The Effect On Anti-apoptosis Of Stanniocalcin-1 In MDBK Cells Induced By Cadmium

In mammals, stanniocalcin-1(STC-1)is a widespread glycoprotein hormone that have a variety of biological effects in the body. STC-1, a key endocrine factor, regulates serum calcium and phosphate homeostasis though the intestine and kidney. In the kidney, STC-1 can inhibit the absorption of calcium, while increase the absorption of phosphorus. In a variety of cells and tissues, the antiapoptotic molecular mechanism of STC-1 is controversial. Cadmium is a heavy metal element with accumulation toxicity. Cadmium poisoning people has been paid more attention and the studies on the cadmium poisoning of mammals are still lacking. Compared with people, mammals have a higher tolerance to cadmium poisoning, which is indetectable. The kidney, the major accumulation sites of cadmium, is damaged by cadmium. Therefore,the study of the function of STC-1 on madin-darby bovine kidney(MDBK) cells induced by cadmium can provide reference for the study of the function on STC-1 and the research on the effect of STC-1 to cells apoptosis induced by cadmium can safeguard mammalian healthy and standardize the animal food safety testing.The expression of STC-1 was detected by RT-PCR and Western-Blot in logarithmic phase MDBK cells induced by cadmium chloride. After MDBK cells were induced by a certain concentration of cadmium chloride and different concentrations of h STC-1 recombinant protein, the m RNA expression of the Caspase-3, Bcl-2 and uncoupling proteins-2(UCP-2) were detected by RT-RCR and the apoptosis rate and the reactive oxygen species(ROS) production were detected by flow cytometry(FCM). The results were as following:1.The mRNA and protein expression level of STC-1 in MDBK cells induced by different concentrations of cadmiumMDBK cells induced by the different concentrations of cadmium: The STC-1 gene of cadmium chloride induced groups were up-regulated significantly compared with the control group and had dose and time-dependent manner. After 2h, the STC-1 gene was up-regulated significantly. The group induced by 20μg/m L cadmium chloride was up-regulated more than 53 folds at 8h(P<0.01) and the group induced by 30μg/m L and 50μg/m L cadmium chloride was up-regulated more than 26 and 16 folds at 8h(P<0.05).MDBK cells induced by the different concentrations of cadmium: In comparision with the control group, the STC-1 protein of groups induced by 20 μg/m Lcadmium chloride after 2h, 4h, 8h and 12 h were up-regulated 1.6, 17, 26.1 and 26.2 folds.2.The effect of h STC-1 recombinant protein on the apoptosis of MDBK cells induced by cadmium chlorideThe apoptosis of MDBK cells induced by a certain concentration of cadmium chloride and different concentrations of h STC-1 recombinant protein: Compared with the control group induced only by a certain concentration of cadmium chloride, the expression level of the Caspase-3, Bcl-2 and UCP-2 gene of experimental groups induced by a certain concentration of cadmium chloride and different concentrations of h STC-1 recombinant protein were different. the Caspase-3 gene of experimental groups were down-regulated. After 4h, it was down-regulated 0.11 and 0.18 folds at 0.5 and 2μM h STC-1 recombinant protein(P<0.05). After 8h, it was down-regulated 0.13 and 0.24 folds at 0.5 and 2μM h STC-1 recombinant protein(P<0.05). The Bcl-2 gene of experimental groups were up-regulated. After 2h and 4h, it was up-regulated 3.2 and 2.4 folds at 0.5μM h STC-1 recombinant protein(P<0.05). The UCP-2 gene of experimental groups were up-regulated. After 2h, it was down-regulated 1.9 folds at 2μM h STC-1 recombinant protein(P<0.05). After 8h, it was down-regulated 3 folds at 0.5μM h STC-1 recombinant protein(P<0.05). Therefore, h STC-1 recombinant protein, 0.5-2μM, plays a major role on up-regulating Bcl-2 and UCP-2 gene on the early phase MDBK cells induced by cadmium and h STC-1 recombinant protein and down-regulating Caspase-3 on the later phase MDBK cells induced by cadmium and h STC-1 recombinant protein.The apoptosis of MDBK cells induced by a certain concentration of cadmium chloride and different concentrations of h STC-1 recombinant protein: In comparision with the control group, the rate of apoptosis of 0.5, 2 and 10μM h STC-1 recombinant protein groups were 8.39%, 4.23% and 4.64% compared with the control group 13.95%, And the generated level of ROS were 9.88, 3.53 and 8.58 compared with the control group 19.8. Therefore, h STC-1 recombinant protein, 2-10μM, can reduce the rate of apoptosis and the level of ROS.Conclusion: The significantly up-regulated STC-1 acts as a marker to identificate cadmium poisoning. The molecular mechanism of h STC-1 recombinant protein inhibiting MDBK cells apoptosis induced by cadmium are up-regulating Bcl-2 and UCP-2 gene on theearly phase of MDBK cells induced by cadmium and down-regulating the expression of Caspase-3 and the level of ROS on the later phase of MDBK cells. This experiment provide a reference for further studying the biological functions of STC-1.