内毒素攻击大鼠肺泡巨噬细胞时HO-1对高尔基体应激的影响*

2022-03-28 05:59李玉婷李香云史佳李翠余剑波
中国病理生理杂志 2022年3期
关键词:肺泡氧化应激试剂盒

李玉婷, 李香云, 史佳, 李翠, 余剑波

内毒素攻击大鼠肺泡巨噬细胞时HO-1对高尔基体应激的影响*

李玉婷, 李香云, 史佳, 李翠, 余剑波△

(天津医科大学南开临床学院,天津市中西医结合医院·南开医院麻醉与重症医学科,天津 300100)

探讨内毒素诱导大鼠肺泡巨噬细胞损伤时血红素加氧酶1(HO-1)对高尔基体应激的影响。体外培养大鼠肺泡巨噬细胞,采用脂多糖(LPS)诱导大鼠肺泡巨噬细胞建立细胞损伤模型。使用CCK-8法检测细胞活力;使用DCFH-DA探针检测细胞内活性氧簇(ROS)的生成;使用生物化学方法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平;使用TUNEL染色和凋亡相关蛋白caspase-3/7活性检测试剂盒检测细胞凋亡;使用RT-qPCR和Western blot法检测HO-1和高尔基体磷蛋白3(GOLPH3)的表达;使用Western blot法检测高尔基体结构相关蛋白GM130、golgin-97和mannosidase II的表达。使用小干扰RNA(siRNA)沉默后,重复以上检测。LPS刺激肺泡巨噬细胞下调细胞活力、SOD活性及GM130、golgin-97和mannosidase II表达水平,上调ROS和MDA含量及HO-1和GOLPH3表达水平,并导致TUNEL标记阳性细胞数增多,caspase-3/7活性增强(<0.05);基因沉默后,细胞活力、SOD活性及GM130、golgin-97和mannosidase II表达显著下降,ROS和MDA含量及GOLPH3表达显著上升,TUNEL标记阳性细胞数增多,caspase-3/-7活性显著增强(<0.05)。内毒素诱导大鼠肺泡巨噬细胞损伤时,HO-1可减轻氧化应激和高尔基体应激反应,减少细胞凋亡。

高尔基体应激;血红素加氧酶1;脂多糖;肺泡巨噬细胞;氧化应激

急性肺损伤(acute lung injury, ALI)是临床常见的危重症,具有高发病率和高死亡率。肺泡巨噬细胞可释放细胞因子和趋化因子以招募炎症细胞,引发炎症风暴,在ALI后期可释放抗炎因子,抑制炎症细胞浸润以介导肺部炎症消退,并通过诱导增殖信号传导以促进组织修复。因此,巨噬细胞可能是治疗ALI的合适靶点之一[1-3]。

高尔基体是细胞死亡途径中应激的传感器和下游效应器之一[4]。应激状态下,高尔基体结构和功能被破坏,离子稳态失衡,导致细胞氧化还原平衡改变,细胞死亡增加[4-5]。高尔基体磷蛋白3(Golgi phosphoprotein 3, GOLPH3)作为高尔基体应激的传感器,在氧化应激过程中迅速上调,并向下游传递应激信号,诱导细胞内活性氧簇(reactive oxygen species, ROS)产生,促进高尔基体解体和细胞凋亡[4, 6-7]。高尔基体结构相关蛋白高尔基体基质蛋白130(Golgi matrix protein 130, GM130)、高尔基体蛋白97(golgin-97)和II类甘露糖苷酶(mannosidase II)对维持高尔基体结构极为重要,在氧化应激期间表达下降,可致高尔基体结构碎裂及功能受损,最终引起细胞凋亡[4, 6, 8-10]。因此,是否可通过调控高尔基体应激反应减轻LPS诱导的肺泡巨噬细胞急性损伤值得进一步探究。

本研究组前期研究表明,血红素加氧酶1(heme oxygenase-1, HO-1)是一种潜在的应激诱导蛋白,其在急性肺损伤中可通过改善线粒体动力学平衡及抑制内质网应激,以发挥内源性肺保护作用[11-13]。但HO-1在肺泡巨噬细胞内对高尔基体应激的调控,目前尚不清楚。本项工作拟通过构建脂多糖(lipopolysaccharide, LPS)诱导的肺泡巨噬细胞急性损伤模型,探究HO-1对高尔基体应激的调控作用,为研究其在急性肺损伤中的作用提供参考资料。

