中华急诊医学杂志  2018, Vol. 27 Issue (1): 66-71
血管紧张素转换酶2改善肝细胞炎症分子机制
肖红丽, 刘晓亚, 王艳, 王国兴, 阴赪宏     
100050 北京,首都医科大学附属北京友谊医院急诊科(肖红丽、王艳、王国兴);100026 北京,首都医科大学附属北京妇产医院内科(刘晓亚、阴赪宏)
摘要: 目的 探讨血管紧张素转换酶(angiotensin-converting enzyme, ACE)2通过抑制P38促分裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)/激活蛋白(activator protein, AP)-1通路改善脂多糖(lipopolysaccharide, LPS)诱导的肝细胞炎症反应的分子机制。方法 培养永生化大鼠BRL肝细胞,随机分为5组:对照组、LPS(10 μg/mL)组、LPS+重组(recombinant, r)ACE2 (LPS处理前30 min予5、10、20 ng/mL rACE2)组、LPS+ACE2抑制剂MLN-4760(LPS处理前30 min予10-7,10-6,10-5 mmol/L MLN-4760)组、LPS+ rACE2(LPS处理前30 min予20 ng/mL rACE2)+P38MAPK抑制剂SB203580(LPS处理前30 min予10-5 mmol/L SB203580)组。于LPS处理后6、12、24 h应用Western blot方法检测ACE2、P38MAPK、磷酸化(p)-P38MAPK、AP-1蛋白表达。实时定量PCR方法检测P38MAPK、AP-1、肿瘤坏死因子-α(TNF-α)mRNA表达。结果 与对照组相比,LPS处理后ACE2、P38MAPK、AP-1蛋白表达呈时间依赖性激活,均于12 h达峰值(均P < 0.05)。与LPS组相比,rACE2组AP-1、P38MAPK、p-P38MAPK、肿瘤坏死因子α表达明显下降(均P < 0.05),20 ng/mL浓度的rACE2对AP-1(0.12±0.002 vs. 0.04±0.005, P < 0.01)、P38MAPK(0.17±0.02 vs. 0.02±0.002,P < 0.01)、p-P38MAPK(0.29±0.01 vs. 0.02±0.01,P < 0.01)的抑制作用最强。MLN-4760组上述因子表达明显升高(均P < 0.05),并呈剂量依赖性。SB203580去除了rACE2对AP-1、TNF-α表达的抑制作用。结论 rACE2通过下调P38MAPK/AP-1信号通路改善LPS诱导的肝细胞炎症。
关键词: 血管紧张素转换酶2     P38促分裂原活化蛋白激酶     脂多糖     肝细胞炎症     分子机制    
The molecular mechanism of angiotensin-converting enzyme 2 alleviating hepatocyte inflammation
Xiao Hongli , Liu Xiaoya , Wang Yan , Wang Guoxing , Yin Chenghong     
Emergency Department, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China (Xiao HL, Wang Y, Wang GX);Department of Internal Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China (Liu XY, Yin CH)
Abstract: Objective To observe the protective mechanism of angiotensin-converting enzyme (ACE) 2 on lipopolysaccharide (LPS) -induced hepatocyte inflammation by inhibiting P38 mitogen activated protein kinase (MAPK)/ activator protein (AP)-1 pathway. Methods Rat liver BRL cells after immortalized culture were randomly divided into five kinds of groups: control group, LPS (10 μg/mL) group, LPS + recombinant(r) ACE2 (5, 10, 20 ng/mL rACE2 for 30 min before cells stimulated with LPS) groups, LPS+ACE2 inhibitor MLN-4760 (10-7, 10-6, 10-5 mmol/L MLN-4760 for 30 min before cells stimulated with LPS) groups, LPS + rACE2 (20 ng/mL rACE2 for 30 min before cells stimulated with LPS) + P38MAPK inhibitor SB203580 (10-5 mmol/L SB203580 for 30 min before cells stimulated with LPS) groups. The changes in protein levels of ACE2, P38MAPK, p-P38MAPK and AP-1 were detected by western blot after LPS exposure for 6, 12 and 24 hours, and the mRNA expressions of P38MAPK, AP-1 and tumor necrosis factor-α were quantified by real-time RT-PCR. Results Compared with control group, the protein levels of ACE2, P38MAPK and AP-1 were up-regulated in LPS-induced hepatic cells in a time-dependent manner, peaking at 12 h after LPS stimulation (all P < 0.05). Compared with LPS group, the mRNA expressions of AP-1, P38MAPK, p-P38MAPK and tumor necrosis factor-α decreased significantly in rACE2 group (all P < 0.05). The dose of 20 ng/mL rACE2 had the best inhibitory effects on the mRNA expression of AP-1 (0.12±0.002 vs. 0.04±0.005, P < 0.01), P38MAPK (0.17±0.02 vs. 0.02±0.002, P < 0.01) and p-P38MAPK(0.29±0.01 vs. 0.02±0.01, P < 0.01)compared with LPS group. The mRNA expressions of AP-1, P38MAPK and p-P38MAPK increased in MLN-4760 group in a concentration dependent manner (all P < 0.05). Furthermore, the inhibitory effects of rACE2 on AP-1 and tumor necrosis factor-α levels were cancelled by SB203580. Conclusion The rACE2 can alleviate the LPS-induced hepatocyte injury by down regulating the P38MAPK/AP-1 signaling pathway.
Key words: Angiotensin-converting enzyme 2     p38 mitogen activated protein kinase     Lipopolysaccharide     Hepatocyte inflammation     Molecular mechanism    

