Effects Of Early Fatty Liver On Liver Lipids Metabolism And Immunity

Fatty liver is a common nutritional and metabolic disease in mammals,which seriously affects the health of mammals.For some mammalian livestock,pets and special economic animals,excessive intake of high-energy diets or metabolic problems of the body will lead to the occurrence of fatty liver.Therefore,it is of great significance for the development of livestock to explore the pathogenesis of fatty liver and explore the targets for the treatment of fatty liver.In this experiment,rats,mouse hepatocytes,and dairy cows were used as the research objects to explore the effects of early fatty liver on liver lipid metabolism and immunity,in order to provide some data support for the study of fatty liver in mammalian livestock,pets and special economic animals.1.Establishment and phenotypic analysis of early stage of fatty liver model in ratsTwenty-four 8-week-old Sprague-Dawley rats were randomly divided into two groups.FL group was fed with high-fat and choline deficiency diet to induce early fatty liver model,and CON group was fed with normal diet,for 44 days.After the successful induction of the model,the serum biochemical indexes and hormone indexes of the two groups were determined.The results showed that the concentrations of NEFA(non-esterified fatty acid,P=0.0015),TG(triglyceride,P=0.0413),LDL(low density lipoprotein,P=0.0029),ALP(alkaline phosphatase,P=0.0059),TP(total protein,P=0.0001),insulin(P=0.0123)and glucagon(P=0.0259)in FL group were significantly higher than those in CON group,while the levels of growth hormone(P=0.0119),calcium(P=0.0138)and IGF-1(insulin-like growth factor 1,P=0.0222)were significantly lower.However,there was no significant difference(P>0.05)in GLU(glucose),HDL(high density lipoprotein),ALB(serum albumin),AST(aspartate aminotransferase),ALT(alanine aminotransferase),magnesium ion,phosphorus ion and LDH(lactate dehydrogenase)between FL group and CON group.The level of insulin resistance index(P=0.0235)increased significantly in FL group.At the end of the trial,the body weight gain of the rats in the FL group was significantly higher than that in the CON group(P=0.0305),but there was no significant change in the organ index of liver,kidney,spleen and heart(P>0.05).2.Effects of early fatty liver on liver lipid metabolism in ratsThrough the wide-target liposome analysis of the livers of the two groups,a total of 982 kinds of lipid metabolites were detected in the liver tissues,of which 474 kinds were significantly different between the two groups.There were significant differences in 42.51%GP,and 97.78% of these differences were significantly down-regulated.65.60% of GL and 29.52% of SL changed significantly,while 94.72% and96.77% of these differentially expressed lipids were upregulated,respectively.The lipid with the biggest difference was TG.The result of KEGG enrichment analysis showed that there were significant differences in seven pathways between the two groups(P<0.05),namely: cholesterol metabolism,vitamin digestion and absorption,fat digestion and absorption,lipolysis regulation of adipocytes,insulin resistance,thermogenesis and glycerol metabolism.Through transcriptome analysis of the livers,it was found that only a small number of genes changed significantly,178 differentially expressed genes between the two groups.Among them,the expression of DNL(de novo lipid synthesis)process-related genes was inhibited,and the expression of Cyp(cytochrome P450)family genes was significantly affected.KEGG enrichment analysis showed that 8 pathways were significantly enriched(P<0.05): steroid biosynthesis,fat digestion and absorption,chemical carcinogenesis,retinol metabolism,bile secretion,cytochrome P450 metabolism of foreign substances,PPAR signaling pathway and cholesterol metabolism.Liposome and transcriptome joint analysis showed that the most prominent features of liver in the early stage of fatty liver were lipid absorption,cholesterol metabolism and metabolic pathway.3.The inflammation expression of blood and multiple organs in early fatty liver of ratsFirst of all,it was found that there was a trend towards NASH in the liver of rats with early fatty liver.There were significant expression differences in related genes: Cxcl13(c-x-c motif chemokine ligand 13),Nfatc4(activated t nuclear factor 