异丙酚通过HNF-1α/apoM途径抑制炎症反应的机制研究
摘要
背景
异丙酚是一种广泛应用于临床麻醉诱导和维持以及重症患者镇静的静脉麻醉药。一系列的研究表明异丙酚具有抗炎抗氧化的特性：可以减少促炎因子的生成包括肿瘤坏死因子a(tumor necrosis factor-a,TNF-a)以及活性氧簇的产生,减少氧自由基的形成,减低脂质的过氧化和抑制血小板的聚集等。然而异丙酚的具体的抗炎机制仍然不是很明确。
高密度脂蛋白具有心血管保护效应,包括促进胆固醇的逆向转运,抗炎和抗氧化特性等。载脂蛋白M(Apolipoprotein M,apoM)是最近发现的与高密度脂蛋白密切相关的脂蛋白家族成员。研究表明apoM是一种负向快速反应蛋白能够发挥减少感染和炎症反应的作用。外界刺激诱发炎症反应发生时,能够减少肝脏和肾脏的apoM的mRNA水平和血清中apoM的分泌,同时也能引起Hep3B细胞apoM的分泌减少。此外,在人类,急性细菌感染或者慢性HIV感染后,血清中的apoM表达水平均降低。
研究表明apoM表达受多种因素调控,其中包括转录因子肝细胞核因子1α(Hepatocyte Nuclear Factor la,HNF-1α)。研究表明, HNF-1α有一个稳定的apoM结合位点,可与apoM增强子区域相结合,这对于肝脏和肾脏的apoM表达是必须的；HNF-1α缺乏的大鼠组织器官和血浆中均检测不到apoM的mRNA表达；肝脏HNF-1α缺陷的大鼠血浆中的apoM表达浓度仅仅是野生型大鼠的一半；早发糖尿病患者由于HNF-1α基因突变血浆中的apoM表达也显著降低。此外研究表明HNF-1α的表达和功能也受到促炎因子的调节：细菌脂多糖(Lipopoly saccharide, LPS)处理后HNF-1α的mRNA水平和蛋白水平以及结合活性都显著下调。
然而,异丙酚是否能影响HNF-1α和apoM的表达还不清楚,HNF-1α和apoM是否可以介导异丙酚抑制LPS诱导的炎症反应还不是很清楚。本研究通过细胞实验和动物实验,观察异丙酚对炎症反应,以及对(?)SNF-1α和apoM的表达的影响并探讨其相关机制。
目的
1、观察异丙酚对脂多糖诱导处理的C57BL/6小鼠肝脏组织中apoM和HNF-1α表达的影响。
2、观察异丙酚对脂多糖诱导处理的HepG2细胞中的apoM和HNF-1α表达的影响。
3、观察HNF-1α是否参与异丙酚调节脂多糖诱导处理的HepG2细胞中apoM的表达。
4、观察异丙酚对脂多糖诱导处理的THP-1细胞的炎症反应的影响。
5、观察apoM是否参与异丙酚抑制脂多糖诱导处理的THP-1细胞的炎症反应。
材料和方法
1、材料
1.1药品
异丙酚(2,6-二异丙基苯酚)和细菌脂多糖(from Escherichia coli055:B5)均购买自Sigma公司(美国)。RNA逆转录试剂盒(The PrimeScript RT Reagent kit, DRR037A)和实时荧光定量PCR试剂盒(the SYBR■Premix Ex TaqTM II kit,DRR820A)均购自于TaKaRa公司(日本)。
其他化学试剂均为医药级别,均来自于试剂供应商。
1.2动物
选取8周龄,雌性C57BL/6小鼠80只,均购买于北京大学实验动物中心,平均体重为20克。随机分为四组每组20只(1)对照组(control组)-腹腔注射磷酸盐缓冲盐水(PBS,pH7.4);(2)细菌脂多糖组(LPS组)-腹腔注射5mg/kg细菌脂多糖；(3)异丙酚组(Prop组)-腹腔注射10mg/kg异丙酚；(4)细菌脂多糖+异丙酚组(LPS+Prop)-腹腔注射5mg/kg细菌脂多糖和10mg/kg异丙酚。所有的动物每五只放在同一笼饲养,室温维持在25℃,采用12h昼夜循环。实验动物购买,饲养以及动物实验程序均得到南方医院实验动物中心批准。
1.3细胞培养
人肝癌组织细胞(HepG2)和人急性单核白血病细胞(THP-1)均购买于American Type Culture Collection(美国弗吉尼亚州,马纳萨斯)。HepG2细胞生长于370C,5%CO2,含有10%新生胎牛血清的DMED培养基中,培养箱中静置培养,取对数生长期细胞进行实验。THP-1细胞生长于37℃,5%CO2,含有10%新生胎牛血清的RPMI-1640培养基中,培养箱中静置培养。取对数生长期细胞进行实验,在每次实验前用100nM佛波酯(phorbol12-myristate13-acetate,PMA)5%C2,370C孵育THP-1细胞72h,诱导使其分化成巨噬细胞,细胞贴壁分别接种到6孔板、12孔板或者60毫米培养皿中,等到细胞生长到汇合度为80-90%时进行处理。在实验前用PBS清洗两次,最后一次用不含抗体的无血清的PBS冲洗。
2、方法
2.1RNA分离提取和实时定量PCR(real-time PCR)
根据Invitrogen公司RNA提取试剂盒操作说明提取小鼠组织或培养细胞中的总RNA。然后将1μg总RNA用逆转录试剂盒(TaKaRa)反转录成20μl的cDNA。实时荧光定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。采用△△Ct值法,以GAPDH表达为内参定量其它基因的表达。
2.2Western blot分析
根据总蛋白提取试剂盒说明书提取小鼠组织和培养细胞的总蛋白,采用BCA法蛋白定量试剂盒(KeyGen Biotechnologies,中国南京)进行蛋白定量。采用SDS-聚丙烯酰胺凝胶电泳分离蛋白,每个点样孔30μg总蛋白。蛋白质转膜后,孵育一抗。采用兔抗apoM多克隆抗体(BD Biosciences, San Jose, CA,美国),兔抗iNOS多克隆抗体(Santa Cruz Biotechnology,Inc.Santa Cruz,CA,美国),兔抗HNF-1α多克隆抗体和兔抗β-actin-单克隆抗体(Abcam, Cambridge, MA,美国)。孵育结束后以HRP-兔抗山羊IgG二抗进行孵育,再采用化学荧光蛋白显色方法(ECL Plus Western Blot Detection System;Amerisham Biosciences,Foster City,CA,美国)进行显色曝光。
2.3细胞因子检测
采用100nmol/L氟波脂(PMA)诱导孵育THP-1细胞72小时使其分化成巨噬细胞,分别经细菌脂多糖,和脂多糖联合异丙酚处理。收集细胞上清分别采用酶联免疫分析试剂盒(Amersham Biosciences)检测肿瘤坏死因子-α(TNF-a),白细胞介素-1β (IL-1β)和白细胞介素-6(IL-6)的表达情况。
2.4小分子干扰RNA(siRNA)转染
特异性siRNA-HNF-1α和siRNA-apoM的RNA干扰片段以及长度为21个核苷酸的siRNA阴性对照片段均购自广州锐博生物科技有限公司。采用脂质体2000(Invitrogen)转染培养细胞(2×106/well)。转染48小时后采用实时定量PCR和western blot检测目的基因mRNA和蛋白表达情况。
2.5重组质粒构建
包含HNF-1α的PCR-XL-TOPO载体和PIRES2-EGFP载体购自Invitrogen公司。在HNF-1α上游添加EcoRI酶切位点扩增得到EcoRI-HNF-1α片段；在EGFP下游添加：Xhol酶切位点,在上游添加IRES连接基因,扩增得到IRES-EGFP-XhoI。进行PCR反应,PCR反应结束后,分别凝胶电泳回收EcoRI-HNF-la和IRES-EGFP-XhoI目的条带。通过重叠PCR将上述两个片段进行连接,合成含有目的基因HNF-la和EGFP的片段EcoRI-HNF-1a-IRES-EGFP-XhoI.重叠PCR反应结束后凝胶电泳回收EcoRI-HNF-1α-IRES-EGFP-XhoI的目的条带。双酶切上述重叠PCR的回收产物与pCDNA3.1(+)载体。并采用连接酶切回收的HNF-1α-IRES-EGFP片段和pcDNA3.1(+)载体。再将感受态大肠杆菌DH5α解冻后与含重组质粒连接反应液充分混合进行重组质粒的转化。利用PCR及酶切鉴定重组质粒,并用测序引物作测序鉴定。鉴定正确无误后,利用超纯质粒试剂盒提取pcDNA3.1-HNF-1α以备使用。采用脂质体2000将pcDNA3.1-HNF-1α转染目的基因。
结果
1、异丙酚对脂多糖诱导处理的小鼠肝脏组织中apoM和HNF-1α表达的影响
采用实时定量PCR检测各组各时间点apoM和HNF-1α的mRNA表达,析因分析结果表明,对于apoM和HNF1α的mRNA表达,不同处理组和各处理组不同处理时间之间存在交互效应(F=141.603,P=0.000;F=118.214,P=0.000)。经脂多糖处理后,6小时,12小时和24小时apoM的mRNA表达水平均显著下调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；异丙酚处理后,6小时,12小时和24小时apoM的mRNA表达水平均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；经脂多糖和异丙酚联合处理后,6小时,12小时和24小时apoM的mRNA表达水平改变,与0小时比较差异没有统计学意义(P=1.000；P=1.000；P=0.732)。经脂多糖处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平均显著下调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；异丙酚处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；脂多糖和异丙酚联合处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平改变,与0小时比较差异没有统计学意义(P=1.000；P=1.000；P=1.000)。而且脂多糖对apoM和HNF-1α的mRNA表达抑制作用在24小时最强(41.400±7.301vs100.200±4.207,P=0.000;38.400±8.019vs100.200±9.257,P=0.000);异丙酚对apoM和HNF-1α的mRNA表达增强作用也具有时间依赖性,随着时间延长而增强,在24小时增加最明显(256.400±8.355vs99.200±8.438,P=0.000；288.800±13.255vs99.800±9.203,P=0.000)。采用western-blot检测各处理因素处理24小时后,各组apoM和HNF-1α的蛋白表达：脂多糖处理后,apoM和HNF-1α的蛋白表达水平均显著下调,与对照组比较差异有统计学意义(P=0.000,P=0.000)；异丙酚处理后,apoM和HNF-1α的蛋白表达水平则均显著上调,与对照组比较差异有统计学意义(P=0.000,P=0.000)；脂多糖和异丙酚联合处理后,apoM和HNF-1α的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=0.079,P=0.090)。