内毒素即细菌脂多糖(lipopolysaccharide, LPS),广泛存在于革兰阴性菌等微生物细胞壁中,内毒素血症时大量炎症介质激活导致肝脏损伤,与危重症患者预后密切相关[1-2]。内毒素血症时全身及肝脏局部肾素血管紧张素系统(renin angiotensin system, RAS)高度活化[3-4]。近期发现,RAS除了包含血管紧张素转换酶(angiotensin converting enzyme, ACE)-血管紧张素(angiotensin, Ang)Ⅱ-血管紧张素Ⅱ 1型受体(angiotensin Ⅱ type 1 receptor, AT1)经典轴外,还存在新轴ACE2-Ang(1-7)-Mas受体,其与经典轴具有相反的生物学效应,如抗炎、扩张血管等[5]。肿瘤坏死因子(tumor necrosis factor, TNF)-α是LPS致肝损害最关键的促炎介质[6]。激活蛋白(activator protein, AP-1)是调节TNF-α产生的重要转录因子[7]。P38丝裂素活化蛋白激酶(mitogen-activated protein kinase, MAPK)负责将细胞外信号传递给细胞浆和细胞核。研究表明,LPS可激活肝脏P38MAPK使其磷酸化[8],并激活AP-1[9]。Ang(1-7)通过Mas调节P38 MAPK [10]。因此推测ACE2通过抑制P38MAPK/AP-1通路减轻内毒素血症肝脏炎症反应的分子机制。

1 材料与方法 1.1 实验材料和试剂

永生化大鼠BRL肝细胞株购自中科院上海细胞库。Dulbecco's必需培养基(美国Invitrogen),10%胎牛血清(美国Invitrogen)。LPS、重组ACE2(Recombinant, rACE2)和MLN-4760购自美国Sigma公司,SB 203580购自上海碧云天生物技术有限公司。总RNA提取试剂盒(美国Invitrogen公司)、cDNA反转录试剂盒(美国ABI公司),美国Bio-Rad公司Smartspec plus核酸蛋白测定仪,SYBR Green染料法行荧光定量(quantitative real-time,PCR)检测,按美国ABI公司7500-PCR Real time PCR system默认程序进行PCR扩增。BCA蛋白定量试剂盒(sunbio)、基因表达测定使用PowerSYBR Green PCR Master Mix、High-Capacity Cdna、Reverse Transcription Kits试剂盒(美国ABI公司)。P38MAPK一抗:兔P38 MAPK的单克隆抗体(美国CST公司,稀释1:800);p-P38 MAPK一抗:兔p-P38 MAPK的单克隆抗体(英国Abcam公司,稀释1:800):AP-1一抗:兔AP-1的单克隆抗体(美国CST公司,稀释1:800);β-actin单克隆抗体(英国Abcam公司,稀释1:800),二抗购美国Santa公司。

