| Objective:To investigate the effect and potential mechanism of asialoglycoprotein receptor 1（ASGR1）in lipid uptake in hepatocytes.Methods:（1）Assay kit was used to detect the serum lipid indexes of Apo E-/-mice fed with standard diet（SD）and high fat diet（HFD）:total cholesterol（TC）,triglyceride（TG）,high-density lipoprotein cholesterol（HDL-C）,low-density lipoprotein cholesterol（LDL-C）.The expression of ASGR1,proprotein convertase subtilisin kexin type 9（PCSK9）,low-density lipoprotein receptor（LDLR）in the liver of Apo E-/-mice fed with HFD was detected by immunohistochemical method.（2）The two kinds of hepatocytes were treated with different concentrations of LDL（0,10,25,50,75μg/m L）.The expression of ASGR1 m RNA and protein were detected by Quantitative Real-time Reverse Transcription-Polymerase Chain Reaction（RT-q PCR）and Western blot.Then 25μg/m L LDL was used to treat the two kinds of hepatocytes for 0 h,6 h,12 h,24 h,48 h.The expression of ASGR1 protein was detected by Western blot.（3）The possible upstream transcription factors of ASGR1 and its binding sites were analyzed and screened by bioinformatics.The expression of m RNA in hepatocytes treated with 25μg/ml LDL was detected by RT-q PCR.（4）ASGR1 si RNA was introduced into hepatocytes,and the expression of ASGR1 in hepatocytes was detected by RT-q PCR and Western blot.Cholesterol accumulation was detected by oil red O test and Boron-dipyrromethene（Bodipy）fluorescence staining after hepatocytes combinately treated with LDL and ASGR1 si RNA.Hepatocytes transfected with ASGR1 si RNA and incubated with Dil-LDL for 4 h,its ability of Dil-LDL uptake was detected by fluorescence microscope.（5）ASGR1 si RNA was transfected into hepatocytes,and Western blot was used to detect the protein expression of LDLR and PCSK9.Results:（1）The results of assay kit test showed that the contents of TC,TG,HDL-C and LDL-C in serum of Apo E-/-mice fed with HFD were higher than those of SD group.The results of immunohistochemistry showed that the expression of ASGR1 and LDLR was increased,while PCSK9 was decreased in the liver of Apo E-/-mice fed with high fat.（2）The m RNA and protein of ASGR1 were upregulated by LDL in a concentration-dependent manner detected by real-time fluorescence quantitative PCR and westernblot.And protein of ASGR1 was increased in time-dependent manner.（3）Candidated 8 transcription factors related to ASGR1 were screened according to the results of bioinformatics analysis.Motif software further analyzed that the proteins of 4transcription factors may have binding sites with ASGR1 promoter sequence.The m RNA levels of these transcription factors did not change significantly after LDL treatment.（4）The expression of m RNA and protein of ASGR1 in hepatocytes was inhibited after ASGR1 si RNA introduction.The results of oil red O staining and Bodipy fluorescence staining showed that the lipid accumulation in hepatocytes in ASGR1 si RNA+LDL group was significantly higher than that in LDL alone.In Dil-LDL experiment,obviously increased uptake of Dil-LDL in hepatocytes in ASGR1 low expression group was observated using fluorescence microscope.（5）ASGR1 inhibition upregulated the level of LDLR protein in LDL-treated hepatocytes,while reduced PCSK9 protein.Conclusion: 1.ASGR1 protein expression was significantly increased in liver of Apo E-/-mice fed with HFD.LDL upregulated the m RNA and protein expression of ASGR1 in hepatocytes.2.ASGR1 inhibition can promote LDL uptake by hepatocytes,and its mechanism may be related to down-regulation of PCSK9 and increase of LDLR expression. |
