柳 軍，王 雪，尚 靖，穆冬姸

中國(guó)藥科大學(xué)新藥篩選中心，南京210009

山楂酸減少L02細(xì)胞脂質(zhì)累積作用的研究

柳 軍*，王 雪，尚 靖，穆冬姸

中國(guó)藥科大學(xué)新藥篩選中心，南京210009

非酒精性脂肪性肝在發(fā)達(dá)國(guó)家和發(fā)展中國(guó)家中是一種常見的肝臟疾病。引起非酒精性脂肪性肝病的最常見原因有肥胖、糖尿病和高膽固醇。盡管非酒精性脂肪性肝病的發(fā)病率日益升高，事實(shí)上至今尚未發(fā)現(xiàn)可以用于治療的藥物。山楂酸是一種五環(huán)三萜化合物，已報(bào)道具有多種藥理特性，包括抗炎、抗氧化以及免疫調(diào)節(jié)作用。我們的前期研究表明，山楂酸能夠抑制高脂飼料誘導(dǎo)大鼠非酒精性脂肪性肝病的生成。本研究中，我們將探討山楂酸體外對(duì)肝細(xì)胞(LO2)脂質(zhì)累積的抑制作用。結(jié)果表明，山楂酸可抑制游離脂肪酸(FFA)誘導(dǎo)的LO2細(xì)胞脂質(zhì)累積，進(jìn)一步研究表明，山楂酸可抑制LO2細(xì)胞的SCAP mRNA水平和蛋白水平的表達(dá)。

山楂酸;肝細(xì)胞脂肪變性;LO2細(xì)胞;固醇調(diào)節(jié)元件結(jié)合蛋白裂解激活蛋白

Introduction

Non-alcoholic fatty liver disease(NAFLD)is fatty inflammation of the liver when this is not due to excessive alcohol use.It is reported that NAFLD relates to insulin resistance and the metabolic syndrome，and may respond to treatments originally developed for other insulin resistant states，such as weight loss，metformin and thiazolidinediones［1］. Non-alcoholic steatohepatitis (NASH)is the most extreme form of NAFLD，which is regarded as a major cause of cryptogenic cirrhosis of the liver［2］.

MA(Fig.1)occurs naturally in a wide variety of plant species such as Leguminosae，Boraginaceae and Asteraceae.We have previous reported that MA is capable of lowering serum glucose and preventing high fat diet induced fatty liver in rats［3］.However，the molecular/ cellular mechanisms of MA on lipid metabolism remain unknown.

Sterol regulatory element binding protein cleavage activating protein(SCAP)is regarded as an important regulator in synthesis and absorbance of lipid［4］，and playing a critical role in regulating the balance of lipid metabolism.

In this study，steatosis models of hepatocytes were established by adding free fatty acids(FFA，oleate/palmitate，2∶1)to the growing L02 cell.The lipid droplets in the hepatocytes were observed with oil red staining and the contents of triglyceride in hepatocytes were measured with analyzed kit.The mRNA and protein levels of SCAP were measured as well.The over expression of SCAP in model cell groups could lead to disturbance of lipid metabolism and probably participate the process of steatosis of hepatocytes.Treatment with MA showed that MA can decrease effectively lipids accumulation in FFA-induced L02 cells.Furthermore，MA can regulate the lipid metabolism through regulating the mRNA and protein expression of SCAP in L02 cells.

Fig.1 The structure of MA

Materials and Methods

Cell culture

Human normal liver L02 cells(Cell Bank，Type Culture Collection of Chinese Academy of Sciences)were cultured in RPMI1640 supplemented with 10% heat inactivated FBS，100 U/mL penicillin，100(g/mL streptomycin，and 2 mM glutamine.Cells were incubated at 37℃ in a humidified atmosphere(5%CO2).FFA(oleate/palmitate，2∶1)were mixed with FFA-free BSA and added into medium to a final concentration of 1 mM.

Chemicals and reagents

Roswell Park Memorial Institute 1640(RPMI-1640)，fetal bovine serum(FBS)，horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from GIBCO-BRL.Rabbit monoclonal antibody against actin was from Sigma.Nitrocellulose membranes (Hybond)，and an enhanced chemiluminescence detection system were all obtained from Amersham Pharmacia.DNA Taq polymerase was obtained from Fermentas(Shanghai，China)，M-MLV reversetranscriptase was obtained from Promega(Madison，WI).TRIzol reagent was obtained from Gibco(Gaithersburg，MD，USA).All other reagents unless indicated were from Sigma Chemical Co. MA was obtained by semi-synthesis starting from readily available oleanolic acid as previously described.［5，6］MA was stored at room temperature until use.

Oil red O staining

Oil red O staining was used specifically for lipid droplet accumulation.L02 cells were stained by the Oil Red O method.［7］Cells were treated with 1 mM of FFA together with MA(1 μM and 10 μM)for 24 h.After treatments，cells were washed three times with iced PBS and fixed with 10%formalin for 60 min.After fixation，cells were washed and stained with Oil Red O solution (stock solution，3 mg/mL in isopropanol;working solution，60%Oil Red O stock solution and 40%distilled water)for 60 min at room temperature.After staining，cells were washed with water to remove unbound dye.Quantitative analysis of Oil Red O content levels，isopropanol was added to each sample，and shaken at room temperature for 5 min.The samples were read spectrophotometrically at 510 nm by Tecan reader (Megallan Company，Swiss).

