Effects Of Cyadox On MRNA Expression Of Insulin-Like Growth Factor-1 And Epidermal Growth Factor In Pig Liver

Cyadox is a new derivative of quinoxaline-N-dioxide. It is used as an antimicrobial agent for pigs, calves and poultry, but the mechanism of its promoting growth is not known. In order to elucidate the mechanism of its promoting growth further, effects of cyadox on the expression of IGF-1 and EGF mRNA in the primary culture piglet hepatocytes and livers were studied in this article by relative quantity RT-PCR methods.Two-step collagenase perfusion technique established by Seglen was adopted and was a little modified (Seglen, 1976). Hepatocytes were inoculated on 24 wells culture plank at 5×10⁵/well. Adding 900μL culture medium in each well and cultured in 37℃5% CO₂ incubator. Cells were divided into PBS control, pig somatotropin (pST) control (0.01ng/mL, 0.1ng/mL, 1.0ng/mL, 10ng/mL, 100ng/mL) and cyadox group (50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL) after 80% converged and continued culturing for 48h. Then total RNA of the cells was distilled. Hepatocytes were cultured in another plank by the same method for 48h. PST 100ng/mL and cyadox 400ng/mL were appended in the medium, the same volume of PBS was added in PBS control. Total RNA of cells were distilled after 4h, 8h, 12h, 24h and 48h. All total RNA were used to reverse transcription PCR and the products were separated by 1% agarose, then analyzed the relative grey absorption by SYGENE gel imagine system. All groups were selected 3 wells to count the cells. The results show that 200ng/mL of cyadox can enhance the expression of IGF-1 mRNA in pig hepatocytes significantly but not of EGF mRNA. 400ng/mL and 800ng/mL of cyadox can promote the expression of IGF-1 mRNA and EGF mRNA significantly (P<0.05). 400ng/mL of cyadox was added to the cultures for 8h can markedly enhance the expression of EGF-1 mRNA (P<0.05), but not of IGF-1 mRNA (P>0.05). Cultured for 12h, 24h, 48h can markedly enhance the expression of IGF-1 mRNA arid EGF mRNA (P<0.05). The result of cell counting shows that cyadox does not effect the proliferation of hepatocytes obviously.9 Chang-Da piglets (castrated males) were used for the test, weighed 10.0±2.0kg, were randomly divided into 3 groups. Piglets were raised by nutrition criterion of America NRC(1998). In pST control, piglets was administered 4mg/d of pST via i.m at 9:00 AM. Blank feed was intaked by piglets of blank control. Piglets of test group were feed to the feed containing 200mg/kg of cyadox. All groups were allowed ad-libitum feed intake. The test lasted for ten days, and sampled by slaughter after 4h of the administered of the last day, samples was immediately freezed in liquid nitrogen and then transferred to store at -70 °C in refrigeratory. The expression of EGF and IGF-1 mRNA in the livers was determined by relative quantity RT-PCR. Cyadox can promote body weight of piglets received via feed significantly (PO.05) and enhance the expression of EGF and IGF-1 mRNA of their livers significantly (PO.05).Conclusions: promoting growth of cyadox may correlate to its regulating the transcription of insulin-like growth factor-1 and epidermal growth factor. The investigation present has primarily established a biological technique to study the relationship of mechanism of antimicrobial promoting growth of cyadox and the expression of the polypeptide growth factors in animals, and made a foundationment of elucidating the mechanism of antimicrobial promoting agent further and developing the new products.