2、异丙酚对HepG2细胞apoM和HNF-1α表达的影响
由于apoM和HNF-1α在小鼠肝脏组织中的表达受脂多糖的负向调节,并且apoM的表达受HNF-1α的直接调节。‘我们通过实时定量PCR方法检测脂多糖和异丙酚对HepG2细胞apoM和HNF-1α表达的影响：脂多糖诱导处理后,apoM的mRNA表达在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.048；P=0.002；P=0.000):HNF-1α的mRNA表达在6h,12h和24h也均显著下调,与0小时比较差异有统计学意义(P=0.019；P=0.000；P=0.000)；异丙酚处理后,apoM的mRNA表达在6h,12h和24h均显著上调,与0小时比较差异有统计学意义(P=0.016；P=0.000；P=0.000):HNF-1α的mRNA表达在6h,12h和24h也均显著上调,与0小时比较差异有统计学意义(P=0.042;P=0.000;P=0.000)。然后我们采用westem blot方法检测脂多糖和异丙酚对HepG2细胞apoM和HNF-1α蛋白表达的影响：脂多糖处理后,apoM的蛋白表达水平在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.043；P=0.001；P=0.000);HNF-1α的蛋白表达水平在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.030；P=0.000；P=0.000)。相反,当异丙酚处理后,apoM的蛋白表达水平在6h,12h和24h均显著上调,与0小时比较差异有统计学意义(P=0.004;P=0.000;P=0.000);HNF-1α的蛋白表达水平在6h,12h和24h也均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)。
3、异丙酚通过HNF-1α途径调节HepG2细胞的apoM表达
首先采用HNF-1α-siRNA转染HepG2细胞,使转染后的HepG2细胞的HNF-la表达降低。Western blot结果显示与对照组比较,siRNA组HNF-1α的表达下降达到89%(0.113±0.060vs1.003±0.096),差异有统计学意义(F=115.062,P=0.000)。然后检测异丙酚对HNF-la-siRNA转染后的HepG2细胞apoM蛋白表达的影响：实验分组如下：对照组：0μM异丙酚+0ng/ml脂多糖,LPS组：10ng/m1脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖。在经过negative control处理的HepG2细胞中,LPS组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.006);Prop组apoM蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000)；LPS+Prop组apoM蛋白表达水平与对照组比较差异没有统计学意义(P=1.000)。在HNF-1α的表达被HNF-1α-siRNA干扰后,LPS组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.005);Prop组apoM蛋白表达水平改变与对照组比较差异没有统计学意义(P=0.623);LPS+Prop组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.012)。接下来,我们采用HNF-1α重组过表达质粒使HNF-1α表达增加,观察异丙酚对于HepG2细胞apoM蛋白表达的影响。用重组过表达质粒上调HNF-1α的表达可以达到613%(6.137±0.172vs1.007±0.131),与对照组比较差异有统计学意义(F=1337.813,P=0.000)。重组过表达质粒转染的HepG2细胞,实验分组如下：对照组：OμM异丙酚+0ng/ml脂多糖,LPS组：lOng/ml脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖。Prop组apoM的表达被显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组apoM的表达被显著上调,与对照组比较差异有统计学意义(P=0.000); LPS组的apoM的表达水平变化,与对照组比较差异没有统计学意义(P=1.000)。
4、异丙酚抑制THP-1巨噬细胞的炎症反应
将THP-1巨噬细胞随机分为四组：Control组：0μM异丙酚+0ng/mlLPS, LPS组：10ng/mlLPS, Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/mlLPS分别孵育24小时。结果,LPS组apoM的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.003); Prop组apoM的蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组apoM的表达水平改变,与对照组比较差异没有统计学意义(P=1.000)。同样,脂多糖诱导处理后,LPS组HNF-1α的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.012); Prop组HNF-1α蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组HNF-1α的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=0.555)。接下来用western blot方法检测了iNOS蛋白表达水平,结果：LPS组iNOS的蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000);Prop组iNOS的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000);LPS+Prop组iNOS的蛋白表达水平显著上调,与LPS组比较差异有统计学意义(P=0.000)。之后我们采用ELISA方法检测THP-1巨噬细胞中炎症因子TNF-α,IL-1β和IL-6的表达变化。实验分组如下,Control组：0μM异丙+0ng/mlLPS,LPS组：10ng/mlLPS,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/mlLPS,分别孵育24小时。ELISA结果：LPS组TNF-α, IL-1β和IL-6的表达水平显著增加,与对照组比较差异有统计学意义(P=0.000,P=0.000,P=0.000);Prop组TNF-α,L-1β和IL-6的表达水平改变,与对照组比较差异没有统计学意义(P=1.000,P=1.000,P=1.000);LPS+Prop组的TNF-α,IL-1β和IL-6的表达水平显著下降,与LPS组比较差异有统计学意义(P=0.000,P=0.000,P=0.000)。
5、异丙酚通过apoM途径抑制THP-1巨噬细胞的炎症反应
采用apoM-siRNA转染THP-1巨噬细胞,经过apoM-siRNA处理的THP-1巨噬细胞apoM的蛋白表达下降了86%(0.140±0.076vs1.003±0.086),与对照组比较差异有统计学意义(F=97.836,P=0.000)。实验分组如下,处理因素：Control组：OμM异丙酚+0ng/ml脂多糖,LPS组：10ng/m脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖,分别孵育24小时。首先检测iNOS的蛋白表达：negative control处理条件下,THP-1巨噬细胞中,LPS组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); Prop组iNOS蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000)；LPS+Prop组iNOS的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.000),与LPS组比较差异有统计学意义(P=0.000)。在apoM-siRNA转染的THP-1巨噬细胞,LPS组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000), Prop组iNOS蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000);LPS+Prop组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000),与LPS组比较差异没有统计学意义(P=0.843)。接下来将被apoM-siRNA和negative control转染的THP-1巨噬细胞随机分为五组(1)Control组,(2)siRNA-NC+异丙酚(50μM)组,(3)siRNA-apoM+异丙酚(50μM)组,(4)siRNA-NC+异丙酚(50μM)+脂多糖(10ng/ml)组,(5)siRNA-apoM+异丙酚(50gM)+脂多糖(10ng/ml)组,五组细胞分别在37℃孵育24小时。采用ELISA方法分析各组炎症因子TNF-a,IL-1β和IL-6的表达情况。异丙酚与脂多糖联合处理用apoM-siRNA转染的THP-1巨噬细胞后,TNF-α、IL-1p和IL-6的表达均被升高,与siRNA-NC+异丙酚+脂多糖组比较差异有统计学意义(P=0.000,P=0.000,P=0.000)。