材料和方法

1 细胞和主要试剂

大鼠肺泡巨噬细胞系NR8383购自中国科学院。F12培养液和青链双抗购自HyClone;胎牛血清购自四季青公司;LPS购自Sigma;-小干扰RNA(small interfering RNA, siRNA)购自GE Dharmacon;转染试剂Lipofectamine 3000购自Invitrogen;qPCR引物由湖北百奥生物科技有限公司合成;逆转录试剂盒和实时荧光定量PCR试剂购自北京艾德莱生物科技有限公司;caspase-3/7试剂盒购自大连美仑生物技术有限公司;超氧化物歧化酶(superoxide dismutase, SOD)试剂盒和丙二醛(malondialdehyde, MDA)试剂盒购自南京建成生物研究所;细胞计数试剂盒8(Cell Counting Kit-8, CCK8)购自上海生工生物工程公司;TUNEL细胞检测试剂盒和DCFH-DA探针购自碧云天生物技术研究所;BCA蛋白定量试剂盒购自北京艾德莱生物科技有限公司;HO-1抗体、GOLPH3抗体和GM130抗体购自PTG;golgin-97抗体购自CST;mannosidase II抗体购自Santa;β-actin抗体、HRP标记的山羊抗兔抗体和HRP标记的山羊抗鼠抗体购自三箭生物技术有限公司。

2 主要方法

2.1细胞培养及分组将NR8383细胞培养于含10%胎牛血清和1%青链双抗的F12培养液,置于37 ℃、5% CO2饱和湿度的培养箱中。每2 d更换1次培养液。待细胞密度为80%左右时离心收集并传代。将细胞接种于12孔板,密度为5×107/L的细胞,采用随机数字表法分为4组(=3):对照(control)组、LPS组、LPS+Scr-siRNA组和LPS+-siRNA组。对照组细胞正常培养,其余3组参考文献[14]给予10 mg/L LPS制备肺泡巨噬细胞内毒素攻击模型。

2.2细胞转染及造模依据试剂说明,使用Lipofectamine 3000进行Scr-siRNA和siRNA转染。LPS+Scr-siRNA组细胞中加入Scr-siRNA和Lipofectamine 3000转染试剂,LPS+-siRNA组加入-siRNA和Lipofectamine 3000转染试剂,培养24 h后,加入LPS 10 mg/L继续培养24 h。

2.3细胞活力检测96孔板接种NR8383细胞,细胞接种量每孔5 000个。细胞贴壁后按照试验分组进行处理,再按照CCK-8试剂盒说明书操作,选择酶标仪于450 nm波长检测吸光度()值并计算相对细胞活力。

2.4ROS的检测DCFH-DA用无血清培养液稀释至10 μmol/L。细胞收集后悬浮于稀释的DCFH-DA中30 min,每2~3 min混合一次,最后在无血清培养液中洗涤细胞3次。以485 nm和535 nm的激发波长和发射波长计算DCF荧光。

2.5SOD活性和MDA含量的测定收集处理后的细胞,严格按照SOD和MDA试剂盒说明书操作,采用721分光光度仪检测值。

2.6caspase-3/7活性的检测收集处理后的细胞,根据说明,使用caspase-3/7活性检测试剂盒对样本进行caspase-3/7活性的测定。

2.7TUNEL染色收集处理后的细胞,采用4%多聚甲醛固定细胞,根据TUNEL凋亡试剂盒说明书进行TUNEL染色,再采用DAPI染色液复染细胞核。置于荧光显微镜下观察凋亡细胞,并通过计算TUNEL阳性细胞核数量占DAPI染色核数量的比例,计算凋亡细胞的百分率。

2.8RT-qPCR采用Trizol法[15]提取出NR8383细胞中的总RNA。逆转录合成cDNA,置于-20 ℃保存备用。RT-qPCR扩增目的基因mRNA。反应条件:95 ℃ 5 min;95 ℃ 10 s,60 ℃ 30 s,40个循环。采用荧光定量PCR仪测定Ct值,采用2-ΔΔCt法对目的基因的相对表达量进行分析。相应引物序列见表1。

表1 RT-qPCR引物序列

2.9Western blot实验收集细胞后,采用全蛋白试剂提取试剂盒提取总蛋白并以BCA法测定蛋白浓度。取40 μg样品于12% SDS-PAGE分离、转至PVDF膜、5%的脱脂奶粉37 ℃封闭1 h、TBST洗膜后,分别加入抗HO-1(1∶1 000)、GOLPH3(1∶1 000)、GM130(1∶200)、golgin-97(1∶1 000)、mannosidase II(1∶200)和β-actin(1∶8 000)抗体,4 ℃孵育过夜。TBST洗膜4次,加入山羊抗兔或抗鼠Ⅱ抗(1∶10 000),室温下孵育1 h。TBST洗脱4次后,置于ECL系统中显影,采用ImageJ软件分析蛋白条带灰度值,以目的蛋白条带灰度值与β-actin条带灰度值的比值反映目的蛋白相对表达水平。

