| Non-alcoholic fatty liver disease is a clinicopathologic syndrome of fatty degeneration of liver cells caused by no excessive alcohol intake.Non-alcoholic fatty liver disease will not only lead to pathological changes in the liver,but also lead to metabolic disorders and cardiovascular diseases.Goose fatty liver has no obvious pathological changes in the case of severe fatty degeneration,suggesting that it has some protective mechanism.Previous studies have found that sphingolipids(sphingomyelin,ceramide,sphingosine,etc.)are closely related to the occurrence and development of liver diseases.Ethanolamine can be converted into CDP-choline,and then combined with ceramide or sphingosine to form sphingomyelin.Therefore,ethanolamine may affect the inflammatory reaction during the formation of fatty liver by participating in the metabolism of sphingolipids.The preliminary study results of this project showed that the contents of ethanolamine and sphingosine in the liver of Landes geese in the overfeeding group were significantly lower than those in the control group,and the geese not only possessed ethanolamine kinases(ETNK1,ETNK2),but also choline kinases(CHKA,CHKB);and more importantly,compared with the normal liver,The mRNA and protein expression of inflammatory response markers TNF-α and MCP-1 in goose fatty liver were significantly inhibited.It is inferred that during the formation of goose fatty liver,CDP-choline promotes the conversion of ceramide and sphingosine to sphingomyelin,reduces the contents of pro-inflammatory factors ceramide and sphingosine in the liver,and thus inhibits the inflammatory response caused by high steatosis.This project firstly obtained goose fatty liver through overfeeding in goose in vivo(non-inflammatory model)to verify the role of ethanolamine in goose fatty liver,and then studied the regulatory effect of ethanolamine on inflammatory response and the mechanism of inhibiting inflammatory response on the cellular level(goose primary hepatocytes and HepG2 cells).Finally,the inhibitory effect of ethanolamine on fatty liver inflammation in mice induced by high fat was further verified.The main research results are as follows:(1)Association between the content of cholamine derivatives and sphingolipids in fatty goose liver and the expression of inflammatory factorsThirty 70-day-old male Landes geese were divided into control group and overfeeding group.Compared with the control group,the body weight,liver weight and abdominal fat weight of Landes geese in overfeeding group were significantly increased(P<0.05).After fluorescence quantitative PCR detection,it was found that the mRNA expression of TNF-αand MCP-1 in liver of Landes geese in overfeeding group were significantly decreased(P<0.05).The content of ceramide in liver of Landes geese decreased,while the contents of sphingomyelin and CDP-choline increased.The results suggest that ethanolamine may participate in sphingolipid metabolism by regulating CDP-choline production and inhibit inflammation during goose fatty liver formation.(2)Ethanolamine can participate in sphingolipid production under the action of CHKA and inhibit the occurrence of inflammation in goose primary hepatocytesThe primary hepatocytes of Landes goose embryos were isolated and cultured for 22 days.The non-alcoholic fatty liver disease model of goose primary hepatocytes was established by palmitic acid and ethanolamine was added into the model cells.The results showed that the expression of inflammatory factors TNF-α and MCP-1 in goose primary hepatocytes were significantly increased by 1 mM palmitic acid(P<0.05),while the mRNA and protein levels of TNF-α and MCP-1 in goose primary hepatocytes were significantly decreased by ethanolamine supplementation(P<0.05).The mRNA and protein expression levels of TNF-α and MCP-1 were not significantly changed(P>0.05)after the addition of ethanolamine after silenced the expression of ethanolamine kinase gene by siRNA.The contents of sphingosine and ceramide in inflammatory cells were significantly increased(P<0.05),the contents of ceramide and sphingosine were significantly decreased by ethanolamine addition(P<0.05),and the content of sphingosine was significantly increased(P<0.05);when the CHKA gene was silenced and ethanolamine was added,the contents of ceramide,sphingosine and sphingosphingomyelin had no significant changes compared with the control group(P<0.05).The results showed that ethanolamine was involved in the synthesis of sphingolipids under the action of CHKA in primary goose hepatocytes,and inhibited the occurrence of non-alcoholic fatty liver disease inflammation.(3)Ethanolamine can participate in sphingolipid production under the action of CHKA and inhibit the occurrence of inflammation in HepG2 cellsThe mRNA expression levels of TNF-α and MCP-1 in HepG2 cells induced by palmitic acid were detected by fluorescence quantitative PCR.The mRNA expressions of TNF-α and MCP-1 in HepG2 cells were significantly increased(P<0.05)with palmitic acid concentrations of 0.125 mM and 0.25 mM,but there were no significant changes(P>0.05)in the mRNA expressions of TNF-α and MCP-1 after ethanolamine supplementation.Compared with palmitic acid+ethanolamine group,when CHKA gene was silted,the contents of ceramide,sphingosine and sphingosphingomyelin in cells were not significantly changed after ethanolamine addition(P>0.05).The results showed that ethanolamine was involved in the synthesis of sphingolipid metabolism under the action of CHKA in HepG2 cells induced by palmitic acid,and inhibited the occurrence of inflammation in HepG2 cells.(4)Adding ethanolamine to high-fat diet can inhibit liver inflammation in mouseMouse were induced by high fat diet to establish non-alcoholic fatty liver disease model,and ethanolamine was added to high fat diet to detect the related indexes.The results showed that compared with the control group,the body weight,liver and abdominal fat weight in high fat group were significantly increased(P<0.05).Compared with the high fat group,the body weight,liver and abdominal fat weight in the three groups supplemented with ethanolamine were significantly decreased(P<0.05).By measuring the blood glucose tolerance of mice,the contents of alanine transaminase and aspartate transaminase and liver sections,it was found that the liver damage of mice in the high-fat group was increased,but no significant liver damage was observed in the three groups supplemented with cholamine.Compared with the control group,the mRNA and protein expressions of TNF-α and MCP-1 in liver tissue of high fat group were significantly increased(P<0.05);compared with the high fat group,the mRNA expression and protein expression of TNF-α and MCP-1 in the 3 groups supplemented with ethanolamine were significantly decreased(P<0.05);compared with the control group,the contents of ceramide and sphingosine in the liver of mice in the high fat group were significantly increased(P<0.05),and the contents of ceramide and CDP-choline were significantly decreased(P<0.05).Compared with the high fat group,the contents of ceramide and sphingosine in the three groups supplemented with ethanolamine were significantly decreased(P<0.05).The contents of ceramide and CDP-choline were significantly increased(P<0.05);Compared with the high fat group,mRNA expressions of CHKA and ETNK1 and ETNK2 genes were significantly increased in the ethanolamine group(P<0.05),while mRNA expressions of ASAH1 and CERS1 genes were significantly decreased(P<0.05).The results showed that adding ethanolamine to high-fat diet in mice could activate the mechanism of ethanolamine participating in sphingolipid metabolism and inhibit the occurrence of inflammation.Ethanolamine is involved in sphingolipid under the action of CHKA,and inhibits the occurrence of inflammation by promoting the conversion of ceramide and sphingosine to sphingolipin.In conclusion,CDP-choline generated by ethanolamine under the action of CHKA in animal body promotes the conversion of ceramide and sphingosine to sphingosine,reduces the contents of ceramide and sphingosine in liver,and inhibits the occurrence of inflammation. |
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