1.2 实验方法 1.2.1 细胞培养及分组

永生化大鼠BRL肝细胞株培养在Dulbecco's必需培养基中,培养基包括10%胎牛血清、100 U/mL青霉素、100 μg/mL链霉素。细胞培养在37 ℃、含5% CO2的潮湿的空气中。细胞接种后24 h开始实验。LPS用于诱导BRL细胞炎症反应以制作脓毒症模型。首先,采用10 μg/mL的LPS干预0、6、12、24 h,以决定诱导炎症反应的最佳时间点。随后,细胞分为5组:对照未干预组;LPS干预组;LPS+rACE2组,在LPS干预前30 min预先予rACE2干预,分别分为低浓度5 ng/mL,中浓度10 ng/mL,高浓度20 ng/mL;LPS+MLN-4760组,在LPS干预前30 min预先予MLN-4760干预,分别分为低浓度10-7 mmol/L,中浓度10-6 mmol/L,高浓度10-5 mmol/L;LPS+ rACE2+SB203580组,在LPS干预前30 min预先予rACE2 (20 ng/mL)和P38MAPK抑制剂SB 203580 (10-5 mmol/L)干预。在LPS干预后12 h收集细胞。

1.2.2 RNA提取、反转录和实时定量PCR

应用TRIzol(一步法)总RNA提取试剂盒提取培养的细胞总RNA,采用分光光度计法测定提取的总RNA含量及纯度,吸光度260/280为1.8~2.0。采用100 μL反转录反应体系,内含5×buffer、4×dNTP、Oligo(dT)15 Primer、M-MLV、RNA酶抑制剂,应用Power SYBR Green PCR Master Mix和ABI 7500荧光定量PCR仪进行PCR反应,引物序列(表 1),反应条件95℃反应5 min,随后95 ℃反应15 s,60 ℃反应1 min,共40个循环。以2-ΔΔCT方法进行数据分析。

表 1 AP-1、P38MAPK、TNF-α PCR引物序列 Table 1 PCR primer sequence of AP-1, P38MAPK and TNF-α
指标 引物序列
AP-1 forward:5'-CTA CAA ACT CCT GAA ACC CAC C-3';
reverse:5'-TCT GAT CCC TGA CCC GAA A-3')
P38MAPK forward:5'-GGA CCT AAA GCC CAG CAA-3';
reverse:5'-CAG CCC ACG GAC CAA ATA-3'
TNF-α forward: 5'-GGT GCC TAT GTC TCA GCC TCT T-3';
reverse: 5'-GCA CCT CCA CTT GGT GGT TT-3'
GAPDH forward: 5'-GGC ACA GTC AAG GCT GAG AAT G-3';
reverse: 5'-ATG GTG GTG AAG ACG CCA GTA-3'
1.2.3 Western blot分析

经不同处理的BRL细胞,按照操作说明用细胞裂解液提取细胞蛋白,用BCA蛋白检测试剂盒测定蛋白浓度。取25 μg样本用8%SDS-聚丙烯酰胺凝胶电泳(电压120 V),分离出不同相对分子质量的蛋白;将蛋白转移至PVDF膜上,390 mA,1 h;37 ℃下用含5%脱脂牛奶的Tris缓冲液(TBST)孵育3 h以封闭膜上非特异性区域;TBST洗膜3次,每次5 min;用一抗在4 ℃孵育PVDF膜过夜,所用一抗为:兔抗鼠P38 MAPK单克隆抗体(1:800)、兔抗鼠p-P38MAPK单克隆抗体(1:800)、兔抗鼠AP-1单克隆抗体(1:800)和anti-β-actin单克隆抗体(1:800);室温下用TBST洗膜3次,每次5 min;用TBST稀释辣根过氧化物酶连接的山羊抗兔或山羊抗鼠二抗(1:6 000),室温孵育1 h;室温下用TBST洗膜3次,每次10 min;用ECL液显色检测蛋白信号,Bio-Rad凝胶成像系统进行图片采集,并用Image lab图像分析软件分析各阳性条带的积分灰度值,设内参照β-actin,计算各目标蛋白的相对含量。

