| In intensive sheep farming,in order to pursue short-term economic benefits,producers feed lambs with high-energy（HE）diets to make them grow quickly.This increases body fat content,decreases polyunsaturated fatty acids（PUFAs）content in mutton and antioxidant capacity in lambs.Spirulina is characterized by high protein,low fat,comprehensive nutrition,and rich inγ-linolenic acid（GLA）.Spirulina can improve the body’s immunity and antioxidant capacity,and reduce the lipids in the blood.However,whether spirulina can relieve oxidative stress and excessive fat deposition caused by HE diets,and improve meat quality in Hu sheep needs further research.Autophagy takes part in lipid metabolism.Activated autophagy can decrease lipids by decomposing lipid droplets and regulating proteins related to lipid metabolism.AMPK is involved in many important physiological processes,such as energy balance,antioxidation,autophagy and lipid-lowering.LKB1,an upstream kinase of AMPK,regulates autophagy by phosphorylating AMPK.The AMPK-m TOR axis is one of the key pathways involved in autophagy.However,whether spirulina can relieve excessive fat deposition caused by HE via LKB1-AMPK-m TOR autophagy pathway remains unknown.In this study,firstly,we evaluated effects of spirulina supplementation on the growth performance,oxidation indexes,meat quality,lipid metabolism,and autophagy in Hu sheep fed HE diets in vivo study.Then,we used PA to treat AML-12 cells to create a HE model,and added spirulina’s active ingredient GLA to analyse effect of GLA on lipid metabolism in PA-treated AML-12 cells,and focus on exploring its mechanism in vitro study.The study consisted of following three parts:1.Effects of spirulina supplementation on production performance and oxidation indexes in Hu sheep fed HE diets108 Hu lambs（90±3 d,27.57±1.78 kg）were randomly selected and divided into two different energy levles dietary groups:standard energy（ST）group and HE group.Then lambs in each energy dietary group were randomly divided into three subgroups:0%,1%or3%spirulina supplementation groups.After 74 d,blood was collected.36 sheep（6 sheep for each group）were slaughtered.The weight of internal organs and body fat content（GR）were recorded,liver and longissimus thoracis et lumborum（LTL）were collected.Serum biochemical indexes were detected by automatic analyser.Oxidation-antioxidation indexes were detected by ELISA and q RT-PCR.The results showed that HE diets significantly increased body fat,glucose,insulin,aspartatetransaminase（AST）,alanineaminotransferase（ALT）,free fatty acids concentrations in serum,and ROS content in liver（P<0.05）,HE diets significantly decreased activities and gene expression levels of GPX and CAT in lambs（P<0.05）.Spirulina supplementation significantly increased average daily gain（ADG）and carcass weight,increased total protein,albumin,and immunoglobin（Ig G）concentrations in serum,and increased activities and expression levels of SOD,GPX and CAT（P<0.05）.In conclusion,spirulina improved growth performance,and alleviated oxidative stress in Hu sheep fed HE diets.2.Effects of spirulina supplementation on meat quality and lipid metabolism,and preliminary exploration of its mechanismThe experiment design was the same as experiment 1.Gas chromatography,q RT-PCR and Western-blot were used to detect fatty acid composition,lipid metabolism-and autophagy-related indicators.The results showed that HE diets significantly increased p H24hand L*values in LTL,increased cholesterol（TC）and triacylglycerol（TG）contents in serum,saturated fatty acids（SFAs）content and SCD expression level in liver（P<0.05）.HE diets significantly decreased shear force in LTL,and LC3,Beclin-1,AMPK expression levels in liver（P<0.05）.Spirulina supplementation significantly increased crude protein content in LTL and polyunsaturated fatty acids（PUFAs）contents in liver,and increased PPARα、PPARγ、Beclin-1、LKB1 and AMPK expression levels（P<0.05）.Spirulina supplementation decreased TG and TC contents in serum,ACC and ELOVL2 gene expression levels in LTL（P<0.05）.In conclusion,spirulina supplementation could decrease lipid content,increase PUFAs content in liver,and up-regulate gene expression levels involved in autophagy and LKB1-AMPK–m TOR pathway.3.Effect and mechanism of GLA on lipid metabolism in AML-12 treated by palmitic acidThis section was conducted to further explore the mechanism of spirulina’s active ingredient GLA on lipid metabolism in HE-treated hepatocytes in vitro study.AML-12 cells were taken as the research object.PA and GLA were dissolved with BSA and DMSO.Cells were divided into following four groups:BSA group,DMSO group,PA group,PA+GLA group.ELISA,Flow cytometry,q RT-PCR and Western-blot were used to detect lipid metabolism-,oxidation-,apoptosis-,autophagy-and LKB1-AMPK-m TOR pathway-related indexes.The results showed that 400μM PA significantly increased TG and TC contents,and decreased lipid metabolism-related proteins PPARα,CPT1α,and FASN expression levels（P<0.05）;PA significantly decreased antioxidant-related genes SOD and GPX expression levels and T-AOC activity（P<0.05）,and significantly increased apoptosis cells percentage（P<0.05）.PA inhibited autophagy and LKB1-AMPK-m TOR pathway,as indicated by decreases in expression levels of Beclin-1,Atg5 and LKB1（P<0.05）.On the contrary,100μM GLA reduced lipid accumulation in AML-12 treated by PA,as indicated by decreases in TG and TC contents,GLA increased fatty acidβ-oxidation level,as indicated by increases in m RNA and protein expression levels of CPT1αand PPARα（P<0.05）.GLA relieved oxidative stress caused by PA（P<0.05）.GLA activated autophagy and LKB1-AMPK-m TOR pathway,as indicated by increases in expression levels of Beclin-1,Atg5 and LKB1（P<0.05）.In conclusion,the results revealed that GLA regulated lipid metabolism via LKB1-AMPK-m TOR autophagy pathway in AML-12 cells treated by PA. |
