| Copper is a necessary element that is essential for biological function and metabolism. But due to the effect of promoting growth and antibacterial in livestock, the high copper has been added blindly, causing the animal food poisoning of animal origin, environmental pollution and so on. Because of the small size, high-activity in cells and easily-absorbed, nanosized CuO may be used in the feed, as copper source not the copper sulfate. The study was conducted to deterimine the effect of level and the source of the dietary copper on copper metabolism in the Swine Intestinal Epithelial Cells (SIEC)After thaw, SIEC were conventional cultured, the experiment was designed on the5-8generation cells. The cells were stratified to one of the following treatment:control, copper sulfate groups, nanosized CuO groups. The copper sulfate groups were classified into four grades:5,10,20,40μg/mL groups and the nanosized groups also followed in the same way.24h after treatmentment with copper, cells were harvested. The activities of the superoxide dismutase (CuZn-SOD) was measured by colorimetric method and concentrations of the metallothionein (MT) were determined by elisa, the contents of the mRNA of the copper regulatory(ATOXl, CTR1, COX17, MT, CuZn-SOD) were detected by Quantitative real-time PCR. Cell proliferation at12,24,36,48,60h was measured by MTT.The SIEC cells is monolayer columnlike epithelia, irregular polygon-shaped, both the nanosized CuO and copper sulfate groups, proliferation and viability decreased in a concentration-dependent manner. The activities of the CuZn-SOD inreased in40μg/mL nanosized CuO and5μg/mL copper sulfate groups (P<0.05) VS controls, but the40μg/mL copper sulfate groups was opposite(P<0.05). The concentrations of the metallothionein in the nanosized CuO groups had increased(P<0.05). The concentrations of the metallothionein were increased in10,20,40μg/mL nanosized CuO groups compared to copper sulfate groups at the same levels. Compared to the control group, in the nanosized CuO groups a increase of CTR1mRNA in cell at copper levels of5μg/mL was observed(P<0.01), but above this level in CuO groups reduced significantly were observed vs copper sulfate groups (P<0.01). The content of MT mRNA was also rise in the40μg/mL nanosized CuO and20,40μg/mL copper sulfate groups (P<0.05), besides10μg/mL nanosized CuO groups is more obviously(P<0.01). The mRNA of CuZn-SOD had backfall in the10,20,40ug/mL nanosized CuO groups vs copper sulfate groups and controls (P<0.05). The ATOX1mRNA was decreased in the nanosized CuO group and copper sulfate groups vs controls (P<0.01), a significant reduction was observed in the5μg/mL copper sulfated groups vs nanosized CuO groups at the same level. Compared to controls, the mRNA of COX17was found no significant differencein in the two groups (P>0.05)In the copper sulfated, copper element shipped into cells in the shape of copper ion by CTR1proteins. Comparaed to copper sulfats, parts of nanosized CuO entered into cells in the shape of copper ion by CTR1proteins, the others in the shape of nano particles. Low dependence on the CTR1due to the activity-high, that led to the low transcription oc CTR1. One pathway after copper element entered into cells was combine with metallothionein, just because amount of copper element in the cells led to a increase of content of metallothionein mRNA. Activation of the antioxidant system, when copper ions entered into cells, led to increase of activity of CuZn-SOD, besides a increase of the concentrations of the ATOX1mRNA in5μg/mL nanosized CuO promoted the copper ions out of the cells, above this level a decrease of the concentrations of the ATOX1mRNA could accelerate the incorporation of the copper ions and GSH. The experimrnt of MTT found little restrain in the nanosized CuO vs copper sulfate. Based on these, the nanosized CuO had an advantage over copper sulfate at copper metabolism. |
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