结论
1、异丙酚可以上调C57BL/6小鼠肝脏组织中的apoM和HNF-1α的mRNA和蛋白表达水平,并且具有时间依赖性。
2、异丙酚可以上调HepG2细胞的apoM和HNF-1α的mRNA和蛋白表达水平,并且具有时间依赖性。
3、异丙酚通过HNF-1α途径上调HepG2细胞的apoM的表达。
4、异丙酚能够抑制THP-1巨噬细胞的炎症反应。
5、异丙酚通过apoM途径抑制THP-1细胞的炎症反应,发挥抗炎作用。
Background
Propofol (2,6-diisopropylphenol) is probably the most widely used intravenous hypnotic agent in daily practice. However, its anti-inflammatory properties have seldom been addressed. In this study, we evaluated the anti-inflammatory activity and mechanisms of propofol on lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro and found that propofol markedly inhibited LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and expression of inducible nitric oxide synthase (iNOS). At the same time, the expression of hepatocyte nuclear factor-la (HNF-la) and apolipoprotein M (apoM) was inhibited by treatment with LPS and LPS-induced down-regulation of HNF-la expression and apoM expression could be compensated by propofol treatment. However, propofol could not compensate LPS-induced down-regulation of apoM expression by treatment with HNF-la siRNA and the suppressive effect on LPS-induced pro-inflammatory cytokines production by propofol was significantly compensated by treatment with apoM siRNA. These results provide evidence that propofol may first up-regulate apoM expression by enhancing HNF-la expression and then inhibit pro-inflammatory cytokine production in LPS-stimulated cells. Therefore, our study may be useful in understanding the critical effect of propofol in patients with systemic inflammatory response syndrome.
Aims
1. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated C57BL/6mice.
2. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated HepG2cells.
3. To study wether HNF-la is involved in the propfol induced apoM expression in LPS treated HepG2cells.
4. To study the anti-inflamation effects of propofol on LPS treated THP-1cells.
5. To study propofol excert anti-inflammation effects whether through the apoM pathway in LPS treated THP-1cells.
Materials and Methods
Propofol (2,'6-di-isopropylphenol) and lipopolysaccharides from Escherichia coli055:B5were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PrimeScript RT Reagent kit (Perfect Real Time; catalog no. DRR037A) and the SYBR(?) Premix Ex TaqTM II kit (Tli RNaseH Plus; catalog no. DRR820A) were obtained from TaKaRa Bio, Inc.(Shiga, Japan). All other chemicals were of pharmaceutical grade and purchased from commercial suppliers.
Animals
Eight-week-old, female C57BL/6mice (Laboratory Animal Center of Peking University, Beijing, China) with a mean body mass of20g were randomized into four groups:(1) PBS-received intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS, pH7.4);(2) LPS-received i.p.5mg/kg of LPS;(3) Propofol-received i.p.10mg/kg of propofol; and (4) LPS+Propofol-received i.p.5mg/kg of LPS plus10mg/kg of propofol. All animals were housed five per cage at25oC on a12-h light/dark cycle. The animal care and experimental procedures were approved by the Animal Experimental Committee at Nanfang Hospital (Guangdong, China).
Cell Culture
Human hepatocytes (HepG2) and acute monocytic leukemia (THP-1) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The HepG2cells were cultured in25-cm2vented flasks containing Dulbecco's modified Eagle's medium (DMEM) with10%fetal calf serum (FCS) under standard culture conditions (5%CO2,37℃). THP-1cells were maintained in Roswell Park Memorial Institute (RPMI)1640medium containing10%FCS and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA) under standard culture conditions (5%CO2,37℃). Prior to the experiment, cells were washed twice with PBS and once with serum-free medium without antibiotics. Experimental media contained DMEM or RPMI1640with0.2%human serum albumin and one or more additives (i.e., LPS, propofol, etc.) at the concentrations described in the figure legends.
RNA isolation and quantitative real time PCR (qPCR)
Total RNA from mouse tissues or cultured cells was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacture's instructions. qPCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City. CA, USA). Melt curve analyses of all qPCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered. Quantitative measurements were determined using the△Ct method and expression of glyceraldehyde3-phosphate dehydrogenase was used as an internal control.
