The Mechanism And Significance Of Abnormal Hemolytic Lecithin Metabolism In The Course Of Liver Cancer

Background and objectivesLiver is an important metabolic organ and has important physiological functions in human bodyto participate in biological transformation.In various metabolic pathways,liver metabolism involves a variety of biomolecules to generate different small molecules.Liver metabolic function must be affected during the process of normal liver-cirrhosis-liver cancer.Liver function indicators often include alpha-fetoprotein,ALT,AST,TBIL,GGT,AKP,and these indicators are often negative in the development of early liver disease,but once they are positive liver diseases often turn into cancer at late stage.Therefore,it is urgent to search for early biomarkers and sensitive detection methods.Lipid metabolism is an important physiological function of liver.Cholesterol can be metabolized to produce bile acids,and the bile acid metabolism is abnormal when liver diseases appeared.In addition,when the ratioes of cholesterol to bile acid and to lecithin increase,cholesterol is precipitated to form gallstones due to satiation,and lysolecithin is produced from lecithin under the action of phospholipase A.Therefore,bile acids,hemolytic lecithin and unsaturated fatty acids could be considered the core metabolites.To quantitatively determine the levels of these core metabolites may reflect the development of liver disease stage.However,these substances are easily and quickly metabolized and the routine detection method is not sensitive,so the clinical study has not examined the concentration of these substances as a study of liver disease development stage.In this thesis,we have developed a method using quantitative determination of the concentration of these metabolites in blood and urine by UPLC-MS / MS mass spectrometry.It is rapid,convenient and accurate,and possibly provides evidence for evaluating the development stage of liver diseases.Many studies have found that lyso-phosphatidylcholine(LPC)has abnormal changes during the process from normal liver to the development of cancer,but its mechanism and its relationship with the occurrence of liver cancer are still unclear.Phosphatidylcholine(PC)is generated from LPC under the action of lyso-phosphatidylcholine acyltransferase 1(LPCAT1),and further catalyzed to generate LPC and polyunsaturated fatty acids under the action of phospholipase A2(PLA2),and to generate LPC and cholesterol ester under the action of lecithin cholesterol acyl transferase(LCAT).LPC is an important link of these metabolic pathways,so we will combine LPCAT1,LCAT,PLA2 with LPC to find the reasons for abnormal LPC.In addition,in the upstream pathways of these enzymes,there are involved in AKT,ERK,JNK and other pathways.We also do the relevant experiments to verify the relationship between these pathways and LPC.ELISA,immunohistochemistry,Western blot and mass spectrometry are used to explore the reasons for abnormal LPC,and we hope to explore whether it is related to the occurrence and development of liver cancer.Research methods1.Collection of blood and urine specimens from patients with different liver diseases:the blood and urine samples were collected from thehepatitis,cirrhosis,liver cancer patients in the Eastern Hepatobiliary Surgery Hospital from 2013 to 2016,and stored in the-80 degrees.The blood and urine samples from healthy controls were synchronously collected.2.Methods of serum mass spectrometry in patients with different liver diseases:Blood and urine samples from normal,hepatitis,cirrhotic,hepatocellular carcinoma patients were examined at the mass spectrometry analysis center of Shanghai Jiaotong University to explore the stability and sensibility under different storage conditions and the effects of different detection methods.3.Protease levels in serum samples detected by ELISA:the sera from normal,liver cirrhosis and liver cancer patients in Eastern Hepatobiliary Surgery Hospital were used to detect the levels of LCAT,LPCAT1,sPLA2-ⅡA,cPLA2α.4.Immunohistochemical detection of related protease markers: The liver normal tissues,liver cancer tissues and adjacent cirrhosis tissues,liver cancer tissues and adjacent hepatitis tissues were paraffin-embedded and the sections were detected by immunohistochemistry for the expression of LPCAT1,LCAT,cPLA2α,SPLA2-ⅡA.5.Expression changes of related proteins in various cell lines: The normal cells of L02 and WRL-68,the hepatic stellate cell line LX2,the hepatoma cell lines Hep-3B,HepG2,Huh-7,QGY-7701,SMMC-7721,HCC-LM3 were cultured and extracted total protein.The expression of LPCAT1,LCAT,cPLA2α and sPLA2-ⅡA,and their related upstream substances of AKT,ERK and JNK were detected by Western blot in cell lines.6.Mass spectrometry of intracellular and extracellular LPC: Mass spectrometry method was used to quantitatively detect the changes of LPC in the cultured cells and culture media,and the results were compared with the expression of LPC by western blot.Research results1.Mass spectrometry methodology: The UPLC-MS/MS was used to detect the changes of lysolecithin,bile acid,unsaturated fatty acid in blood and urine.The method is rapid,sensitive and easy to operate.We compared the differences of metabolites in plasma and serum and found that the plasma more closes to the actual state of the whole blood.Therefore,our research suggests that plasma is the better as biological samples for detection of the metabolites.2.Comparison of the expression of LPCAT1,LCAT,PLA2 in immunohistochemistry and ELISA: Detection of LPCAT1,LCAT,cPLA2 in clinical specimens found that these biomarkers play an important role in the abnormal changes of LPC in process of liver disease.The difference of SPLA2-Ⅱ A expression in cancer and adjacent tissues is not obvious.3.Analysis of the results from mass spectrometry and Western blot in cell lines: The expression of LCAT,LPCAT1,cPLA2 α and SPLA2 were analyzed by cytology.The results showed that the changes of LCAT content in cell culture medium affected the change of LPC in the course of liver disease and changes of LPCAT1 content in the course of liver disease affect the changes of LPC content.The trend of cPLA2 α and its phosphorylation was opposite to that of LPC.4.Other protein which associated with LPC-related upstream pathways : The expression of total AKT,P-AKT,total ERK,P-ERK and JNK protein was significantly different from that of intracellular LPC,but P-JNK and P-P38 were correlated with LPC.ConclusionsIn this study,we used UPLC-MS / MS to quantitatively detect the changes of core metabolites in blood and urine from patients with liver diseases,and found the method is generally fast,convenient,high sensitivity,and that plasma is better than sera for determination of the core metabolites.To investigate the reasons of LPC changes in patients with liver diseases,we concluded that LPCAT1,LCAT,cPLA2α are closely related to the changes of LPC.We thought that the changes of LPC content in LCAT-dominated extracellular(serum and culture medium)in the course of liver disease and LPCAT1-mediated intracellular(tissue)LPC content.CPLA2α,P-cPLA2α,sPLA2-ⅡA had little effect on the abnormal changes of LPC in the process of liver disease.This mechanism of LPC changes dominated by different enzymes inside and outside the cell needs further exploration.In addition,P38,AKT,P-AKT,ERK,P-ERK,JNK and LPC are not related to the LPC changes,P-P38 and P-JNK have certain correlation with it.This also needs further exploration.