression of barrier function and immunityCells were treated with different zinc sources (nano-ZnO, ZnO and ZnSO4) and concentrations (0,6,12,24,48,96μmol/L) in the presence of ETEC (ETEC K88, multiplicity of infection were 100) for 3 hours. Cells without zinc and ETEC were set as normal control, cells treated with ETEC were set as negative control. The results showed that addition with ETEC significant reduced mRNA expression of Claudin-1, ZO-1 and Occludin (P<0.05). In contrast with ETEC group,24μmol/L nano-ZnO up-regulated Claudin-1 and Occludin mRNA expression significantly (P<0.05). However, addition with 96μmol/L nano-ZnO promoted ETEC-induced gene expression of ZO-1 significantly (P<0.05). Treating ZnSO4 with levels of 6,12,24 and 48μmol/L did not differ from that of untreated control cells (P>0.05). Compared with ZnO and ZnSO4, Claudin-1 and Occludin mRNA expression were higher than 24μmol/L nano-ZnO group significantly. It suggested that 24μmol/L nano-ZnO revised the damage induced by ETEC, but 96μmol/L nano-ZnO and ZnSO4 aggravated damage of barrier function.Treating with ETEC up-regulated the gene expression of IL-1β, IL-6 and TNF-α significantly (P<0.05). It suggested that ETEC can induce intestinal inflammation. 24μmol/L nano-ZnO significant reduced IL-1β and TNF-α mRNA expression induced by ETEC (P<0.05). However,96μmol/L nano-ZnO down-regulated the expression of IL-1β, IL-6 and TNF-α mRNA compared with ETEC treatment (P<0.05). All the zinc treatment up-regulated the gene expression of TGF-β induced by ETEC, and nano-ZnO with the concentrations of 6,12,24,48μmol/L and ZnO with concentrations of 12,24,48,96μmol /L reached the significant level (P<0.05). It suggested that moderate concentration of zinc can alleviate inflammatory response induced by ETEC. It also suggested that cell cytokines showed different susceptibility to different forms of zinc, and means that zinc show anti-inflammatory effects with different way.In summary, effects of ETEC on cell vitality of IEC-6 cells have time and dose effects, and infection models of ETEC on small intestinal epithelial cells were preliminary established with multiplicity of infection of 100 and time of 3h. Nano-ZnO, ZnO and ZnSO4 can inhibit the growth of ETEC with different extent, and differ in the forms, concentrations and time.96μmol/L zinc can significant inhibit ETEC adhesion, and the effect of nano-ZnO is better than ZnO and ZnSO4.24μmol/L nano-ZnO can alleviate damage of tight junction and inflammatory response by regulate gene expressions of cytokines. However,96μmol/L nano-ZnO and ZnSO4 accelerated the damage of tight junction and inflammatory response. |
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