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浙江大学学报(医学版)  2022, Vol. 51 Issue (4): 405-414    DOI: 10.3724/zdxbyxb-2022-0159
专题报道     
槟榔多酚对大鼠肺微血管内皮细胞低氧损伤的保护作用
贺嘉馨1,2,霍妍1,2,孙月梅1,2,程俊飞1,2,赵以览1,2,李文斌1,2,*,王荣1,2,*
1. 中国人民解放军联勤保障部队第九四〇医院药剂科 全军高原医学实验室,甘肃 兰州 730050
2. 兰州大学药学院,甘肃 兰州 730000
Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells
HE Jiaxin1,2,HUO Yan1,2,SUN Yuemei1,2,CHENG Junfei1,2,ZHAO Yilan1,2,LI Wenbin1,2,*,WANG Rong1,2,*
1. Department of Pharmacy, the 940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army, Key Laboratory of the Plateau Medicine, Lanzhou 730050, China;
2. School of Pharmacy, Lanzhou University, Lanzhou 730000, China
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摘要:

目的:探究槟榔多酚对大鼠肺微血管内皮细胞(PMVEC)低氧损伤的保护作用。 方法:采用氧化应激指标丙二醛和超氧化物歧化酶筛选肺部缺氧损伤细胞模型最适条件;采用细胞计数试剂盒法筛选槟榔多酚最适给药剂量。将PMVEC分为常氧对照组、模型对照组和槟榔多酚组,采用BCA法检测蛋白浓度并测定PMVEC中的氧化应激水平,蛋白质印迹法检测炎症和凋亡相关蛋白的表达,免疫荧光染色法检测闭合蛋白和闭锁小带1等紧密连接蛋白的表达,Transwell小室实验检测跨内皮细胞膜电阻,罗丹明荧光染料检测PMVEC屏障的通透性。 结果:1%氧浓度培养PMVEC细胞 48?h后成功构建肺部缺氧损伤细胞模型 ;20?μg/mL的槟榔多酚可以显著逆转缺氧损伤细胞模型中PMVEC的存活率下降以及氧化应激水平的升高(均 P<0.05);槟榔多酚对于低氧诱导下PMVEC中炎症相关蛋白核因子κB(NF-κB)和核因子E2相关因子(Nrf)2的上调有显著抑制作用(均P<0.05);槟榔多酚可以下调低氧诱导下PMVEC中凋亡相关蛋白胱天蛋白酶(caspase)3、Bcl-2相关X蛋白(Bax)的表达(均P<0.05),从而减轻低氧诱导下细胞的凋亡;槟榔多酚显著上调低氧诱导下PMVEC闭合蛋白和闭锁小带1的表达(均P<0.05),提高跨膜电阻值并降低罗丹明透过量,改善肺微血管内皮屏障的通透性。结论:槟榔多酚可以通过减轻PMVEC的氧化应激损伤、降低炎症相关蛋白表达、减少细胞凋亡及提高细胞屏障的通透性,改善PMVEC的低氧损伤。

关键词: 槟榔多酚低氧肺损伤细胞凋亡紧密连接蛋白氧化应激肺微血管内皮细胞    
Abstract:

Objective: To investigate the protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells (PMVECs). Methods: Malondialdehyde and superoxide dismutase (SOD) were used to determine the optimal modeling of lung hypoxic injury cells. CCK-8 method was used to detect cell viability for determining the effective dose of areca nut polyphenols. Rat PMVECs were divided into control group, hypoxia model group and areca nut polyphenols group. BCA method was used to detect the protein concentration of each group, and the oxidative stress level in PMVECs was measured. Western blotting was used to detect the expression of inflammatory and apoptosis-related proteins. Immunofluorescence staining was used to detect the expression of occludin and zonula occludens (ZO) 1. Transwell chamber was used to detect transendothelial electrical resistance, and rhodamine fluorescent dye was used to detect PMVECs barrier permeability. Results: The hypobaric hypoxia-induced cell injury model was established by culturing PMVECs for 48?h at 1% oxygen concentration. The 20?μg/mL areca nut polyphenols significantly reversed the survival rate and the oxidative stress of PMVECs in hypoxia model group (all P<0.05). Areca nut polyphenols had significant inhibitory effect on the up-regulation of inflammation-related proteins, including nuclear factor-κB (NF-κB) and nuclear factor-E2-related factor (Nrf) 2 in hypoxia model group (allP<0.05). And areca nut polyphenols could reduce hypoxia-induced PMVECs apoptosis by down-regulating the expressions of apoptosis-related proteins, including cysteine aspartic acid specific protease (caspase) 3, Bcl-2 associated X protein (Bax) in PMVECs (allP<0.05). In addition, areca nut polyphenols effectively improves the transendothelial electrical resistance and barrier permeability of PMVECs through elevating the expression of occludin and ZO-1 (allP<0.05).Conclusion: Areca nut polyphenols can inhibit the hypoxic damage of PMVECs by reducing oxidative stress and apoptosis down-regulating the expression of inflammatory proteins and reducing membrane permeability.