SCAP gene analysis with RT-PCR

L02 cells were treated with 1mM FFA together with various concentrations of MA for 24 h.Total RNA of each group was extracted from L02 cells with homogenization in TRIzol reagent(Gibco，Maryland，USA)according to the manufacturer＇s instructions.Reverse transcription and cDNA-PCR process were done according to general RT-PCR method.PCR amplification was carried out in the thermal cycler using a protocol of initial denaturing step at 95℃ for 10 min;followed 35 cycles of 95℃for 1 min，60℃ for 40 s and 72℃ for 40 s;and final cycle of 72℃ for 10 min.The PCR products were run on 2%agarose gel in 1×TBE buffer and measured as height value by using Quantity One main program (BIO-RAD，USA).Sequences of the deoxyribonucleotide for PCR are as table 1.

Table 1 Oligonucleotide sequences of primers

Western blot analysis

Total cell lysates were prepared and processed as described previously［8］.The expression levels of SCAP were detected with specific antibodies.(-actin was used as loading control.The densities of immunoblot bands were analyzed using Quantity One main program (BIO-RAD，USA).

Statistical analysis

All values are expressed as means±standard error of the mean(SEM).Student's t-test was used to determine the significance of differences in multiple comparisons.Values of P＜0.05 were considered statistically significant.

Results

Fig.2 Effects of MA on intracellular lipid accumulation in L02 cells

MA inhibit L02 cellular lipid accumulation

Previous studies have suggested that MA have hypolipidemia effects in high fat diet induced fatty liver rats［3］.To evaluate the hypolipidemia effects in L02 cells，1 mM FFA(oleate/palmitate，2∶1)was used in the present study.We examined whether MA was able to prevent FFA-induced lipid accumulation in L02 cells using oil red O staining.As shown in Fig.2，the intracellular lipid content could be reduced significantly by treatment with MA.

Gene Expression of SCAP

A single transcript(257 bp)was observed in all groups.RT-PCR analysis of SCAP showed a significant increased in FFA-overloaded cells，while the expression of SCAP mRNA in MA-treated cells was decreased when compared with model cells.(Fig.3)

Fig.3 Effect of MA on the mRNA levels of SCAP in L02 cells

MA inhibit the protein levels of SCAP in L02 cells Compared with the initial level，L02 cells already had increased levelsof SCAP after 24h at highFFA conditions.However，these levels reduced undercoincubation with MA.(Fig.4)

Fig.4 Effect of MA on SCAP expression in L02 cells

Discussions

The causes ofNon-alcoholic fatty liver disease (NAFLD)remain unknown.However，it is related to insulin resistance and the metabolic syndrome［10］.Recent studies have shown that NAFLD develops in a relatively short period of irreversible liver damage;some patients can progress to cirrhosis of the liver［11］.

SCAP is regarded as an important regulator in synthesis and absorbance of lipid［4］.Sterol regulatory element binding proteins(SREBPs)are a family of membranebound transcription factors that control the synthesis of cholesterol and fatty acids in animal cells［12］.SCAP activates SREBP，and initiates the release of NH2-terminal fragments from cell membranes;the liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids［13］.This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors(statins)and high calorie diets(insulin).

FFA plays an important role in the pathogenesis of NAFLD.As a part of our studies that we have reported that MA，as a natural product to be an agent for lowing blood glucose and improvement insulin resistance and has therapeutic effect on fatty liver as we reported previously［8，9］.Whether MA has any beneficial effects on FFA-induced hepatic toxicity remains to be characterized.In this study，we found that the mRNA expression levels and protein expression levels of SCAP increased after adding FFA on cultured liver cells for 24 h.The model group cells increased steatosis，and total lipid，triglyceride and cholesterol content increased significantly.Oil red O staining of liver cells shows that fatty degeneration also increased.After treating with MA，the lipid accumulation will decrease(Fig.1).The present study provided the first evidence for the protective effect of MA on FFA-induced lipotoxicity in L02 cells.MA not only reduced FFA-induced lipid accumulation，but also significantly inhibited SCAP expression(Fig.3 and 4).In summary，MA inhibits the lipid accumulation in L02 cells and maybe as a natural and effect agent in treating NAFLD.

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November 25，2011;Accepted February 28，2012

This work was supported by the Fundamental Research Funds for the Central Universities(No.JKP2011004)

Effect of Maslinic Acid on Attenuating Lipid Accumulation in L02 Cell

LIU Jun*，WANG Xue，SHANG Jing，MU Dong-yan
National Drug Screening Center，China Pharmaceutical University，Nanjing 210009，China

Non-alcoholic fatty liver disease(NAFLD)is the most common liver disorder both in western industrialized countries and in developing countries.The most common causes of NAFLD are obesity，diabetes，and high cholesterol levels.Despite the increasing prevalence of NAFLD，the therapeutic agents are remained to be discovered.Maslinic acid (MA)has a variety of pharmacological properties including anti-inflammatory，antioxidant，and immune-modulating activities.In our previous study，we have reported that MA has protected NAFLD effect in the high-cholesterol induced hyperlipidemia rats.In the present study，we examined the effects of MA on attenuating lipid accumulation in human normal liver L02 cells.The study showed that MA can prevent free fatty acid(FFA)induced lipid accumulation in L02 cells.Furthermore，MA can suppress the mRNA expression and protein levels of SCAP induced by FFA in L02 cells.

maslinic acid;steatosis hepatocyte;L02 cell;SCAP

1001-6880(2012)10-1355-04

*Corresponding author Tel:86-25-83271043;E-mail:junliu@cpu.edu.cn

R966

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