1.3 统计学方法

采用SPSS 16.0统计软件分析,计量资料以均数±标准差(x±s)表示,多组间比较采用One-Way ANOVA方差分析(SNK-q),以P < 0.05为差异有统计学意义。

2 结果 2.1 LPS诱导AP-1和p38MAPK表达随时间动态改变

为说明LPS对信号通路介导的炎症反应的影响,采用LPS(10 μg/mL)干预肝细胞不同的时间(6、12、24 h)。与对照组比较(0.16±0.04),ACE2表达于LPS干预后6 h(0.37±0.05)和12 h(0.55±0.05)明显升高,24 h(0.10±0.01)明显降低(F=53.25,P < 0.01)(图 1A)。P38MAPK表达在12 h明显高于对照组(0.42±0.09 vs. 0.25±0.03,F=14.30,P < 0.01)(图 1B)。与对照组比较(0.013±0.000),AP-1表达于LPS干预后6 h(0.054±0.003)和12 h(0.068±0.002)明显升高,于24 h(0.006±0.001)明显下降(F=779.42,P < 0.01)(图 1C)。可见,ACE2、P38MAPK和AP-1蛋白表达均在LPS干预后12 h达到高峰,24 h后均较12 h明显下降,因此,选取LPS处理后12 h为研究分子机制的时间点。

与对照组比较,aP < 0.05;与LPS 6 h组比较,bP < 0.05;与LPS 12 h组比较,cP < 0.05 图 1 LPS对ACE2(A),P38MAPK(B),AP-1 (C)表达的影响 Figure 1 The effect of LPS on the expression of ACE2 (A), P38MAPK (B) and AP-1 (C)
2.2 rACE2抑制LPS诱导的AP-1和p-P38MAPK表达

用5、10、20 ng/mL三个浓度rACE2分别对大鼠BRL肝细胞预处理30 min,然后给予LPS干预诱导培养12 h。结果表明,与未用rACE2预处理的细胞相比,rACE2预处理的细胞AP-1蛋白表达显著下降, 由未预处理时的0.12±0.002下降至5 ng/mL rACE2预处理后的0.07±0.006、10 ng/mL rACE2预处理后的0.06±0.009、20 ng/mL rACE2预处理后的0.04±0.005(F=91.28,P < 0.01,图 2A)。图 2B显示AP-1 mRNA在各组表达情况,LPS对其影响并不显著,但高浓度rACE2可明显降低LPS处理组的AP-1 mRNA水平(0.60±0.24 vs. 1.27±0.51,F=1.751,P < 0.05)。

A:rACE2对AP-1蛋白表达的影响;B:rACE2或MLN-4760对AP-1 mRNA表达的影响;C:rACE2对P38MAPK和p-P38MAPK蛋白表达的影响;D:rACE2或MLN-4760对P38MAPK mRNA表达的影响;与对照组比较,aP < 0.05;与LPS组比较,bP < 0.05;与LPS+rACE2 5 ng/mL组比较,cP < 0.05;与LPS+ rACE2 10 ng/mL组比较,dP < 0.05 图 2 rACE2对LPS诱导的AP-1,P38MAPK和p-P38MAPK表达的影响 Figure 2 The effect of rACE2 on LPS-induced expression of AP-1, P38MAPK and p-P38MAPK