Key words: Areca nut polyphenols    Hypoxic lung injury    Apoptosis    Tight junction proteins    Oxidative stress    Pulmonary microvascular endothelial cells
收稿日期: 2022-04-14 出版日期: 2022-11-16
CLC:  R96  
基金资助: 国家自然科学基金(82173738); 中央高校基本科研业务费专项(3192020009); 甘肃省自然科学基金(20JR10RA014); 军队后勤医学科技青年培育计划(20QNPY070)
通讯作者: 李文斌,王荣   
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引用本文:

贺嘉馨,霍妍,孙月梅,程俊飞,赵以览,李文斌,王荣. 槟榔多酚对大鼠肺微血管内皮细胞低氧损伤的保护作用[J]. 浙江大学学报(医学版), 2022, 51(4): 405-414.

HE Jiaxin,HUO Yan,SUN Yuemei,CHENG Junfei,ZHAO Yilan,LI Wenbin,WANG Rong. Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells. J Zhejiang Univ (Med Sci), 2022, 51(4): 405-414.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0159        https://www.zjujournals.com/med/CN/Y2022/V51/I4/405

图1  PMVEC免疫荧光染色鉴定结果

组 别

n

丙二醛(nmol/mg)

SOD(U/mg)

常氧对照组

3

0.57±0.05

28.8±3.7

缺氧 24?h模型组

3

1.22±0.19 **

24.2±1.8 *

缺氧 48?h模型组

3

1.44±0.29 **#

20.8±1.1 **#

缺氧 72?h模型组

3

1.58±0.25 **#

16.1±1.4 **##

表1  不同缺氧时间PMVEC中丙二醛及SOD水平
图2  各组PMVEC中活性氧的荧光染色结果

组 别

n

丙二醛(nmol/mg)

SOD (U/mg)

活性氧 (×10 5AU)

常氧对照组

3

0.65±0.07

28.9±3.7

61.8±2.5

模型对照组

3

1.59±0.10 **

22.0±1.2 **

136.1±6.4 **

槟榔多酚组

3

1.18±0.07 #

25.4±0.9 #

92.0±5.7 ##

表2  各组氧化应激相关指标检测结果比较
图3  各组PMVEC中NF-κB及Nrf-2的表达 ( =3) nA:各组蛋白表达电泳图;B:各组蛋白表达水平比较. 与常氧对照组比较, <0.01;与模型对照组比较, <0.01. PMVEC:肺微血管内皮细胞;NF-κB:核因子κB;Nrf:核因子E2相关因子.
图4  各组PMVEC中凋亡相关蛋白的表达 ( =3) nA:各组蛋白表达电泳图;B:各组蛋白表达水平比较. 与常氧对照组比较, <0.01;与模型对照组比较, <0.05, <0.01. PMVEC:肺微血管内皮细胞;HIF:缺氧诱导因子;caspase:胱天蛋白酶;Bcl:B细胞淋巴瘤;Bax:Bcl-2相关X蛋白.
图5  各组PMVEC中闭合蛋白及ZO-1荧光染色图 A:闭合蛋白表达;B:ZO-1表达. 蓝色荧光指示细胞核, 红色荧光指示闭合蛋白和ZO-1阳性表达. 低氧处理后模型对照组细胞中闭合蛋白和ZO-1荧光表达及扩散面积明显减小, 槟榔多酚给药后荧光强度有所增强, 部分逆转了模型对照组中闭合蛋白、ZO-1荧光强度的减弱, 且闭合蛋白的变化较ZO-1更为显著. 标尺 =50 μm. PMVEC:肺微血管内皮细胞;DAPI:4′,6-二脒基-2-苯基吲哚;ZO:闭锁小带.
图6  各组PMVEC中闭合蛋白和ZO-1的表达( =3) nA:各组蛋白表达电泳图;B:各组蛋白表达水平比较. 与常氧对照组比较, <0.01;与模型对照组比较, <0.05, <0.01. PMVEC:肺微血管内皮细胞;DAPI:4′,6-二脒基-2-苯基吲哚;ZO:闭锁小带.

组 别

n

跨膜电阻值(Ω/cm 2)

罗丹明透过量(μmol/cm 2)

常氧对照组

3

38.3±2.1

0.056±0.002

模型对照组

3

20.3±1.7 **

0.076±0.003 **

槟榔多酚组

3

29.7±2.6 *#

0.067±0.003 *#

表3  各组肺微血管内皮细胞单层细胞屏障通透性比较
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