Study On The Role Of ApoM Gene In The Formation Of Non-alcoholic Fatty Liver Disease

Objective:The mouse and cell model of ApoM gene knockout was established to observe the pathological morphological changes of liver tissue and liver cells after the knockout of ApoM gene,and then explore the possible molecular mechanism of these changes.Methods:Knockout ApoM gene with CRISPR/Cas9 method,builds systemic ApoM gene knockout mice model and monoclonal liver cell line model.Using transmission electron microscopy,HE staining,oil red O staining,DAPI staining,FILIPIN total free cholesterol fluorescence staining,blood biochemical index detection,antibody microarrays and other techniques and methods to observe the pathological changes of liver tissue after ApoM gene deletion and the morphological changes of liver cells,as well as the changes of related blood lipid biochemical indexes.At the same time,RT-PCR and Western blot methods were used to detect the expression changes of the key proteins in the RNA and protein levels,which were closely related to lipid metabolism,inflammation and apoptosis,and then discussed the possible molecular mechanism of ApoM gene deletion in liver tissue and hepatocyte pathological morphological changes.Results:1.Successfully constructed the systemic ApoM gene knockout mice model and ApoM gene knockout monoclonal liver cell model.2.ApoM^-/-mouse liver tissue has a mild non-alcoholic fatty lesion,and the electron microscopy showed that the lipid cells in the liver increased and were accompanied by lipid droplets of varying sizes.Oil red O staining and totally free cholesterol FILIPIN fluorescence staining showed a mild fat accumulation in the liver.At the same time,ApoM^-/-monoclonal hepatocyte oil red O staining also showed the same pathological changes,with lipid accumulation in the liver cells and a mild fatty change.ApoM^-/-mouse liver tissue electron microscopy showed mitochondrial swelling,and DAPI staining of monoclonal liver cells showed an increase in cell nucleus,and the results indicated that there was a possibility of apoptosis.In addition to ApoM^-/-mouse liver tissue HE staining showed inflammatory cell infiltration.Blood lipid and biochemical indicator detection results show ApoM^-/-mice with TG and LDL-C in the blood higher level compared with the control of wild mice（P<0.05）,in which the TG has a significant difference（P<0.01）.However,there was no significant difference between TC and HDL-C level compared with the control of wild mice（P>0.05）.There was no statistical difference between TG,TC,HDL-C and LDL-C level in liver tissue homogenation（P>0.05）.3.The key factors involved in lipid metabolism in the liver tissues of ApoM^-/-mice were detected from both mRNA level and protein level,and the results showed that LDLR,PPARα,PPARγ,LXRα,FASN,ABCA1 and SREBP2 were both higher in mRNA levels and protein levels than in control wild-type mice（P<0.05）.The expression of ACACA at the mRNA level was significantly lower than that for the control of wild-type mice（P<0.01）,while the expression of the protein level was higher than that for the control of wild-type mice（P<0.05）.The expression of SREBP1 at the mRNA level was not significant（P>0.05）,while the expression of the protein level was significantly higher than that for the control of wild-type mice（P<0.01）.The expression of PCSK9 in protein level was not significant compared with that of control wild-type mice（P>0.05）.The protein levels of the key factors involved in lipid metabolism in ApoM^-/-monoclonal liver cells were detected,and the results showed that LDLR,PPARα,PPARγ,LXRα,FASN,ABCA1,ACACA,SREBP1,and SREBP2 were all higher in protein levels than in the control group（P<0.05）,which was consistent with the detection results of liver tissue protein levels.The above results suggest that after the ApoM gene knockout,the expression of key proteins in the lipid metabolism pathway can be changed in both the cellular level and the overall level.4.Use ApoM^-/-mice liver tissue and ApoM^-/-monoclonal liver cell model at the protein level of the three classic way to participate in lipid metabolism