Study On The Expression Of Carbohydrate And Lipid And Their Metabolic Related Factors In Rana Oil

In order to better survive the hibernation period,Rana dybowskii stores energy in the pre-hibernation,and its oviduct will specifically expand before hibernation and form rana oil.Rana oil is a traditional health food in China,which has been paid more and more attention because of its multiple medical and health functions.However,there are few reports of in-depth studies on the nutrient composition of rana oil and its metabolic factors.This subject took the oviduct of Rana dybowskii in the pre-ibernation as the research object,and detects the content of carbohydrate and lipids in the rana oil and its metabolic related factors from the level of gene and protein to study the nutritional value of the rana oil.The oviduct of Rana dybowskii during pre-hibernation and non-hibernation(the breeding period)were selected as the research objects.The distribution of glycogen and fat in rana oil was observed by PAS staining and oil red staining respectively in different periods,and was quantified by the determination of glycogen and triglyceride.The m RNA expression level of carbohydrate and lipid metabolism related factors in rana oil was compared by transcriptomics and real-time quantitative PCR.The expression level of glycogen synthesis-related factors in rana oil was determined by immunohistochemistry and Western blotting to compare the protein expression level and immunolocalization of carbohydrate and lipid metabolism related factors in rana oil,and to analyze the metabolite level of carbohydrate and lipid metabolism pathway in rana oil by metabonomics.The results showed that:(1)the weight,wall thickness and gland cells of rana oil were significantly higher than those of the oviduct in the breeding period(The breeding period as a representative of non-pre-hibernation);(2)the PAS staining showed that the glycogen mainly accumulated in the glandular cells in the pre-hibernation period and the content was high,and the glycogen content in the pre-hibernation period(12.83 ± 0.04520)was significantly higher than the glycogen content in the in the breeding period(4.235 ± 0.3026);the oil red staining showed that the fat was mainly accumulated in epithelial cells during pre-hibernation,and its triglyceride content(4.808 ± 0.7838)was significantly lower than the triglyceride content during reproduction(31.17 ± 4.186);(3)transcriptomics analysis showed that the levels of glucose metabolism-related genes GYS,pgm,gpi and other genes were up-regulated and pck gene expression was down-regulated in pre-hibernation;(4)immunohistochemical results showed that glycogen synthesis-related factors GLUT1,total-GYS,p-GYS,total-GSK-3β,and p-GSK-3β were expressed in glandular cells and epithelial cells in the pre-hibernation;immunoblotting results showed that GLUT1,total-GYS,and p-GSK-3β,which are positively related to glycogen synthesis,were highly expressed in rana oil during the pre-hibernation;(5)metabolomics analysis showed that the content of α-ketoglutarate,a metabolite of sugar metabolism-related pathways in rana oil,increased,and the content of α-D-glucose6-phosphate,cis aconitic acid,and citric acid decreased;lipid metabolism unsaturated fatty acids such as linoleic acid,α-linolenic acid,and lecithin are elevated in related pathways.The above results indicate that,first,the gene level of glycolysis-related factors is up-regulated during the pre-hibernation period,and the gene level of gluconeogenesis-related factors is down-regulated.The glycogen content of rana oil was increased during the pre-hibernation period,and the factors positively related to glycogen synthesis had high expression.Factors that are negatively related to glycogen synthesis have low expression,indicating that rana oil can accumulate a large amount of glycogen during the pre-hibernation period.Second,the triglyceride content in the oviduct was lower during the pre-hibernation period,and the gene level of factors positively related to lipid metabolism was increased,and some fatty acid content increased,indicating that the triglycerides in rana oil during the pre-hibernation period decomposed to form a large amount of fatty acids.