| Traditionally,the function of fat is only used for storing lipid and providing energy.But according to the recent researches,fat is an endocrine organ which owns active metabolism and complex function.It can synthesize and release all kinds of adipokines,and then regulate a variety of functions in the body.Zinc-α2-glycoprotein(ZAG),as a novel adipokine,has already attracted widespread attentions.The researches found that ZAG can promote lipolysis of adipose tissue,accelerate oxidation of fatty acids,and then reduce adipose tissue weight.Researchers also found that ZAG can alleviate insulin resistance.However,up to now,there is hardly any researches about the relation between ZAG and inflammation.Therefore,this study took Duroc ×(Landrace × Large White)male growing pigs as models,used LPS intramuscular injection as treatment method,and explored the changes of ZAG and hepatic lipometabolism of pigs during inflammation.Then,the study took ZAG overexpression mice and ZAG knockout mice as models,used LPS intraperitoneal injection as treatment method,and revealed the effects of ZAG on hepatic inflammation and lipometabolism and its mechanisms.This study not only contributions to know more new function about ZAG,but also provides a new target and approach to anti-inflammatory researches.1 The effects of LPS treatment on ZAG and hepatic lipometabolism of pigsIn this study,twelve Duroc ×(Landrace × Large White)male growing pigs were selected and randomly assigned to two groups,namely control group(CON)and lipopolysaccharide group(LPS).CON group pigs were intramuscularly injected with equivalent volume of physiological saline and LPS group pigs were intramuscularly injected with 15 μg/kg body weight LPS.After 6 h treatment,blood,liver and back fat were collected to further examine.The results showed that body temperature of pigs significantly increased after LPS treatment(P<0.05).Plasma cortisol,TNFa and IL1β abundance significantly increased after LPS treatment(P<0.05).LPS treatment significantly increased hepatic TLR4,pNFκB p65 and TNFa protein expression(P<0.05).LPS treatment significantly decreased hepatic ZAG gene expression(P<0.05)but significantly increased its protein expression(P<0.05).LPS treatment significantly increased hepatic β3-AR gene expression(P<0.05)but had no effect on hepatic PKA protein expression.LPS treatment had no effect on adipose ZAG and adiponectin gene expression but significantly increased their protein expression(P<0.05).LPS treatment had no effect on adipose leptin expression.Hepatic ACC and SCD1 gene and protein expression significantly decreased after LPS treatment(P<0.05).LPS treatment had no effect on hepatic HSL gene expression and lipolytic enzymes activity,but significantly decreased hepatic ATGL gene expression(P<0.05).LPS treatment had no effect on hepatic PCK1 and PC gene expression,but significantly decreased hepatic PCK2 and G6PC gene expression(P<0.05).LPS treatment had no effect on hepatic PCK1,PCK2 and G6PC protein expression.From the above results,it can be concluded that LPS induced acute inflammation of pigs.And during inflammation,hepatic and adipose ZAG protein expression of pigs significantly increased,and hepatic lipometabolism became disordered.2 The effects of ZAG overexpression on hepatic inflammation of LPS-treated mice and its mechanismsIn this study,twenty-four C57BL/6 male mice were selected and randomly assigned to three groups,namely control group(CON),lipopolysaccharide group(LPS)and lipopolysaccharide and ZAG overexpression group(LZO).LPS was intraperitoneally injected,and the dose of treatment was 1 mg/kg body weight,the time of treatment was 24 h.ZAG overexpression plasmid was injected by tail vein twice,and the interval time was 24 h.After treatment,blood and liver were collected to examine.The results showed that LPS treatment significantly increased liver weight and liver index(P<0.05),and after ZAG overexpression,compared with LPS group,liver weight and liver index showed no difference.LPS treatment had no effect on plasma and hepatic ZAG protein abundance,and after ZAG overexpression,compared with LPS group,plasma and hepatic ZAG protein abundance significantly increased(P<0.05).LPS treatment significantly increased plasma IL1α and IL1β protein abundance(P<0.05),and after ZAG overexpression,compared with LPS group,plasma IL1α and IL1β protein abundance significantly decreased(P<0.05).LPS treatment significantly increased hepatic IL1α,IL1β and IL10 gene expression(P<0.05),and after ZAG overexpression,compared with LPS group,hepatic ILla and IL1β gene expression significantly decreased(P<0.05),hepatic IL10 gene expression significantly increased(P<0.05).LPS treatment significantly increased hepatic IL1α and IL1β protein expression(P<0.05),and after ZAG overexpression,compared with LPS group,their protein expression significantly decreased(P<0.05).LPS treatment significantly increased hepatic F4/80 and CD3 protein abundance(P<0.05),and after ZAG overexpression,compared with LPS group,their protein abundance significantly decreased(P<0.05).LPS treatment significantly increased hepatic BAX gene expression(P<0.05),and after ZAG overexpression,compared with LPS group,its gene expression significantly decreased(P<0.05).Compared with LPS group,BCLXL and MCL1 gene expression significantly increased after ZAG overexpression(P<0.05).LPS treatment significantly increased the ratio of BAX protein expression and BCL2 protein expression(P<0.05),and after ZAG overexpression,compared with LPS group,this ratio significantly decreased(P<0.05).LPS treatment significantly increased