4),Slit2(slit guidance ligand 2)and apoa4(apolipoprotein A4)between the two groups,and inflammatory related PPAR pathway was significantly enriched(P<0.05).During the development of insulin resistance,it is easy to cause non-infectious chronic inflammation of the whole body.So the concentrations of Ig A(immunoglobulin a),Ig G(immunoglobulin g),Ig M(immunoglobulin m),IL-1β(interleukin-1 β),IL-2(interleukin-2),IL-6(interleukin-6),IL-10(interleukin-10)and TNF-α(tumor necrosis factor α)in serum,spleen,kidney and duodenum were measured,in order to explore the occurrence of inflammation in some important tissues and organs outside the liver.It was found that there were different degrees of inflammation in these tissues and organs,especially in the spleen.Because the spleen is the largest immune organ of the body,the transcriptome analysis of the spleen was carried out.A total of 78 differentially expressed genes were screened in the spleen.KEGG enrichment analysis showed that two pathways were significantly enriched(P<0.05): PPAR signal pathway,porphyrin and chlorophyll metabolism pathway.GO(gene ontology)analysis showed that heme biosynthesis and organic acid transport were affected.4.Effects of early fatty liver on hepatic lipophagy in ratsThe livers of the two groups were tested by immunofluorescence test to detect the co-localization of autophagy-associated protein LC3 B and lipid droplet membrane protein ADRP.At the same time,autophagy-associated protein p62 and ADRP were detected by Western Blotting method.5.Study on the regulatory mechanism of PKLR gene on fatty liverThis study attempted to find a key gene regulating the occurrence of fatty liver,and PKLR gene was selected as the target gene for a series of experiments to verify the regulatory effect of this gene on fatty liver.The early fatty liver model of NCTC-1469 cells was induced by adding 0.25 m M sodium oleate for12 hours.si RNA-PKLR was used to suppress the PKLR gene,and p CMV6-PKLR overexpress PKLR gene.It was found that overexpression of PKLR gene could increase the area of lipid droplets in cells,while after suppressed the PKLR gene,the area of lipid droplets decreased significantly after the same time induced by sodium oleate.When the expression of PKLR gene was interfered,the expression of adipogenic genes(Acacb,Elovl6,FASN,SCD)were significantly down-regulated(P<0.05),but after overexpression of PKLR gene,the expression of these genes up-regulated(P<0.05).Finally,cows suspected of having fatty liver and normal cows were selected by scoring the body condition,which were all in the early lactation period.Before morning feeding,the tail vein blood was collected,and the serum was collected to determine the content of β-hydroxybutyric acid and NEFA in the serum.12 dairy cows(5 normal dairy cows,7 suspected fatty liver dairy cows)were subjected to liver biopsy sampling,and then oil red O staining was performed to accurately identify the occurrence of fatty liver in dairy cows.The livers of 3normal dairy cows and 3 dairy cows with fatty liver were randomly selected for follow-up experiments.RNA was extracted from the livers of the selected 6 dairy cows,and the expression levels of adipogenic genes and PKLR genes were detected.The results showed that compared with normal cows,the expression of adipogenic genes(Acacb,Elovl6,FASN,SCD)and PKLR genes in the liver of dairy cows with fatty liver was significantly up-regulated(P<0.05).To sum up,early stage of fatty liver has a significant effect on body weight gain of rats,but has little effect on organ indexes of various organs,and has great impacts on serum biochemical indexes and serum hormone levels.In the early stage of fatty liver,a small amount of genetic changes in the liver can cause strong changes in liver lipids.The occurrence of fatty liver not only affects the liver,but also causes systemic inflammation.The effect of early fatty liver on spleen mainly affects the synthesis of heme and the change of immune function,and is accompanied by the changes of lipids transport and metabolism in spleen.In the early stage of fatty liver,the liver can make a self-regulating response to the surge of lipids,including inhibiting the synthesis of fatty acids and promoting lipophagy.PKLR gene can regulate the occurrence of fatty liver,which may be achieved by affecting the synthesis of lipids.