Western blot analyses
Proteins were extracted from mouse tissues or cultured cells using RIPA buffer (Biocolor Ltd., Belfast, Northern Ireland, UK), quantified using the BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China), and then subjected to western blot analyses (10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis;30μg protein per lane) using rabbit anti-APOM antibodies (BD Biosciences, San Jose, CA, USA), rabbit polyclonal anti-iNOS antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and rabbit polyclonal anti-HNF-la-and β-actin-specific antibodies (Abcam, Cambridge, MA, USA). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amerisham Biosciences, Foster City, CA, USA).
Measurement of cytokine production
The THP-1cells were differentiated into macrophages with100nM PMA for72h and incubated in the absence or presence of LPS with or without propofol. After stimulation and sample treatment, the cell-free supernatants of the THP-1macrophages were collected and used for cytokine analysis. The concentrations of TNF-a, IL-1β, and IL-6were measured in duplicate using a BiotrakTM enzyme-linked immunosorbent assay (ELISA) kit (Amersham Biosciences).
Transfection with small interfering RNA (siRNA)
The siRNAs against HNF-la and APOM and irrelevant21-nucleotide control siRNAs were purchased from RiboBio Co., Ltd.(Guangzhou, China). Cells (2×106/well) were transfected with siRNA targeting HNF-la, APOM, or the control in the absence or presence of appropriate plasmids using Lipofectamine2000(Invitrogen). qPCR and western blotting were performed48h post-transfection.
Construction of recombinant plasmids
The PIRES2-EGFP and PCR-XL-TOPO vectors (containing HNF-la which was assembled by the chemically synthesized oligos through PCR) were purchased from Invitrogen. Segments of EcoRI-HNF-la and IRES-EGFP XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-HNF-la-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent E. coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-HNF-la. The plasmid transfection process was performed using Lipofectamine2000transfection reagent according to the manu-facturer's instructions.
Results
1. Effects of propofol on APOM and HNF-la expression in LPS-treated mice
We first investigated the role of propofol on APOM and HNF-la expression in hepatocytes of LPS-treated mice by qPCR and western blotting. As shown in Figures1A and C, APOM RNA and protein levels were markedly down-regulated by LPS treatment. In contrast, APOM expression was significantly up-regulated by propofol treatment. Moreover, propofol obviously compensated LPS-induced down-regulation of APOM expression in mouse hepatocytes. As shown in Figures IB and D, the transcript and protein levels of HNF-la were down-regulated by LPS treatment while up-regulated by propofol treatment, as propofol obviously compensated LPS-induced down-regulation of HNF-la expression in mouse hepatocytes.
2. Effects of LPS and propofol on expression of apoM and HNF-la in HepG2cells
Since the expression of apoM and HNF-la was negatively regulated by LPS in mouse hepatocytes and apoM expression was directly regulated by transcription factor HNF-la, we next examined LPS effects on apoM and HNF-la expression in HepG2cells by qPCR (Figures2A and2B) and western blotting (Figures2C and2D). As shown, expression levels of apoM and HNF-la were decreased by LPS treatment at the transcriptional and translational levels. The inhibitory effects of LPS on apoM and HNF-la expression were increased with cell culture time and the strongest effects were seen after24h. We also observed the effects of propofol on APOM and HNF-la expression in HepG2cells by qPCR (Figures2E and2F) and western blotting (Figures2G and2H). We showed that expression of APOM and HNF-la was enhanced by propofol at the transcriptional and translational levels in a time-dependent manner in HepG2cells.
3. HNF-la is involved in apoM expression regulation by propofol in LPS-stimulated HepG2cells
Since both apoM and HNF-la expression were inhibited by LPS while enhanced by propofol, we further investigated the involvement of HNF-la in apoM expression regulation following propofol treatment in LPS-stimulated HepG2cells. First, we examined the effects of HNF-la siRNA on apoM expression treated with propofol in LPS-stimulated HepG2cells. As shown in Figure3, treatment with siRNA targeting HNF-la downregulated HNF-la protein expression by89%in HepG2cells. The up-regulation of apoM expression via propofol treatment was nearly restored by HNF-la siRNA treatment, whereas propofol could not compensate LPS-induced down-regulation of apoM expression in HepG2cells. Next, we observed the effects of recombinant plasmids over-expressing HNF-la (pcDNA-HNF-1α) on apoM expression following propofol treatment in LPS-stimulated HepG2cells. As shown in Figure3, treatment with pcDNA-HNF-la upregulated HNF-la protein expression by613%in HepG2cells. Suppression of apoM expression by LPS was markedly compensated by treatment with pcDNA-HNF-la and propofol significantly enhanced apoM expression in LPS-stimulated HepG2cells.
4. Anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophages
We explored the effects of propofol on apoM and HNF-la expression in LPS-stimulated THP-1macrophages by western blotting. As shown in Figure4A, apoM levels were markedly down-regulated by LPS treatment. In contrast, apoM expression was significantly up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of apoM expression in THP-1 macrophages. Likewise, as shown in Figure4B, HNF-la levels were down-regulated by LPS treatment while up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of HNF-la expression in THP-1macrophages. In order to study the anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophages, we analyzed iNOS expression via western blotting. As shown in Figure4C, iNOS levels were markedly up-regulated by LPS treatment, whereas propofol notably inhibited the enhancement effect induced by LPS in THP-1macrophages. To evaluate the effects of propofol on pro-inflammatory cytokine production, including TNF-a, IL-1β and IL-6in the culture medium, THP-1macrophages were treated with LPS only or LPS with propofol. As shown in Table1, LPS treatment dramatically increased pro-inflammatory cytokine secretion into the culture medium as compared to the control group. However, pro-inflammatory cytokine production was decreased following propofol treatment in LPS-stimulated THP-1macrophages.
5. apoM is involved in the anti-inflammatory effects of propofol in LPS-stimulated THP-1macrophages.Next, we investigated the role of apoM in propofol-induced anti-inflammatory effects on LPS-stimulated THP-1macrophages using apoM siRNA. As shown in Figure5, treatment with siRNA targeting apoM for48h downregulated apoM protein expression by86%in THP-1macrophages. The iNOS-induced up-regulation of LPS expression could not be compensated by propofol after treatment with apoM siRNA in THP-1macrophages. We also observed the effects of APOM siRNA on expression of pro-inflammatory cytokines by propofol in LPS-stimulated THP-1macrophages. As shown in Table2, the suppression of pro-inflammatory cytokines expression by propofol was markedly compensated by treatment with APOM siRNA in LPS-stimulated THP-1macrophages.
Conclusion
1. apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in LPS-treated C57BL/6mice.
2. Expression of apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in HepG2cells.
3. HNF-la is involved in apoM expression by propofol in LPS-stimulated HepG2cells.
4. Propofol has anti-inflammatory effects in LPS-stimulated THP-1macrophages.
5. apoM is involved in the anti-inflammatory effects of propofol in LPS treated THP-1macrophages cells.