3 统计学处理

采用SPSS 24.0软件进行分析。正态分布的计量资料以均数±标准差(mean±SD)表示。多组均数间比较采用单因素方差分析(one-way ANOVA),组间两两比较采用Tukey´s事后检验进行比较。以<0.05为差异有统计学意义。

结果

1 肺泡巨噬细胞活力和氧化应激水平的变化

与对照组相比,LPS诱导肺泡巨噬细胞活力下降,ROS和MDA含量增加,SOD活性下降(<0.05);沉默-后,与LPS组相比,肺泡巨噬细胞活力显著下降,ROS和MDA含量显著增加,SOD活性显著下降(<0.05),见图1。

Figure 1.Cell viability, ROS content, MDA content and SOD activity in alveolar macrophages. A: the viability of alveolar macrophages was detected by CCK-8 assay; B: the level of ROS was analyzed by DCFH-DA probing; C: the change of MDA content was detected by MDA kit; D: the change of SOD activity was analyzed by SOD kit. Mean±SD. n=3. *P<0.05 vs control group; #P<0.05 vs LPS group.

2 HO-1和高尔基体应激蛋白GOLPH3表达的变化

RT-qPCR和Western blot结果显示,与对照组相比,LPS诱导的肺泡巨噬细胞HO-1表达增加(<0.05),高尔基体应激蛋白GOLPH3 mRNA和蛋白表达增加(<0.05);沉默后,其在肺泡巨噬细胞中的表达显著下调(<0.05),见图2、3,GOLPH3表达高于LPS组(<0.05),见图4。

Figure2.The transfection efficiency of HO-1 siRNA in alveolar macrophages. The HO-1 protein level was detected by Western blot. Mean±SD. n=3. *P<0.05 vs control group.

Figure 3.HO-1 expression levels in alveolar macrophages. A: HO-1 mRNA level in each group was measured by RT-qPCR; B: HO-1 protein expression in each group was measured by Western blot. Mean±SD. n=3. *P<0.05 vs control group; #P<0.05 vs LPS group.

Figure 4.GOLPH3 expression levels in alveolar macrophages. A: GOLPH3 mRNA expression of each group was measured by RT-qPCR; B: GOLPH3 protein expression of each group was measured by western blot. Mean±SD. n=3. *P<0.05 vs control group; #P<0.05 vs LPS group.

3 高尔基体结构相关蛋白表达的变化

LPS诱导的肺泡巨噬细胞中,高尔基体结构相关蛋白GM130、golgin-97及mannosidase II表达水平下降(<0.05);沉默-后,与LPS组相比,GM130、golgin-97及mannosidase II表达水平显著下降(<0.05),见图5。

Figure 5.The expression of Golgi structure-related proteins (GM130, golgin-97 and mannosidase II) in alveolar macrophages measured by Western blot. Mean±SD. n=3. *P<0.05 vs control group; #P<0.05 vs LPS group.

4 肺泡巨噬细胞凋亡的变化

与对照组相比,LPS诱导的肺泡巨噬细胞中TUNEL标记阳性细胞数增多,caspase-3/7活性增强(<0.05);沉默-后,与LPS组相比,TUNEL标记阳性细胞显著增多,caspase-3/7活性显著增强(<0.05),见图6。

Figure 6.The apoptosis of alveolar macrophages in each group. A: the apoptosis of alveolar macrophages was detected by caspase-3/7 activity determination; B: the apoptosis of alveolar macrophages was detected by TUNEL assay (green: TUNEL-positive nuclei; blue: DAPI-stained nuclei; scale bar=50 μm). Mean±SD. n=3. *P<0.05 vs control group; #P<0.05 vs LPS group.

讨论

ALI是以弥漫性肺泡损伤、肺水肿、免疫细胞浸润和肺顺应性下降为特征的临床综合征,严重时可致患者死亡[16]。内毒素是ALI的常见原因,可激活巨噬细胞引发炎症[17]。正常情况下,肺泡巨噬细胞具有维持肺免疫稳态和抑制固有免疫的双重作用[2]。在ALI病程中,肺泡巨噬细胞主要通过早期启动炎症和晚期抑制炎症免疫反应以发挥关键作用[16, 18]。研究表明,肺泡巨噬细胞功能障碍和凋亡增加会抑制肺部中性粒细胞清除,导致炎症消退延迟和组织损伤[1, 18]。因此,肺泡巨噬细胞在ALI中的重要作用表明肺泡巨噬细胞可能是治疗ALI的潜在靶点。

