| IEC-6culture line was used as a model to study the absorption of zinc in different zinc sources (nano-ZnO, feed grade-ZnO and ZnSO4) and protective effects against apoptosis. This study included a total of three tests:Firstly, the effects of different levels and sources of zinc on gene expression and zinc absorption rate of IEC-6cells. Secondly, establishment of IEC-6cells apoptosis model induced by H2O2. At last, protective effect of different levels and sources of zinc on H2O2-induced IEC-6cells apoptosis.Test1:Cells were treated with different zinc sources (nano-ZnO, feed grade-ZnO and ZnSO4) and concentration (0,25,50,100μmol/L) or2μmol/L N,N,N’,N’-tetrakis (2-phridylmethyl) ethylenediamine (TPEN) for24hours. The results indicate:2μmol/L TPEN decreased the CCK-8OD significantly (P<0.05). Nano-ZnO and ZnO with lower concentrations (25μmol/L) increased the CCK-8OD significantly and the former is better than the later significantly. The results showed that the ability to enhance cell proliferation of nano-ZnO is better than ZnO under low concentrations. The zinc zinc absorption rate nano-ZnO was higher than that treated with feed grade ZnO and ZnSO4significantly (P<0.05). The zinc absorption rate of nano-ZnO peaked at3hours within12hours and that is much higher under37℃than that under4℃(P<0.05). It suggested that energy-dependent endocytosis is the primary uptake mechanism for the internalization of nano-ZnO. Zinc deficiency down-regulated the expression of ZnT-1, ZnT-4, MT, DMT-1mRNA, up-regulated the expression of ZnT-7mRNA (P<0.05), while Nano-ZnO up-regulated the expression of ZnT-1, ZnT-5, MT, DMT-1mRNA. ZnSO4is similar with Nano-ZnO in this respect. The three zinc source had no significant effect on the expression of ZnT4(P>0.05). It suggested that the nano-ZnO could conduct its biological effects by Zn+as ZnSO4.Test2:IEC-6cells were treated with different levels H2O2(0、50、100、200、400、800μmol/L) for4hours. The results indicate: H2O2could induce the cell apoptosis.200μmol/L H2O2enhanced the apoptotic rate, LDH activity and reduced the CCK-8OD significantly (P<0.05).Test3: Cells were treated with different zinc sources (nano-ZnO, feed grade-ZnO and ZnSO4) and concentration (0,25,50,100μmol/L) in the presence of H2O2(200μmol/L) for4hours. The results indicate:25umol/L nano-ZnO and ZnSO4could inhibit the increase of H2O2-induced IEC-6cells apoptotic rate, LDH activity and Caspase-3enzyme activity (P<0.05), and the inhibiton effect of former is better than that of later significantly (P<0.05). But higher concentration of nano-ZnO and ZnSO4aggravate cell apoptosis significantly (P<0.05). It suggested that low concentration (25μmol/L) nano-ZnO could protect the IEC-6cells from H2O2-induced cell apoptosis effectively. Low concentration (25μmol/L) nano-ZnO and ZnSO4down-regulated the expression of Caspase-3,8mRNA and up-regulated the ratio of Bcl-2/Bax mRNA expression (P<0.05) while high concentration (100μmol/L) of nano-ZnO and ZnSO4up-regulated the expression of Caspase-3.8mRNA and down-regulated the ratio of Bc1-2/Bax mRNA expression (P<0.05). The different levels of nano-ZnO and ZnSO4down-regulated the expression of Caspase-9mRNA (P<0.05). It suggested that the protective effect of nano-ZnO and ZnSO4is worked through the influence of mitochondria initiated apoptotic pathway while the negative effects of high concentration (100μmol/L) zinc is worked through the influence of Fas ligend pathway.In summary, the absorption rate of nano-ZnO is higher than that of feed grade ZnO, and the ability to enhance cell proliferation of nano-ZnO is better than ZnO under low concentrations (25μmol/L). The Zn+of three zinc source can enter the cell by zinc transport protein, and conduct their biological effects. The low concentration nano-ZnO could protect the IEC-6cells from H2O2-induced cell apoptosis effectively through the influence of mitochondria initiated apoptotic pathway. It is more effectively than ZnSO4and feed grade ZnO. But higher concentration of nano-ZnO aggravates cell apoptosis. |
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