异丙酚是一种广泛应用于临床麻醉诱导和维持以及重症患者镇静的静脉麻醉药。一系列的研究表明异丙酚具有抗炎抗氧化的特性：可以减少促炎因子的生成包括肿瘤坏死因子a(tumor necrosis factor-a,TNF-a)以及活性氧簇的产生,减少氧自由基的形成,减低脂质的过氧化和抑制血小板的聚集等。然而异丙酚的具体的抗炎机制仍然不是很明确。
高密度脂蛋白具有心血管保护效应,包括促进胆固醇的逆向转运,抗炎和抗氧化特性等。载脂蛋白M(Apolipoprotein M,apoM)是最近发现的与高密度脂蛋白密切相关的脂蛋白家族成员。研究表明apoM是一种负向快速反应蛋白能够发挥减少感染和炎症反应的作用。外界刺激诱发炎症反应发生时,能够减少肝脏和肾脏的apoM的mRNA水平和血清中apoM的分泌,同时也能引起Hep3B细胞apoM的分泌减少。此外,在人类,急性细菌感染或者慢性HIV感染后,血清中的apoM表达水平均降低。
研究表明apoM表达受多种因素调控,其中包括转录因子肝细胞核因子1α(Hepatocyte Nuclear Factor la,HNF-1α)。研究表明, HNF-1α有一个稳定的apoM结合位点,可与apoM增强子区域相结合,这对于肝脏和肾脏的apoM表达是必须的；HNF-1α缺乏的大鼠组织器官和血浆中均检测不到apoM的mRNA表达；肝脏HNF-1α缺陷的大鼠血浆中的apoM表达浓度仅仅是野生型大鼠的一半；早发糖尿病患者由于HNF-1α基因突变血浆中的apoM表达也显著降低。此外研究表明HNF-1α的表达和功能也受到促炎因子的调节：细菌脂多糖(Lipopoly saccharide, LPS)处理后HNF-1α的mRNA水平和蛋白水平以及结合活性都显著下调。
然而,异丙酚是否能影响HNF-1α和apoM的表达还不清楚,HNF-1α和apoM是否可以介导异丙酚抑制LPS诱导的炎症反应还不是很清楚。本研究通过细胞实验和动物实验,观察异丙酚对炎症反应,以及对(?)SNF-1α和apoM的表达的影响并探讨其相关机制。
目的
1、观察异丙酚对脂多糖诱导处理的C57BL/6小鼠肝脏组织中apoM和HNF-1α表达的影响。
2、观察异丙酚对脂多糖诱导处理的HepG2细胞中的apoM和HNF-1α表达的影响。
3、观察HNF-1α是否参与异丙酚调节脂多糖诱导处理的HepG2细胞中apoM的表达。
4、观察异丙酚对脂多糖诱导处理的THP-1细胞的炎症反应的影响。
5、观察apoM是否参与异丙酚抑制脂多糖诱导处理的THP-1细胞的炎症反应。
材料和方法
1、材料
1.1药品
异丙酚(2,6-二异丙基苯酚)和细菌脂多糖(from Escherichia coli055:B5)均购买自Sigma公司(美国)。RNA逆转录试剂盒(The PrimeScript RT Reagent kit, DRR037A)和实时荧光定量PCR试剂盒(the SYBR■Premix Ex TaqTM II kit,DRR820A)均购自于TaKaRa公司(日本)。
其他化学试剂均为医药级别,均来自于试剂供应商。
1.2动物
选取8周龄,雌性C57BL/6小鼠80只,均购买于北京大学实验动物中心,平均体重为20克。随机分为四组每组20只(1)对照组(control组)-腹腔注射磷酸盐缓冲盐水(PBS,pH7.4);(2)细菌脂多糖组(LPS组)-腹腔注射5mg/kg细菌脂多糖；(3)异丙酚组(Prop组)-腹腔注射10mg/kg异丙酚；(4)细菌脂多糖+异丙酚组(LPS+Prop)-腹腔注射5mg/kg细菌脂多糖和10mg/kg异丙酚。所有的动物每五只放在同一笼饲养,室温维持在25℃,采用12h昼夜循环。实验动物购买,饲养以及动物实验程序均得到南方医院实验动物中心批准。
1.3细胞培养
人肝癌组织细胞(HepG2)和人急性单核白血病细胞(THP-1)均购买于American Type Culture Collection(美国弗吉尼亚州,马纳萨斯)。HepG2细胞生长于370C,5%CO2,含有10%新生胎牛血清的DMED培养基中,培养箱中静置培养,取对数生长期细胞进行实验。THP-1细胞生长于37℃,5%CO2,含有10%新生胎牛血清的RPMI-1640培养基中,培养箱中静置培养。取对数生长期细胞进行实验,在每次实验前用100nM佛波酯(phorbol12-myristate13-acetate,PMA)5%C2,370C孵育THP-1细胞72h,诱导使其分化成巨噬细胞,细胞贴壁分别接种到6孔板、12孔板或者60毫米培养皿中,等到细胞生长到汇合度为80-90%时进行处理。在实验前用PBS清洗两次,最后一次用不含抗体的无血清的PBS冲洗。
2、方法
2.1RNA分离提取和实时定量PCR(real-time PCR)
根据Invitrogen公司RNA提取试剂盒操作说明提取小鼠组织或培养细胞中的总RNA。然后将1μg总RNA用逆转录试剂盒(TaKaRa)反转录成20μl的cDNA。实时荧光定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。采用△△Ct值法,以GAPDH表达为内参定量其它基因的表达。
2.2Western blot分析
根据总蛋白提取试剂盒说明书提取小鼠组织和培养细胞的总蛋白,采用BCA法蛋白定量试剂盒(KeyGen Biotechnologies,中国南京)进行蛋白定量。采用SDS-聚丙烯酰胺凝胶电泳分离蛋白,每个点样孔30μg总蛋白。蛋白质转膜后,孵育一抗。采用兔抗apoM多克隆抗体(BD Biosciences, San Jose, CA,美国),兔抗iNOS多克隆抗体(Santa Cruz Biotechnology,Inc.Santa Cruz,CA,美国),兔抗HNF-1α多克隆抗体和兔抗β-actin-单克隆抗体(Abcam, Cambridge, MA,美国)。孵育结束后以HRP-兔抗山羊IgG二抗进行孵育,再采用化学荧光蛋白显色方法(ECL Plus Western Blot Detection System;Amerisham Biosciences,Foster City,CA,美国)进行显色曝光。
2.3细胞因子检测
采用100nmol/L氟波脂(PMA)诱导孵育THP-1细胞72小时使其分化成巨噬细胞,分别经细菌脂多糖,和脂多糖联合异丙酚处理。收集细胞上清分别采用酶联免疫分析试剂盒(Amersham Biosciences)检测肿瘤坏死因子-α(TNF-a),白细胞介素-1β (IL-1β)和白细胞介素-6(IL-6)的表达情况。
2.4小分子干扰RNA(siRNA)转染
特异性siRNA-HNF-1α和siRNA-apoM的RNA干扰片段以及长度为21个核苷酸的siRNA阴性对照片段均购自广州锐博生物科技有限公司。采用脂质体2000(Invitrogen)转染培养细胞(2×106/well)。转染48小时后采用实时定量PCR和western blot检测目的基因mRNA和蛋白表达情况。
2.5重组质粒构建
包含HNF-1α的PCR-XL-TOPO载体和PIRES2-EGFP载体购自Invitrogen公司。在HNF-1α上游添加EcoRI酶切位点扩增得到EcoRI-HNF-1α片段；在EGFP下游添加：Xhol酶切位点,在上游添加IRES连接基因,扩增得到IRES-EGFP-XhoI。进行PCR反应,PCR反应结束后,分别凝胶电泳回收EcoRI-HNF-la和IRES-EGFP-XhoI目的条带。通过重叠PCR将上述两个片段进行连接,合成含有目的基因HNF-la和EGFP的片段EcoRI-HNF-1a-IRES-EGFP-XhoI.重叠PCR反应结束后凝胶电泳回收EcoRI-HNF-1α-IRES-EGFP-XhoI的目的条带。双酶切上述重叠PCR的回收产物与pCDNA3.1(+)载体。并采用连接酶切回收的HNF-1α-IRES-EGFP片段和pcDNA3.1(+)载体。再将感受态大肠杆菌DH5α解冻后与含重组质粒连接反应液充分混合进行重组质粒的转化。利用PCR及酶切鉴定重组质粒,并用测序引物作测序鉴定。鉴定正确无误后,利用超纯质粒试剂盒提取pcDNA3.1-HNF-1α以备使用。采用脂质体2000将pcDNA3.1-HNF-1α转染目的基因。
结果
1、异丙酚对脂多糖诱导处理的小鼠肝脏组织中apoM和HNF-1α表达的影响
采用实时定量PCR检测各组各时间点apoM和HNF-1α的mRNA表达,析因分析结果表明,对于apoM和HNF1α的mRNA表达,不同处理组和各处理组不同处理时间之间存在交互效应(F=141.603,P=0.000;F=118.214,P=0.000)。经脂多糖处理后,6小时,12小时和24小时apoM的mRNA表达水平均显著下调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；异丙酚处理后,6小时,12小时和24小时apoM的mRNA表达水平均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；经脂多糖和异丙酚联合处理后,6小时,12小时和24小时apoM的mRNA表达水平改变,与0小时比较差异没有统计学意义(P=1.000；P=1.000；P=0.732)。经脂多糖处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平均显著下调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；异丙酚处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)；脂多糖和异丙酚联合处理后,6小时,12小时和24小时HNF-1α的mRNA表达水平改变,与0小时比较差异没有统计学意义(P=1.000；P=1.000；P=1.000)。而且脂多糖对apoM和HNF-1α的mRNA表达抑制作用在24小时最强(41.400±7.301vs100.200±4.207,P=0.000;38.400±8.019vs100.200±9.257,P=0.000);异丙酚对apoM和HNF-1α的mRNA表达增强作用也具有时间依赖性,随着时间延长而增强,在24小时增加最明显(256.400±8.355vs99.200±8.438,P=0.000；288.800±13.255vs99.800±9.203,P=0.000)。采用western-blot检测各处理因素处理24小时后,各组apoM和HNF-1α的蛋白表达：脂多糖处理后,apoM和HNF-1α的蛋白表达水平均显著下调,与对照组比较差异有统计学意义(P=0.000,P=0.000)；异丙酚处理后,apoM和HNF-1α的蛋白表达水平则均显著上调,与对照组比较差异有统计学意义(P=0.000,P=0.000)；脂多糖和异丙酚联合处理后,apoM和HNF-1α的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=0.079,P=0.090)。