高尔基体是细胞内重要的信号中枢,具有重要生物合成和加工、运输、分选等功能[19]。在氧化应激、DNA损伤和营养缺乏等应激条件下,高尔基体通过表达GOLPH3感知并启动应激反应,高尔基体结构相关蛋白GM130、golgin-97等被切割降解,导致高尔基体结构碎裂和功能受损,引起细胞凋亡[4, 6, 10]。高尔基体应激也可向下游传递应激信号,促进细胞凋亡[4]。此外,许多位于高尔基体的促凋亡因子半胱天冬酶-2等可反向诱导高尔基体结构相关蛋白GM130、golgin-97和golgin-160等裂解[4,20-21]。研究表明,一些神经退行性疾病和神经发育疾病与高尔基体应激蛋白GOLPH3表达增加和高尔基体结构相关蛋白裂解密切相关[22-23]。本研究组前期研究表明,高尔基体应激在急性肺损伤中发挥重要作用[24],但在LPS刺激的肺泡巨噬细胞模型中的作用尚不清楚。因此,本实验选择LPS诱导NR838细胞以制备肺泡巨噬细胞急性损伤模型进行实验,以探究高尔基体应激在LPS诱导急性肺损伤的作用机制提供研究依据。结果显示,LPS攻击肺泡巨噬细胞时,细胞氧化应激和高尔基体应激增加,高尔基体结构相关蛋白表达下降,细胞凋亡增加。

HO-1是一种重要的抗氧化酶,在LPS诱导的肺损伤、心肌损伤和肾损伤等多组织损伤中发挥保护性作用[25-27]。本课题组前期研究表明,HO-1可通过促进线粒体融合蛋白1、线粒体融合蛋白2和视神经萎缩蛋白1的表达,抑制线粒体分裂蛋白1和线粒体动力相关蛋白1的表达,以调节线粒体融合分裂平衡,减轻急性肺损伤[12, 28]。此外,HO-1还可通过调节线粒体质量控制,抑制内质网应激以发挥抗炎、抗氧化、抗凋亡作用[13, 29]。本实验结果显示,下调HO-1表达后,LPS诱导的肺泡巨噬细胞氧化应激和高尔基体应激加重,高尔基体结构相关蛋白表达进一步下降,细胞凋亡显著增加,表明HO-1可能通过调控高尔基体应激和氧化应激以减轻LPS诱导的肺泡巨噬细胞损伤,减少细胞凋亡。但HO-1调控高尔基体应激的具体机制,以及在体内实验中HO-1是否可通过调控高尔基体应激,减轻细胞凋亡,以改善急性肺损伤仍需进一步研究。

综上所述,内毒素攻击大鼠肺泡巨噬细胞时,HO-1可以调控高尔基体应激和氧化应激,减少细胞凋亡。

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Role of HO-1 on Golgi stress in LPS-stimulated rat alveolar macrophages

LI Yu-ting, LI Xiang-yun, SHI Jia, LI Cui, YU Jian-bo△

(,,,,300100,)

To evaluate the role of heme oxygenase-1 (HO-1) on Golgi stress in lipopolysaccharide (LPS)-stimulated rat alveolar macrophages.The injury model of rat alveolar macrophages was established by LPS stimulation. CCK-8 assay was applied to measure cell viability, and the DCFH-DA probing was used to detect the generation of reactive oxygen species (ROS). In addition, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were assayed by SOD and MDA kits, and apoptosis was analyzed by TUNEL assay and caspase-3/7 activity determination. The mRNA expression levels of HO-1 and Golgi phosphoprotein 3 (GOLPH3) were detected by RT-qPCR, and the protein expression levels of HO-1, GOLPH3 and Golgi structure-related proteins (GM130, golgin-97 and mannosidase II) were detected by Western blot.small interfering RNA (siRNA) was applied to reduce the expression ofin rat alveolar macrophages.Stimulation with LPS reduced cell viability and SOD activity, increased the levels of ROS and MDA, up-regulated the expression of HO-1 and GOLPH3, impaired the expression of GM130, golgin-97 and mannosidase II, and further increased TUNEL positive rate and caspase-3/7 activityin rat alveolar macrophages (<0.05). Knockdown ofsignificantly inhibited cell viability and SOD activity, increased ROS and MDA levels, decreased the expression of HO-1, GM130, golgin-97 and mannosidase II, increased the expression of GOLPH3, and further increased TUNEL positive rate and caspase-3/7 activity (<0.05).The HO-1 attenuates oxidative stress and Golgi stress response, and prevents apoptosis in LPS-stimulated alveolar macrophages.

Golgi stress; Heme oxygenase-1; Lipopolysaccharides; Alveolar macrophages; Oxidative stress

R329.2+5; R363

A

10.3969/j.issn.1000-4718.2022.03.016

1000-4718(2022)03-0509-08

2021-07-05

2022-01-13

[基金项目]国家自然科学基金资助项目(No.81772106)

Tel: 022-27435873; Email: jianboyu99@sina.com

(责任编辑:林白霜,罗森)

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