与未用rACE2预处理的细胞相比,P38MAPK、p-P38MAPK蛋白表达在rACE2预处理后显著下降:分别由未预处理时的0.17±0.02、0.29±0.01下降至5 ng/mL rACE2预处理后的0.12±0.01、0.17±0.03,10 ng/mL rACE2预处理后的0.09±0.01、0.11±0.02,20 ng/mL rACE2预处理后的0.02±0.002、0.02±0.01(F=61.83,P < 0.01;F=104.87,P < 0.01,图 2C)。高浓度rACE2对P38MAPK mRNA表达的抑制作用更明显(1.59±0.12 vs. 2.09±0.16,F=35.34,P < 0.01)(图 2D)。

2.3 MLN-4760加剧了LPS诱导的AP-1和p-P38MAPK表达

分别用10-7 mmol/L、10-6 mmol/L、10-5 mmol/L三个浓度的ACE2抑制剂MLN-4760分别对大鼠BRL肝细胞预处理30 min,然后给予LPS干预诱导培养12 h。结果表明,与LPS处理组相比(0.067±0.001),低中高浓度的MLN-4760分别显著增加了AP-1蛋白表达(0.087±0.006、0.090±0.01、0.103±0.001,F=48.43,P < 0.01),且中、高浓度MLN-4760组AP-1蛋白表达水平显著高于低浓度组(P < 0.05、P < 0.01,图 3A)。AP-1mRNA在各组的表达趋势与AP-1蛋白水平一致(图 2B)。与LPS组比较,MLN-4760低中高浓度组P38MAPK蛋白表达递增(0.44±0.05 vs. 0.97±0.12、1.18±0.18、1.56±0.02,F=105.18,P < 0.01)、p-P38MAPK蛋白表达亦增加(2.11±0.04 vs. 3.14±0.20、3.28±0.24、3.67±0.43,F=26.68,P < 0.01,图 3B)。

A:MLN-4760对AP-1蛋白表达的影响;B:MLN-4760对P38MAPK、p-P38MAPK蛋白表达的影响;与对照组比较,aP < 0.05;与LPS组比较,bP < 0.05;与LPS+MLN-4760 10-7 mol/L组比较,cP < 0.05;与LPS+ MLN-4760 10-6 mol/L组比较,dP < 0.05 图 3 MLN-4760对LPS诱导的AP-1,P38MAPK和p-P38MAPK表达的影响 Figure 3 The effect of MLN-4760 on LPS-induced expression of AP-1, P38MAPK and p-P38MAPK
2.4 SB 203580去除ACE2对LPS诱导的AP-1和TNF-α表达的抑制作用

进一步采用P38MAPK的抑制剂SB-203580干预,明确ACE2通过P38MAPK途径发挥抗炎效应。与LPS组(0.78±0.05)比较,LPS+rACE2(20 ng/mL)(0.45±0.07)明显抑制了AP-1蛋白的表达(P < 0.05),LPS+rACE2+SB 203580组(0.75±0.08)AP-1蛋白的表达明显高于LPS+rACE2组(F=48.01,P < 0.05,图 4A)。如图 4BC所示,与LPS组比较,rACE2降低了LPS诱导的AP-1mRNA(18.02±10.29 vs. 6.92±2.86)、TNF-α mRNA(1.18±0.13 vs. 0.63±0.39)的表达,与SB 203580合用后rACE2对AP-1(13.11±7.45,F=4.58,P < 0.05)、TNF-α(1.00±0.06,F=4.66,P < 0.05)的抑制作用被消除。

A:AP-1蛋白表达。B:AP-1 mRNA表达。C:TNF-α mRNA表达。与对照组比较,aP < 0.05;与LPS比较,bP < 0.05;与LPS+ rACE2 20 ng/mL比较,cP < 0.05 图 4 rACE2和P38MAPK抑制剂SB 203580对AP-1和TNF-α表达的影响 Figure 4 The effects of rACE2 and P38MAPK inhibitor SB 203580 on the expression of AP-1 and TNF-α
3 讨论

