亚洲免费av电影一区二区三区,日韩爱爱视频,51精品视频一区二区三区,91视频爱爱,日韩欧美在线播放视频,中文字幕少妇AV,亚洲电影中文字幕,久久久久亚洲av成人网址,久久综合视频网站,国产在线不卡免费播放

        ?

        山楂酸減少L02細(xì)胞脂質(zhì)累積作用的研究

        2012-11-23 16:24:32穆冬姸
        天然產(chǎn)物研究與開發(fā) 2012年10期
        關(guān)鍵詞:可抑制藥科脂肪性

        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.

        1 Farrell GC,Larter CZ.Nonalcoholic fatty liver disease:from steatosis to cirrhosis.Hepatology,2006,43(Suppl 1):S99-S112.

        2 Hai S,Kubo S,Shuto T,et al.Hepatocellular carcinoma arising from nonalcoholic steatohepatitis:report of two cases.Surg Today,2006,36:390-394.

        3 Liu J,Sun HB,Wang XF,et al.Effects of oleanolic acid and maslinic acid on hyperlipidemia.Drug Develop Res,2007,68: 261-266.

        4 Salek L,Lutucuta S,Ballantyne CM,et al.Effect s of SREBF-1a and SCAP polymorphisms on plasma levels of lipids,severity,progression and regression of coronary atherosclerosis and response to therapy with fluvastatin.J Mol Med,2002,80:737-744.

        5 Wen XA,Sun HB,Liu J,et al.Pentacyclic triterpenes.Part 1:The first examples of naturally occurring pentacyclic triterpenes as a new class of inhibitors of glycogen phosphorylases.Bioorg Med Chem Lett,2005,15:4944-4948.

        6 Wen XA,Zhang P,Liu J,et al.Pentacyclic triterpenes.part 2:Synthesis and biological activity of maslinic acid derivatives as inhibitors of glycogen phosphorylase.Bioorg Med Chem Lett,2006,16:722-726.

        7 Hwang JT,Park IJ,Shin JI,et al.Genistein,EGCG,and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase.Biochem Biophys Res Commun,2005,338:94-699.

        8 Gao Y,F(xiàn)eng HC,Walder K,et al.Regulation of the selenoprotein SelS by glucose deprivation and endoplasmic reticulum stress—SEPS1 is a novel glucose-regulated protein.FEBS Lett,2004,563:185-190.

        9 Bligh EG,Dyer WJ.A rapid method of total lipid extraction and purification.Can J Biochem Physiol,1959,37:911-917.

        10 Adams LA,Angulo P.Treatment of non-alcoholic fatty liver disease.Postgrad Med J,2006,82:315-322.

        11 Cassiman D,Jaeken J.NASH may be trash.Gut,2008,57: 141-144.

        12 Brown MS,Goldstein JL.Proc Natl Acad Sci USA,1999,96: 11041-11048.

        13 Marc IS.Pharmacological regulation of low density lipoprotein receptor expression:current status and future development.Pharmacol Ther,2006,111:424-433

        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

        A

        猜你喜歡
        可抑制藥科脂肪性
        非酒精性脂肪性肝病,需要治療嗎?
        肝博士(2024年1期)2024-03-12 08:38:22
        熱量限制飲食或可抑制腫瘤生長(zhǎng)
        中老年保健(2022年3期)2022-11-21 09:40:36
        非酒精性脂肪性肝病的中醫(yī)治療
        肝博士(2022年3期)2022-06-30 02:49:06
        PC化合物可抑制汽車內(nèi)飾中的BSR噪聲
        成軍:非酒精性脂肪性肝病新藥的研發(fā)
        肝博士(2021年1期)2021-03-29 02:32:02
        中國(guó)藥科大學(xué)2020年1~7月獲授權(quán)專利情況(3)
        中國(guó)藥科大學(xué)2020年1~7月獲授權(quán)專利情況(1)
        中國(guó)藥科大學(xué)2020年1~7月獲授權(quán)專利情況(2)
        中國(guó)藥科大學(xué)2018年1~6月獲授權(quán)專利情況
        可抑制毛刺的鉆頭結(jié)構(gòu)
        无码国产精品久久一区免费| 色视频日本一区二区三区 | 美女被内射很爽的视频网站| 亚洲乱码无人区卡1卡2卡3| 少妇高潮惨叫正在播放对白| 亚洲日韩欧美一区二区三区| 日韩精品综合在线视频| 亚洲精品宾馆在线精品酒店| 精品少妇人妻av一区二区| 亚洲乱码一区二区三区成人小说| 国产午夜福利av在线麻豆| 国产一区二区三区精品免费av| 天干天干天啪啪夜爽爽av| 午夜亚洲国产理论片亚洲2020| 免费人成网站在线播放| 国产精品久久久久久久久久红粉| 99精品免费久久久久久久久日本 | 后入丝袜美腿在线观看| 中文字幕被公侵犯的漂亮人妻| 亚洲国产精品嫩草影院久久| 亚洲一区二区日韩在线| 久久精品国产久精国产爱| 国产chinese男男gay视频网| 亚洲香蕉毛片久久网站老妇人| 国产精品又湿又黄九九九久久嫩草| 狼狼综合久久久久综合网| 国产真人无遮挡作爱免费视频 | 午夜免费观看一区二区三区| 国产国产人免费人成免费视频 | 国产中文色婷婷久久久精品| 先锋中文字幕在线资源| 无码不卡高清毛片免费 | 亚洲一区二区三区四区精品| 人妻 偷拍 无码 中文字幕| 久久无码人妻一区二区三区午夜 | 中文乱码字幕在线亚洲av| 亚洲a∨无码一区二区三区| 亚洲视频天堂| 国产又色又爽的视频在线观看91| 亚洲一区二区三区尿失禁| 欧美午夜一区二区福利视频|