| Objective:(1)Understanding the changes of sphingosine-1 phosphate(S1P)in serum of hepatitis B patients and the healthy controls,and explore its correlation with liver function and liver fibrosis indicators.(2)Investigating whether apolipoprotein M(apoM)can regulate the autophagy in LX-2 cells through the transport of S1 P,discussing the relationship between autophagy and the activation,proliferation and migration capacity of LX-2 cells;(3)Exploring the changes of REDD1 in liver fibrosis and the possible molecular mechanism in apoM-S1 P promoting liver fibrosis.Methods:(1)HITACHI 7600-210 automatic biochemical analyzer was used to detect liver function indexes of normal control group and hepatitis B patients.Chemiluminescence methods were used to detect four hepatic fibrosis parameters,CHI3L1 and other hepatic fibrosis serological indices;The serum S1 P concentration of all subjects were detected by liquid chromatography-tandem mass spectrometry.(2)The protein expression levels of MMP9,FN,LC3 Ⅱ,Beclin1,p62 in Control,S1 P,apoM and apoM-S1 P group were detected by WB;The RNA expression levels of MMP9,FN,were detected by q PCR;The autophagic lysosomes in LX-2 cells were observed by electron microscopy;The dynamic changes of autophagy flow in LX-2 cells were observed by confocal laser microscopy;WB and q PCR were used to detect the protein expression levels of LC3 Ⅱ,Beclin1,p62,MMP9 and FN in the Control group,S1 P group,apoM group and apoM-S1 P group without the autophagy inhibitor 3-MA.The proliferation and migration of LX-2 cells were detected by Ed U assay and cell scratch assay respectively;(3)WB and q PCR were used to detect the expression of REDD1 in Control group,S1 P group,apoM group and apoM-S1 P group.WB was used to detect the protein expression levels of REDD1,m TORC1,LC3 Ⅱ,Beclin1,p62,MMP9 and FN in Control group,S1 P group,apoM group and apoM-S1 P group after REDD1 was silence;(4)SPSS 25.0 software was used for statistical analysis of the experimental results.One-way ANOVA and Kruskal Wallis and other test methods were used to compare the results of this study among multiple groups.Comparison between the two groups was analyzed by t test.Data were expressed as mean(interquartile spacing),mean ±standard deviation(mean ± SD)and mean ± standard error(mean ±SEM).Two-tailed,p<0.05 were considered statistically significant.Results:(1)The serum S1 P concentration of HBs Ag(+)/Anti-HBe(+)/Anti-HBc(+)patients were higher than that of the healthy controls which was statistically significant,while the serum S1 P concentration of HBs Ag(+)/HBe Ag(+)/Anti-HBc(+)patients were higher than that of the healthy controls,but there was no statistical significance;(2)Serum S1 P concentration was not correlated with the liver function indices,but was positively correlated with the liver fibrosis indices of C IV and CHI3L1;(3)apoM-S1 P regulates the expression of autophagy-related proteins and induces the formation of autophagosomes and autophagolysosomes in LX-2 to activate autophagy.(4)Comparing with DMSO+apoM-S1 P treatment,the3-MA+apoM-S1 P treatment showed a weakened effect on the level of autophagy,and further decreased the activation,proliferation and migration of LX-2 cells;(5)REDD1 was elevated in liver tissue from patients with liver fibrosis and in activated LX-2 cells induced by apoM-S1P;(6)Comparing with si NC+apoM-S1 P treatment,si REDD1+apoMS1 P plays a role in up-regulation of the phosphorylation of m TORC1 and down-regulation of the level of autophagy and activation in LX-2 cells.Conclusions:(1)Serum S1 P concentration in HBs Ag(+)/Anti-HBe(+)/Anti-HBc(+)patients increased compared with that of the control group,and positively correlated with C IV and CHI3L1 of liver fibrosis markers;(2)apoM-S1 P promotes the activation,proliferation and migration of LX-2 cells by activating the level of autophagy;apoM-S1 P promotes liver fibrosis by activating the level of autophagy in LX-2 cells via REDD1/m TORC1 pathway. |
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