Effects Of Tannins On The Expression Of Cytochrome P450in Liver Of Plateau Pikas(Ochotona Curzoniae)

A variety of plants in the Qinghai-Tibet alpine ecosystems contain high levels of phenolic secondary compounds, and the level of the phenolic compounds varies depending on the season. Many phenolics compounds, including tannins, have a certain impact on the physiology of animal behavior. Xenobiotic metabolizing enzymes in the animals can abate the harmful effects of plant phenolics on the animal. Cytochrome P450enzymes in animals involved in the metabolism of the secondary compounds taken by animals, while the secondary compounds may also affect the gene expression of these enzymes. Plateau pikas are herbivorous mammals inhabiting in the highest plateau of the world, the Qinghai-Tibet plateau, feeding on plants contain higher phenols for all life. The purpose of this study was to explore the seasonal changes of the gene expression of CYP1A2and CYP2E1in liver of plateau pika and the effect of tannins on two enzymes, to elucidate co-evolution between herbivorous animals and flora in Qinghai-Tibet plateau. We captured wild plateau pikas in different seasons(May, June, July, Auguest). And they were administrated different doses of tannin acid(0,10,20,40mg/kg.bw) for different days(1d,4d,8d). The cDNA fragment of CYP1A2and CYP2E1in liver of plateau pikas was cloned by RT-PCR, and the fluorescence quantitative real-time PCR was used to quantifify the mRNA of cytochrome CYP1A2and CYP2E1, we also detected the changes of the content of two enzymes with Western blot method. The results were as follows.1) We obtained partial cDNA sequences of CYP1A2and CYP2E1gene in the liver of plateau pikas by RT-PCR method/The partial cDNA of CYP1A2is921bp, encoding307amino acid residues and the partial cDNA of CYP2E1is936bp, encoding312amino acid residues. When aligned the two sequences with that of the rat, mouse, and human, the result showed that the similarity of CYP1A2and CYP2E1amino acids in the liver of the plateau pikas with other three species is73.5%,72.5%,73.3%and80.1%,80.8%,76%respectively.2) In field, the gene expression of CYP1A2and CYP2E1in the liver of plateau pikas varied from month to month.From May to August, the mRNA levels of CYP1A2and CYP2E1in the liver of plateau pikas increased firstly and then show a downward trend, and both reached the highest in the June. The results of Western blot showed that the content of CYP1A2and CYP2E1in the liver of plateau pikas also increased firstly and then showed a downward trend, and both reached the highest levels in June.3) The effect of tannic acid on the gene expression of CYP1A2and CYP2E1in the liver of the plateau pikas channged with the dose and time of treatment.The mRNA encoding CYP1A2and CYP2E1in the liver of plateau pikas increased when animals were given medium and low doses of tannic acid for a short time, while decreased when pikas were treated with high doses of tannic acid or with medium and low doses for a longer time. And the higher the concentration of tannic acid or the longer the processing time was, the lower was the level of mRNA encoding CYP1A2and CYP2E1.But the Western blot results showed that the contents of CYP1A2and CYP2E1in the liver of plateau pikas were reduced in all of the groups treated with tannin acid.These results suggested that tannins affect the hepatic CYP1A2and CYP2E1gene expression in plateau pikas. In field, the influence of tannins on the transcription of two enzymes was coordinate with that on translation. The monthly increased tannins could promote the expression of two enzymes in low dose, and inhibit two genes expression when the tannins content high enough. But the effect of tannin acid on the transcription of two enzymes was different from its effect on translation when the animals were administrated with tannic acid.