regulation the key regulation factor in testing,the results show phospho-Akt473,phospho-Akt308,phospho-mTOR and phospho-p70S6K1,phospho-ERK1/2 and Scap protein expression level increased compared with control group,with significant difference（P<0.05）,and phospho-AMPKαand S1P protein expression level decreased,compared with controls with significant difference（P<0.05）.The protein expression levels of Akt,mTOR,ERK 1/2,AMPKαand p70S6K1 were not significantly different from those in the control group（P>0.05）.5.The infiltration of hepatic inflammatory cells in ApoM^-/-mice was significant,inflammatory factor test results show that:the content of the TNF-α,IL-1βand IL-6 was higher than that of the control wild-type mice（P<0.05）,and inflammation control way of the key protein phospho-NF-κBp65,phospho-IRE1α,phospho-IκBαand TNFR1 protein expression levels were significantly higher than that in the control wild-type mice（P<0.05）.Protein expression levels of IκBαand IRE1αwere lower than those of the control of wild-type mice（P<0.05）.There was no significant difference in the protein expression level of NF-κB compared with wild-type mice（P>0.05）.The above indicators were detected in ApoM^-/-monoclonal hepatocytes and found different indicators from those in ApoM^-/-mice liver tissues was that the protein expression levels of IκBαand IRE1αwere not significantly different from controls（P>0.05）.The remaining test results are consistent with the mouse liver level.6.The liver mitochondria of mice were significantly swollen.Apoptotic antibody microarray analysis showed that the expression levels of seven genes changed significantly（P<0.05）.They were:phosphor-S6 Ribosomal Protein（Ser235/236）,phospho-p53（Ser15）and phospho-Bad（Ser112）are up-regulated,phospho-mTOR（Ser2448）,phospho-Erk1/2（Thr202/Tyr204）,phospho-HSP27（Ser78）and phospho-SAPK/JNK（Thr183/Tyr185）are down-regulated.7.ApoM^-/-mouse liver mitochondria-mediated apoptosis pathway phospho-Bad,Bcl-xL,Bax,cleaved-Caspase9,pro-Caspase9,cleaved-Caspase3,pro-Caspase3 and phospho-p53 of which Western blot analysis showed that the expression level was higher than that of the control wild-type mice（P<0.05）,while the protein expression of Bcl-2 was lower than that of the control wild-type mice（P<0.05）.Protein expression of Bad had no significant difference from the wild-type mice（P>0.05）.The above indicators were detected in ApoM^-/-monoclonal hepatocytes and found different indicator from those in ApoM^-/-mice liver tissues were that the protein expression levels of Bcl-x L were lower than that of the control group（P<0.05）.The remaining test results are consistent with the mouse liver level.8.ApoM^-/-mouse liver endoplasmic reticulum stress-mediated pathway cleaved-Caspase3,pro-Caspase3,CHOP,phospho-PERK,phospho-eiF2α,phospho-IRE1αand phospho-JNK of which Western blot analysis showed that the expression level was higher than that of the control wild-type mice（P<0.05）,while the protein expression of IRE1αwas lower than that of the control wild-type mice（P<0.05）.Protein expression of eiF2αand PERK had no significant difference from the wild-type mice（P>0.05）.The above indicators were detected in ApoM^-/-monoclonal hepatocytes and found different indicator from those in ApoM^-/-mice liver tissues were that the protein expression levels of eiF2αwere higher than that of the controls（P<0.05）.The remaining test results are consistent with the mouse liver level.Conclusion:1.The deletion of ApoM gene can cause pathological changes in non-alcoholic fatty liver disease such as fat-storing cells enlarged fat accumulation,swelling of mitochondria,increase of the nuclei,and infiltration of inflammatory cells in hepatic tissue.2.The ApoM gene may induce the occurrence of nonalcoholic fatty liver disease by affecting expression changes that are closely related to lipid metabolism,inflammation and apoptosis,as well as regulation of key proteins on pathways.3.ApoM gene may be through the PPARs/LXRs/SREBPs way affects lipid metabolism,by TNF-α/NF-κB and IKKβ/NF-κB pathway activation of inflammation,mitochondria and endoplasmic reticulum stress affected by apoptosis.