Recombinant Humanized IgG1 Maintain Liver Triglyceride Homeostasis In Apoe-/- Mice Through Arylacetamide Deacetylase And Its Molecular Mechanism

Hyperlipidemia is a common clinical dyslipidmia characterized by elevated plasma triglyceride(TG)and/or cholesterol.The liver is a pivotal organ for maintaining lipid metabolism homeostasis,and excessive lipid deposition in the liver can lead to non-alcoholic fatty liver disease(NAFLD).NAFLD is the most common chronic liver disease.There is still no specific drug for the treatment of NAFLD.The main treatment methods are weight loss and diet adjustment.In our previous studies,we used the plasma of patients with atherosclerosis to screen the dodecapeptide library,and established a library of human single-chain variable fragments(scFvs)by phage display technology.The scFv was recombined into pcDNA3 vector to express human IgGl(hereafter referred to as 14 mAb).Previous results showed that 14 mAb reduced atherosclerotic plaque area by approximately 40%in high-fat diet Apoe-/-mice.14 mAb can directly act on hepatocytes to promote reverse cholesterol transport and bile acid synthesis and excretion.This project intends to further clarify the effect of 14 mAb on the liver on the basis of previous research,and to explore the regulatory effect and regulatory mechanism of 14 mAb on liver lipid metabolism.Apoe-/-mice were fed with high fat for 20 weeks to induce hyperlipidemia.The contents of serum TG,low density lipoprotein cholesterol(LDL-C),high density lipoprotein cholesterol(HDL-C),total cholesterol(TC)and TG content in mouse liver were detected.HE staining and Oil Red O staining were performed on mouse liver tissue.RNA sequencing(RNA-Seq)of mouse liver was performed to analyze key differential genes between groups.In vitro experiments,the primary mouse hepatocytes steatosis model was constructed with palmitic acid and oleic acid(2:1).The expressions of Aadac and FcRn were knocked down by siRNA,and the mRNA and protein expression levels of Aadac,Foxal,p-PKCδ and FcRn were detected by quantitative real-time PCR(qPCR)and Western Blot.The Aadac luciferase reporter gene was constructed to analyze the transcriptional regulation of Aadac through truncation and sites mutation.The transcriptional regulation effect of transcription factor Foxal on Aadac was detected by chromatin immunoprecipitation(ChIP).The results showed that 14 mAb could significantly reduce serum LDL-C and TG levels in high-fat fed Apoe-/-mice,and significantly reduce liver TG content in mice.Mouse liver RNA-Seq results suggested that 14 mAb could significantly increase the expression of Aadac.In vitro experiments showed that 14 mAb could significantly restore the FFA-induced decrease in the expression of Aadac in primary mouse hepatocytes and reduce the deposition of lipid droplets in the cells caused by FFA.Aadac siRNA could significantly reverse this effect.The results of the luciferase reporter gene and ChIP-PCR indicated that the Foxal response element at-182/-172bp on the Aadac promoter plays a key role in the regulation of Aadac expression by 14 mAb.Meanwhile,14 mAb inhibited the phosphorylation of PKCδand increased the expression of Foxal.Knockdown of neonatal Fc receptor(FcRn)in mouse primary hepatocytes reversed the 14 mAb-induced decrease in PKCδphosphorylation and increased Foxal and Aadac expressions.This study confirmed that 14 mAb maintains TG homeostasis in mouse liver through Aadac,and its possible mechanism may be to regulate the expression of Aadac in hepatocytes through the FcRn/PKCδ/Foxal pathway.