Effect And Mechanism Of Osteocalcin On Fatty Liver Hemorrhagic Syndrome Induced By High-fat Diet In Aged Laying Hens

Fatty liver hemorrhagic syndrome(FLHS)is a metabolic disease characterized by lipid metabolism disorder in poultry liver.It is common in laying hens and one of the main non-infectious causes of death in laying hens,causing serious economic losses to the egg industry.Osteocalcin(OCN)is a protein secreted by osteoblasts that regulates energy metabolism.Previous studies suggested that blood OCN content was correlated with the occurrence of fatty liver in laying hens.Therefore,it is speculated that OCN may be involved in the regulation of FLHS in laying hens.In order to study the regulation of OCN on FLHS in laying hens,thirty 68-weekold White Plymouth laying hens were randomly allocated into one of three treatments(n=10),control group(normal diet + saline,ND+ saline),high-fat diet group(high-fat diet group + saline,HFD+ saline),and osteocalcin group(HFD+OCN,3 μg/bird,1time/2 days,i.m.)for 40 days.The body weight of laying hens was weighed at the 1,20 and 40 days,and feed intake was measured at the 19-20 and 39-40 days.The laying rate and qualified egg rate of laying hens were recorded on a daily basis.12 eggs were randomly collected from each group at 9-10,19-20,29-30 and 39-40 days to detect egg quality,yolk crude fat and crude protein content.At day 30,glucose tolerance(OGTT)and insulin tolerance(ITT)tests were performed.At the end of the experiment,all the hens were euthanized followed by cardiac blood collection after anesthesia.The content of aspartate transaminase(AST),alkaline phosphatase(ALP),total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)in plasma were measured using an automatic biochemistry analyser.The plasma concentrations of insulin,adiponectin(ADPN),interleukin-1(IL-1),interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)were detected by ELISA.Abdominal fat,liver and pancreas were weighed,liver hemorrhage was evaluated by five points method,and liver fat content was determined by Soxhlet extraction method.The pancreas and liver tissue microstructure were observed by light microscopes with HE staining,and the liver fibrosis was examined using Masson trichrome staining,while the liver ultrastructure was analyzed by transmission electron microscope.The level of oxidative stress was evaluated using malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)assay kits,respectively.And the m RNA expression of ADPN,PPARα,IL-1,IL-6 and TNF-α in the liver were detected by realtime PCR.The protein expressions of TNF-α,PPARα,JNK and p-JNK were detected by Western blotting.The results showed that OCN had no effect on body weight,feed intake,laying rate and qualified egg rate of laying hens fed HFD.At day 40,the egg yolk color score of laying hens in osteocalcin group was lighter than the control group and the high-fat diet group(P < 0.05),and the crude lipid content of egg yolk was lower than the control group(P < 0.01).Compared with the control group,HFD increased abdominal fat content of laying hens(P < 0.05),decreased liver relative weight(P < 0.05),but did not influence liver fat content and pancreatic weight(P > 0.05),and significantly elevated the content of blood ALP(P < 0.05),while had no change for the AST,TC,TG,LDL-C and HDL-C(P > 0.05).High-fat diet hens had more severe liver hemorrhage and fibrosis(P < 0.05)than control hens,and the hepatocytes were obviously pyknosis,high intracellular electron density,nuclear pyknosis,heterochromatin margin aggregation,perinuclear space widening,severe mitochondrial swelling,damaged membrane,reduced and disappeared mitochondrial cristae,increased rough endoplasmic reticulum,obvious expansion and degranulation.Compared with the high-fat diet group,OCN resisted the decrease of liver relative weight caused by HFD(P = 0.05),but had no significant effect on blood liver function and blood lipid indexes of HFD laying hens(P > 0.05).The layers in osteocalcin group had higher insulin sensitivity,lower liver MDA concentration(P < 0.05),higher GSHPx content(P < 0.05),lower blood TNF-α concentration(P < 0.05),and lower liver TNF-α m RNA and protein expression(P < 0.05)than high-fat diet group.Moreover,OCN relieved the hepatocyte damage induced by HFD,slowed down the pyknosis of hepatocytes,increased the number of autophagy lysosomes and autophagosomes.Compared with the control group,high-fat diet increased the expression of p-JNK protein(P < 0.01)and the ratio of p-JNK/JNK(P < 0.05)in liver,and OCN could inhibit JNK phosphorylation in liver of laying hens fed HFD.In conclusion,high-fat diet(HFD)can promote the development of FLHS in elderly laying hens,while OCN can reduce HFD-induced liver injury,reduce liver bleeding,fibrosis and inflammatory response,inhibit insulin resistance and oxidative stress,and activate liver autophagy by inhibiting JNK/p-JNK signaling pathway.