Abstract:Aim To explore the mechanism of adipophilin regulating neutral cholesterol ester hydrolase (nCEH) expression thus mediating lipid accumulation in RAW264.7 macrophages. Methods The constructed retroviral plasmid vectors with adipophilin of no expression and over expression were transfected into package cell PA317, then collected virus solution was used to infect RAW264.7 macrophages by screening and identifying to obtain RAW264.7 cell lines with adipophilin stably silenced and highly expressed. The mRNA and protein expression levels of nCEH were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. High performance liquid chromatography (HPLC) was taken to detect the contents of intracellular cholesterol ester. Results The cholesterol and cholesterol ester levels were significantly higher in adipophilin over-expression group than in the control group (P<0.01), while dramatically lower than that in the control group when adipophilin was supressed (P<0.01). In addition, adipophilin inhibited nCEH expression. Furthermore, in RAW264.7 macrophages with high adipophilin expression, treatment with 100 nmol/L protein kinase Cδ agonist PMA for 30 min resulted in remarkable reductions of nCEH mRNA and protein expression(P<0.05), on the contrary, treatment with 100 nmol/L PKCδ inhibitor Rottlerin for 30 min increased the expression of nCEH(P<0.05). Conclusion Adipophilin may exert influence over lipid accumulation by inhibiting the expression of nCEH in RAW264.7 macrophages, and PKCδ played an important role in this regulatory mechanism.

Abstract:Aim To explore whether adipophilin interact with acyl-coenzyme A:cholesterol acyltransferse 1 （ACAT1） and neutral cholesterol ester hydrolase （NCEH） in lipid-loaded RAW264.7 cell induced by oxidized low density lipoprotein （ox-LDL）.Methods RAW264.7 cells were incubated with 50 mg/L ox-LDL for different time.The expression of mRNA and proteins of adipophilin,ACAT1 and NCEH were detected by semi-quantitative reverse transcription-polymerase chain reaction and western blot respectively.Interactions between adipophilin and ACAT1 or NCEH were detected by co-immunoprecipitation.Results As the incubation time of ox-LDL was extended in RAW264.7 cells,the expression of mRNA and proteins of adipophilin,ACAT1 and NCEH were significantly increased compared with 0 h group （P<0.05,n＝3）.Co-immunoprecipitation showed that there were interactions between adipophilin and ACAT1 in RAW264.7 macrophages with ox-LDL treatment at 0,0.5,1 h and 3 h,and no interactions at 6 h.There were not interactions between adipophilin and NCEH in RAW264.7 macrophages with ox-LDL treatment at 0,0.5 h and 1 h,and interactions at 3 h and 6 h.Conclusions There were interactions between adipophilin and ACAT1 or NCEH in RAW264.7 macrophages with ox-LDL treatment.It suggests that adipophilin may have synergistic effects with ACAT1 and NCEH in lipid-loaded RAW264.7 cell.

Abstract:Adipophilin, as a lipid droplet-associated protein, is a specific marker in the accumulation of lipid, the overexpression of Adipophilin can promote the accumulation of lipids, which is relevant to the pathological process of atherosclerosis and insulin resistance. Neutral cholesterol ester hydrolase （NCEH） is a microsomal enzyme and richly expressed in human atheromatous lesions, can hydrolyze cholesterol ester （CE） in a neutral pH environment, and plays a rate-limiting role in the reverse transportation of cholesterol esters. Adipophilin and NCEH plays an opposite role in maintaining the dynamic balance between the formation and hydrolysis of lipid drops. Under the pathological state, the dynamic balance between Adipophilin and NCEH is broken, thus the accumulation of cholesterol ester is promoted, and the occurrence and development of atherosclerosis is further accelerated. However, the mechanism of Adipophilin and NCEH in the formation and hydrolysis of lipid drops, and their role in the occurrence and development of atherosclerosis, is still unclear. So, this paper is reviewed about this issue.