hepatic active caspase 3 protein abundance(P<0.05),and after ZAG overexpression,compared with LPS group,its protein abundance significantly decreased(P<0.05).LPS treatment significantly increased TLR4,phosphorylated IκBα,phosphorylated NFκB p65,NFκB p65 protein expression and nuclear NFκB p65 protein abundance(P<0.05),and after ZAG overexpression,compared with LPS group,these protein all significantly decreased(P<0.05).LPS treatment had no effect on β3-AR/PKA/CREB signal pathway,and after ZAG overexpression,compared with LPS group,hepatic β3-AR,PKA and phosphorylated CREB protein expression significantly increased(P<0.05).LPS treatment promoted the combination of NFκB and CBP(P<0.05)and inhibited the combination of CREB and CBP(P<0.05)in hepatocyte nuclear.After ZAG overexpression,compared with LPS group,the combination of NFκB and CBP was decreased(P<0.05),and the combination of CREB and CBP was increased(P<0.05).From the above results,it can be concluded that LPS treatment activated TLR4/NFκB inflammatory signal pathway,promoted the phosphorylation and nuclear translocation of NFκB p65,promoted the combination of NFκB p65 and CBP and eventually induced inflammation.ZAG overexpression activated β3-AR/PKA/CREB signal pathway,promoted the phosphorylation of CREB,promoted the combination of CREB and CBP,competitively inhibited the combination of NFκB p65 and CBP,and eventually reduced inflammation.3 The effects of ZAG overexpression on hepatic lipometabolism of LPS-treated miceIn this study,twenty-four C57BL/6 male mice were selected and randomly assigned to three groups,namely control group(CON),lipopolysaccharide group(LPS)and lipopolysaccharide and ZAG overexpression group(LZO).LPS was intraperitoneally injected,and the dose of treatment was 1 mg/kg body weight,the time of treatment was 24 h.ZAG overexpression plasmid was injected by tail vein twice,and the interval time was 24 h.After treatment,blood and liver were collected to examine.LPS treatment significantly increased plasma cholesterol,triglyceride,non-esterified fatty acid and liver triglyceride(P<0.05),and after ZAG overexpression,compared with LPS group,these four indexes significantly decreased(P<0.05).LPS treatment significantly increased hepatic ACC and FAS protein expression(P<0.05),and after ZAG overexpression,compared with LPS group,their protein expression significantly decreased(P<0.05).LPS treatment and ZAG overexpression had no effect on pHSL and ATGL protein expression.LPS treatment significantly decreased plasma glucose and liver glycogen(P<0.05),and after ZAG overexpression,compared with LPS group,these two indexes significantly increased(P<0.05).LPS treatment significantly decreased hepctic PEPCK enzyme activity and PCK1 protein expression(P<0.05),and after ZAG overexpression,compared with LPS group,these two indexes significantly increased(P<0.05),Compared with LPS group,ZAG overexpression significantly increased hepatic PC enzyme activity and GLUT1,GCK protein expression(P<0.05).Compared with LPS group,after ZAG overexpression,the number of hepatic mitochondria significantly increased(P<0.05).Compared with LPS group,ZAG overexpression significantly improved the hepatic mitochondrial complex III and V enzyme activity(P<0.05)and increased hepatic COX1 and UCP3 protein expression(P<0.05).From the above results,it can be concluded that LPS treatment significantly increased plasma and liver triglyceride abundance,and ZAG overexpression improved mitochondria function,promoted energy metabolism,and then reduced plasma and liver triglyceride.4 The effects of ZAG knockout on hepatic inflammation and lipometabolism of LPS-treated miceIn this study,four C57BL/6 normal female mice were selected as LPS group and four C57BL/6 systemic ZAG knockout female mice were selected as LPS and ZAG knockout group(LZK),LPS was intraperitoneally injected,and the dose of treatment was 1 mg/kg body weight,the time of treatment was 24 h.The results showed that after ZAG knockout,liver index significantly decreased(P<0.05),and body weight and liver weight showed no change.ZAG knockout significantly decreased plasma ZAG protein abundance(P<0.05),and significantly increased plasma IL1α and IL1β protein abundance(P<0.05).Hepatic ZAG gene expression significantly decreased(P<0.05)and hepatic IL1α,IL1β,IL6 and MCP1 gene expression significantly increased after ZAG knockout(P<0.05).ZAG knockout significantly decreased hepaticβ3-AR and PKA protein expression(P<0.05).Plasma triglyceride and non-esterified fatty acid had a trend to increase after ZAG knockout(P=0.08).Afer ZAG knockout,hepatic ACC and FAS protein expression significantly increased(P<0.05),but pHSL and ATGL showed no change.After ZAG knockout,plasma glucose significantly decreased(P<0.05).PEPCK enzyme activity and PCK1 protein expression were both significantly decreased after ZAG knockout(P<0.05).ZAG knockout decreased hepatic mitochondria complex Ⅲand V enzyme activity(P<0.05),and significantly decreased COX1 and UCP3 protein expression(P<0.05).From the above results,it can be concluded that ZAG knockout aggravated inflammation induced by LPS,and further increased plasma triglyceride abundance during inflammation.In conclusion,LPS induced acute inflammation of pigs.And during inflammation,hepatic and adipose ZAG protein expression of pigs significantly increased,and hepatic lipometabolism became disordered.ZAG activated β3-AR/PKA/CREB signal pathway,alleviated inflammation,and improved mitochondria function,promoted energy metabolism,reduced plasma and liver triglyceride. |
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