本研究应用LPS处理肝细胞,发现ACE2、信号通路P38MAPK、炎症因子TNF-α蛋白表达随时间呈动态变化,于LPS处理后12 h达高峰;rACE2能够显著抑制信号通路P38MAPK、p-P38MAPK、AP-1,炎症因子TNF-α mRNA/蛋白的表达水平;ACE2抑制剂MLN-4760显著增强了P38MAPK、p-P38MAPK、AP-1的表达;P38MAPK阻滞剂SB 203580去除了ACE2对AP-1信号通路和TNF-α炎症因子的抑制作用。

动物实验表明,ACE2-Ang(1-7)-Mas受体轴可用于治疗脓毒症。Wösten-van Asperen等[11]研究表明,在LPS诱导的急性呼吸窘迫综合征中,肺泡灌洗液中ACE和AngⅡ的活性升高,ACE2和Ang-(1-7)的活性下降,Ang-(1-7)治疗可减轻炎症介质、显著降低肺损伤评分、改善氧合。Wong等[12]将LPS注入成年大鼠气道中,从大鼠肺脏中分离培养Ⅱ型肺泡上皮细胞,发现ACE2和AT1受体阻滞剂氯沙坦均可阻断LPS诱导的细胞因子反应,如TNF-α、白介素-6, 、白介素-1β。Li等[13]研究显示ACE2/Ang-(1-7)/Mas轴抑制LPS诱导的肺微血管内皮细胞的炎症反应和凋亡。此外,他们发现卡托普利能够抑制LPS诱导的肺损伤,其分子机制包括卡托普利降低ACE与ACE2的比值,抑制P38MAPK、细胞外信号调节激酶1/2、e-Jun氨基末端激酶激活[14]。他们进一步研究显示,ACE2通过Ang-(1-7)/Mas通路抑制细胞外信号调节激酶/核转录因子kappa B激活以改善LPS诱导的急性呼吸窘迫综合征[15]。以上关于ACE2-Ang(1-7)-Mas受体轴与脓毒症的研究多集中于肺损伤方面,Ge等[16]应用血管紧张素转换酶抑制剂卡托普利治疗内毒素血症诱导的肝损伤,发现卡托普利可降低肝脏TNF-α和白介素-6 mRNA的表达,抑制caspases 3、8、9的激活,减轻肝细胞凋亡。本实验通过分别增强和抑制ACE2的表达,观察信号通路相关因子P38MAPK、AP-1以及炎症启动因子TNF-α表达的变化,阐述ACE2抑制LPS诱导的肝细胞炎症反应的保护作用。

TNF-α是LPS致肝损害最关键的促炎介质。Xie等[17]发现在LPS处理的小鼠肝脏TNF-αmRNA表达及血清TNF-α均明显升高。Ceccarelli等[18]研究显示TNF-α是LPS诱导的肝星状细胞中促炎症反应的重要调节因子,P38MAPK抑制剂可能是非酒精性脂肪肝炎症反应的治疗方法。此外,P38MAPK通过调控AP-1活化调节LPS诱导的炎症反应,并且和LPS诱导的肝损伤密切相关[19]

研究发现,p38MAPK为ACE2-Ang-(1-7)-Mas受体轴的下游信号通路。Giani等[20]发现果糖饮食可诱导大鼠心脏/体质量比值、细胞直径、左心室纤维化增加和血管周围胶原沉积,慢性注射Ang-(1-7)可减轻上述反应。并且Ang-(1-7)可降低果糖饮食诱导的P38MAPK磷酸化水平。Tao等[21]应用ACE2激动剂DIZE治疗LPS干预的人视网膜色素上皮细胞,发现DIZE可降低LPS刺激的p38MAPK的磷酸化。这些结果与本实验相一致,本研究应用ACE2预处理肝细胞显著抑制LPS诱导的P38MAPK、p-P38MAPK、AP-1、TNF-α的表达,而这一效应可被P38MAPK抑制剂SB203580阻断。

综上所述,本研究阐明了ACE2通过抑制P38MAPK/AP-1信号通路减轻LPS诱导的肝细胞炎症的分子机制,为脓毒症并发肝损伤的治疗提供分子靶点。

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