2、异丙酚对HepG2细胞apoM和HNF-1α表达的影响
由于apoM和HNF-1α在小鼠肝脏组织中的表达受脂多糖的负向调节,并且apoM的表达受HNF-1α的直接调节。‘我们通过实时定量PCR方法检测脂多糖和异丙酚对HepG2细胞apoM和HNF-1α表达的影响：脂多糖诱导处理后,apoM的mRNA表达在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.048；P=0.002；P=0.000):HNF-1α的mRNA表达在6h,12h和24h也均显著下调,与0小时比较差异有统计学意义(P=0.019；P=0.000；P=0.000)；异丙酚处理后,apoM的mRNA表达在6h,12h和24h均显著上调,与0小时比较差异有统计学意义(P=0.016；P=0.000；P=0.000):HNF-1α的mRNA表达在6h,12h和24h也均显著上调,与0小时比较差异有统计学意义(P=0.042;P=0.000;P=0.000)。然后我们采用westem blot方法检测脂多糖和异丙酚对HepG2细胞apoM和HNF-1α蛋白表达的影响：脂多糖处理后,apoM的蛋白表达水平在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.043；P=0.001；P=0.000);HNF-1α的蛋白表达水平在6h,12h和24h均显著下调,与0小时比较差异有统计学意义(P=0.030；P=0.000；P=0.000)。相反,当异丙酚处理后,apoM的蛋白表达水平在6h,12h和24h均显著上调,与0小时比较差异有统计学意义(P=0.004;P=0.000;P=0.000);HNF-1α的蛋白表达水平在6h,12h和24h也均显著上调,与0小时比较差异有统计学意义(P=0.000；P=0.000；P=0.000)。
3、异丙酚通过HNF-1α途径调节HepG2细胞的apoM表达
首先采用HNF-1α-siRNA转染HepG2细胞,使转染后的HepG2细胞的HNF-la表达降低。Western blot结果显示与对照组比较,siRNA组HNF-1α的表达下降达到89%(0.113±0.060vs1.003±0.096),差异有统计学意义(F=115.062,P=0.000)。然后检测异丙酚对HNF-la-siRNA转染后的HepG2细胞apoM蛋白表达的影响：实验分组如下：对照组：0μM异丙酚+0ng/ml脂多糖,LPS组：10ng/m1脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖。在经过negative control处理的HepG2细胞中,LPS组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.006);Prop组apoM蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000)；LPS+Prop组apoM蛋白表达水平与对照组比较差异没有统计学意义(P=1.000)。在HNF-1α的表达被HNF-1α-siRNA干扰后,LPS组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.005);Prop组apoM蛋白表达水平改变与对照组比较差异没有统计学意义(P=0.623);LPS+Prop组apoM蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.012)。接下来,我们采用HNF-1α重组过表达质粒使HNF-1α表达增加,观察异丙酚对于HepG2细胞apoM蛋白表达的影响。用重组过表达质粒上调HNF-1α的表达可以达到613%(6.137±0.172vs1.007±0.131),与对照组比较差异有统计学意义(F=1337.813,P=0.000)。重组过表达质粒转染的HepG2细胞,实验分组如下：对照组：OμM异丙酚+0ng/ml脂多糖,LPS组：lOng/ml脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖。Prop组apoM的表达被显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组apoM的表达被显著上调,与对照组比较差异有统计学意义(P=0.000); LPS组的apoM的表达水平变化,与对照组比较差异没有统计学意义(P=1.000)。
4、异丙酚抑制THP-1巨噬细胞的炎症反应
将THP-1巨噬细胞随机分为四组：Control组：0μM异丙酚+0ng/mlLPS, LPS组：10ng/mlLPS, Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/mlLPS分别孵育24小时。结果,LPS组apoM的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.003); Prop组apoM的蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组apoM的表达水平改变,与对照组比较差异没有统计学意义(P=1.000)。同样,脂多糖诱导处理后,LPS组HNF-1α的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.012); Prop组HNF-1α蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); LPS+Prop组HNF-1α的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=0.555)。接下来用western blot方法检测了iNOS蛋白表达水平,结果：LPS组iNOS的蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000);Prop组iNOS的蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000);LPS+Prop组iNOS的蛋白表达水平显著上调,与LPS组比较差异有统计学意义(P=0.000)。之后我们采用ELISA方法检测THP-1巨噬细胞中炎症因子TNF-α,IL-1β和IL-6的表达变化。实验分组如下,Control组：0μM异丙+0ng/mlLPS,LPS组：10ng/mlLPS,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/mlLPS,分别孵育24小时。ELISA结果：LPS组TNF-α, IL-1β和IL-6的表达水平显著增加,与对照组比较差异有统计学意义(P=0.000,P=0.000,P=0.000);Prop组TNF-α,L-1β和IL-6的表达水平改变,与对照组比较差异没有统计学意义(P=1.000,P=1.000,P=1.000);LPS+Prop组的TNF-α,IL-1β和IL-6的表达水平显著下降,与LPS组比较差异有统计学意义(P=0.000,P=0.000,P=0.000)。
5、异丙酚通过apoM途径抑制THP-1巨噬细胞的炎症反应
采用apoM-siRNA转染THP-1巨噬细胞,经过apoM-siRNA处理的THP-1巨噬细胞apoM的蛋白表达下降了86%(0.140±0.076vs1.003±0.086),与对照组比较差异有统计学意义(F=97.836,P=0.000)。实验分组如下,处理因素：Control组：OμM异丙酚+0ng/ml脂多糖,LPS组：10ng/m脂多糖,Prop组：50μM异丙酚,LPS+Prop组：50μM异丙酚+10ng/ml脂多糖,分别孵育24小时。首先检测iNOS的蛋白表达：negative control处理条件下,THP-1巨噬细胞中,LPS组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000); Prop组iNOS蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000)；LPS+Prop组iNOS的蛋白表达水平显著下调,与对照组比较差异有统计学意义(P=0.000),与LPS组比较差异有统计学意义(P=0.000)。在apoM-siRNA转染的THP-1巨噬细胞,LPS组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000), Prop组iNOS蛋白表达水平改变,与对照组比较差异没有统计学意义(P=1.000);LPS+Prop组iNOS蛋白表达水平显著上调,与对照组比较差异有统计学意义(P=0.000),与LPS组比较差异没有统计学意义(P=0.843)。接下来将被apoM-siRNA和negative control转染的THP-1巨噬细胞随机分为五组(1)Control组,(2)siRNA-NC+异丙酚(50μM)组,(3)siRNA-apoM+异丙酚(50μM)组,(4)siRNA-NC+异丙酚(50μM)+脂多糖(10ng/ml)组,(5)siRNA-apoM+异丙酚(50gM)+脂多糖(10ng/ml)组,五组细胞分别在37℃孵育24小时。采用ELISA方法分析各组炎症因子TNF-a,IL-1β和IL-6的表达情况。异丙酚与脂多糖联合处理用apoM-siRNA转染的THP-1巨噬细胞后,TNF-α、IL-1p和IL-6的表达均被升高,与siRNA-NC+异丙酚+脂多糖组比较差异有统计学意义(P=0.000,P=0.000,P=0.000)。
结论
1、异丙酚可以上调C57BL/6小鼠肝脏组织中的apoM和HNF-1α的mRNA和蛋白表达水平,并且具有时间依赖性。
2、异丙酚可以上调HepG2细胞的apoM和HNF-1α的mRNA和蛋白表达水平,并且具有时间依赖性。
3、异丙酚通过HNF-1α途径上调HepG2细胞的apoM的表达。
4、异丙酚能够抑制THP-1巨噬细胞的炎症反应。
5、异丙酚通过apoM途径抑制THP-1细胞的炎症反应,发挥抗炎作用。
Background
Propofol (2,6-diisopropylphenol) is probably the most widely used intravenous hypnotic agent in daily practice. However, its anti-inflammatory properties have seldom been addressed. In this study, we evaluated the anti-inflammatory activity and mechanisms of propofol on lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro and found that propofol markedly inhibited LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and expression of inducible nitric oxide synthase (iNOS). At the same time, the expression of hepatocyte nuclear factor-la (HNF-la) and apolipoprotein M (apoM) was inhibited by treatment with LPS and LPS-induced down-regulation of HNF-la expression and apoM expression could be compensated by propofol treatment. However, propofol could not compensate LPS-induced down-regulation of apoM expression by treatment with HNF-la siRNA and the suppressive effect on LPS-induced pro-inflammatory cytokines production by propofol was significantly compensated by treatment with apoM siRNA. These results provide evidence that propofol may first up-regulate apoM expression by enhancing HNF-la expression and then inhibit pro-inflammatory cytokine production in LPS-stimulated cells. Therefore, our study may be useful in understanding the critical effect of propofol in patients with systemic inflammatory response syndrome.
Aims
1. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated C57BL/6mice.
2. To study the effects of propofol on expressions of apoM and HNF-la in LPS treated HepG2cells.
3. To study wether HNF-la is involved in the propfol induced apoM expression in LPS treated HepG2cells.
4. To study the anti-inflamation effects of propofol on LPS treated THP-1cells.
5. To study propofol excert anti-inflammation effects whether through the apoM pathway in LPS treated THP-1cells.
Materials and Methods
Propofol (2,'6-di-isopropylphenol) and lipopolysaccharides from Escherichia coli055:B5were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PrimeScript RT Reagent kit (Perfect Real Time; catalog no. DRR037A) and the SYBR(?) Premix Ex TaqTM II kit (Tli RNaseH Plus; catalog no. DRR820A) were obtained from TaKaRa Bio, Inc.(Shiga, Japan). All other chemicals were of pharmaceutical grade and purchased from commercial suppliers.
Animals
Eight-week-old, female C57BL/6mice (Laboratory Animal Center of Peking University, Beijing, China) with a mean body mass of20g were randomized into four groups:(1) PBS-received intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS, pH7.4);(2) LPS-received i.p.5mg/kg of LPS;(3) Propofol-received i.p.10mg/kg of propofol; and (4) LPS+Propofol-received i.p.5mg/kg of LPS plus10mg/kg of propofol. All animals were housed five per cage at25oC on a12-h light/dark cycle. The animal care and experimental procedures were approved by the Animal Experimental Committee at Nanfang Hospital (Guangdong, China).
Cell Culture
Human hepatocytes (HepG2) and acute monocytic leukemia (THP-1) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The HepG2cells were cultured in25-cm2vented flasks containing Dulbecco's modified Eagle's medium (DMEM) with10%fetal calf serum (FCS) under standard culture conditions (5%CO2,37℃). THP-1cells were maintained in Roswell Park Memorial Institute (RPMI)1640medium containing10%FCS and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA) under standard culture conditions (5%CO2,37℃). Prior to the experiment, cells were washed twice with PBS and once with serum-free medium without antibiotics. Experimental media contained DMEM or RPMI1640with0.2%human serum albumin and one or more additives (i.e., LPS, propofol, etc.) at the concentrations described in the figure legends.
RNA isolation and quantitative real time PCR (qPCR)
Total RNA from mouse tissues or cultured cells was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacture's instructions. qPCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City. CA, USA). Melt curve analyses of all qPCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered. Quantitative measurements were determined using the△Ct method and expression of glyceraldehyde3-phosphate dehydrogenase was used as an internal control.
Western blot analyses
Proteins were extracted from mouse tissues or cultured cells using RIPA buffer (Biocolor Ltd., Belfast, Northern Ireland, UK), quantified using the BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China), and then subjected to western blot analyses (10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis;30μg protein per lane) using rabbit anti-APOM antibodies (BD Biosciences, San Jose, CA, USA), rabbit polyclonal anti-iNOS antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and rabbit polyclonal anti-HNF-la-and β-actin-specific antibodies (Abcam, Cambridge, MA, USA). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amerisham Biosciences, Foster City, CA, USA).
Measurement of cytokine production
The THP-1cells were differentiated into macrophages with100nM PMA for72h and incubated in the absence or presence of LPS with or without propofol. After stimulation and sample treatment, the cell-free supernatants of the THP-1macrophages were collected and used for cytokine analysis. The concentrations of TNF-a, IL-1β, and IL-6were measured in duplicate using a BiotrakTM enzyme-linked immunosorbent assay (ELISA) kit (Amersham Biosciences).
Transfection with small interfering RNA (siRNA)
The siRNAs against HNF-la and APOM and irrelevant21-nucleotide control siRNAs were purchased from RiboBio Co., Ltd.(Guangzhou, China). Cells (2×106/well) were transfected with siRNA targeting HNF-la, APOM, or the control in the absence or presence of appropriate plasmids using Lipofectamine2000(Invitrogen). qPCR and western blotting were performed48h post-transfection.
Construction of recombinant plasmids
The PIRES2-EGFP and PCR-XL-TOPO vectors (containing HNF-la which was assembled by the chemically synthesized oligos through PCR) were purchased from Invitrogen. Segments of EcoRI-HNF-la and IRES-EGFP XhoI were amplified using the template of the PCR-XL-TOPO and PIRES2-EGFP vectors, respectively. EcoRI-HNF-la-IRES-EGFP-XhoI was joined by the two above-mentioned segments using overlap PCR. Gel electrophoresis was performed and the relevant band was excised from the gel, double enzyme-digested by EcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent E. coli DH5a cells for further amplification and use. The recombinant plasmids were verified by sequencing and named pcDNA3.1-HNF-la. The plasmid transfection process was performed using Lipofectamine2000transfection reagent according to the manu-facturer's instructions.
Results
1. Effects of propofol on APOM and HNF-la expression in LPS-treated mice
We first investigated the role of propofol on APOM and HNF-la expression in hepatocytes of LPS-treated mice by qPCR and western blotting. As shown in Figures1A and C, APOM RNA and protein levels were markedly down-regulated by LPS treatment. In contrast, APOM expression was significantly up-regulated by propofol treatment. Moreover, propofol obviously compensated LPS-induced down-regulation of APOM expression in mouse hepatocytes. As shown in Figures IB and D, the transcript and protein levels of HNF-la were down-regulated by LPS treatment while up-regulated by propofol treatment, as propofol obviously compensated LPS-induced down-regulation of HNF-la expression in mouse hepatocytes.
2. Effects of LPS and propofol on expression of apoM and HNF-la in HepG2cells
Since the expression of apoM and HNF-la was negatively regulated by LPS in mouse hepatocytes and apoM expression was directly regulated by transcription factor HNF-la, we next examined LPS effects on apoM and HNF-la expression in HepG2cells by qPCR (Figures2A and2B) and western blotting (Figures2C and2D). As shown, expression levels of apoM and HNF-la were decreased by LPS treatment at the transcriptional and translational levels. The inhibitory effects of LPS on apoM and HNF-la expression were increased with cell culture time and the strongest effects were seen after24h. We also observed the effects of propofol on APOM and HNF-la expression in HepG2cells by qPCR (Figures2E and2F) and western blotting (Figures2G and2H). We showed that expression of APOM and HNF-la was enhanced by propofol at the transcriptional and translational levels in a time-dependent manner in HepG2cells.
3. HNF-la is involved in apoM expression regulation by propofol in LPS-stimulated HepG2cells
Since both apoM and HNF-la expression were inhibited by LPS while enhanced by propofol, we further investigated the involvement of HNF-la in apoM expression regulation following propofol treatment in LPS-stimulated HepG2cells. First, we examined the effects of HNF-la siRNA on apoM expression treated with propofol in LPS-stimulated HepG2cells. As shown in Figure3, treatment with siRNA targeting HNF-la downregulated HNF-la protein expression by89%in HepG2cells. The up-regulation of apoM expression via propofol treatment was nearly restored by HNF-la siRNA treatment, whereas propofol could not compensate LPS-induced down-regulation of apoM expression in HepG2cells. Next, we observed the effects of recombinant plasmids over-expressing HNF-la (pcDNA-HNF-1α) on apoM expression following propofol treatment in LPS-stimulated HepG2cells. As shown in Figure3, treatment with pcDNA-HNF-la upregulated HNF-la protein expression by613%in HepG2cells. Suppression of apoM expression by LPS was markedly compensated by treatment with pcDNA-HNF-la and propofol significantly enhanced apoM expression in LPS-stimulated HepG2cells.
4. Anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophages
We explored the effects of propofol on apoM and HNF-la expression in LPS-stimulated THP-1macrophages by western blotting. As shown in Figure4A, apoM levels were markedly down-regulated by LPS treatment. In contrast, apoM expression was significantly up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of apoM expression in THP-1 macrophages. Likewise, as shown in Figure4B, HNF-la levels were down-regulated by LPS treatment while up-regulated by propofol treatment, which obviously compensated LPS-induced down-regulation of HNF-la expression in THP-1macrophages. In order to study the anti-inflammatory effects of propofol on LPS-stimulated THP-1macrophages, we analyzed iNOS expression via western blotting. As shown in Figure4C, iNOS levels were markedly up-regulated by LPS treatment, whereas propofol notably inhibited the enhancement effect induced by LPS in THP-1macrophages. To evaluate the effects of propofol on pro-inflammatory cytokine production, including TNF-a, IL-1β and IL-6in the culture medium, THP-1macrophages were treated with LPS only or LPS with propofol. As shown in Table1, LPS treatment dramatically increased pro-inflammatory cytokine secretion into the culture medium as compared to the control group. However, pro-inflammatory cytokine production was decreased following propofol treatment in LPS-stimulated THP-1macrophages.
5. apoM is involved in the anti-inflammatory effects of propofol in LPS-stimulated THP-1macrophages.Next, we investigated the role of apoM in propofol-induced anti-inflammatory effects on LPS-stimulated THP-1macrophages using apoM siRNA. As shown in Figure5, treatment with siRNA targeting apoM for48h downregulated apoM protein expression by86%in THP-1macrophages. The iNOS-induced up-regulation of LPS expression could not be compensated by propofol after treatment with apoM siRNA in THP-1macrophages. We also observed the effects of APOM siRNA on expression of pro-inflammatory cytokines by propofol in LPS-stimulated THP-1macrophages. As shown in Table2, the suppression of pro-inflammatory cytokines expression by propofol was markedly compensated by treatment with APOM siRNA in LPS-stimulated THP-1macrophages.
Conclusion
1. apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in LPS-treated C57BL/6mice.
2. Expression of apoM and HNF-la mRNA and proteins expressions were up-regaulated by propofol in HepG2cells.
3. HNF-la is involved in apoM expression by propofol in LPS-stimulated HepG2cells.
4. Propofol has anti-inflammatory effects in LPS-stimulated THP-1macrophages.
5. apoM is involved in the anti-inflammatory effects of propofol in LPS treated THP-1macrophages cells.
引文
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