姜黄素减轻大气细颗粒物对血管内皮细胞损伤的作用

Alleviating effect of curcumin on vascular endothelial cells injury by PM2. 5- induced

目的研究大气细颗粒物(PM2.5)对EA.hy926型人脐静脉内皮细胞损伤的影响及姜黄素的保护作用。方法采集广州市区大气PM2.5并染毒的EA.hy926细胞,分为空白组、模型A组(PM2.5,20μg·m L^(-1))、模型B组(PM2.5,200μg·m L^(-1))、模型C组(PM2.5,400μg·m L^(-1))。模型制备后测得PM2.5剂量200μg·m L^(-1)可显著影响EA.hy926细胞存活率、凋亡及炎症和氧化应激指标,为此药物干预中,PM2.5剂量均选为200μg·m L^(-1)。以下实验分为空白组、模型组(200μg·m L^(-1)PM2.5)、低中高3个剂量实验组(PM2.5+低剂量姜黄素组、PM2.5+中剂量姜黄素组、PM2.5+高剂量姜黄素组),分别以5,10,20μmol·L^(-1)姜黄素预处理细胞1 h后,加200μg·m L^(-1)的PM2.5染毒24h;对照组,以10μmol·L^(-1)的Anthrapyrazolone(SP600125)预处理细胞30 min后,加200μg·m L^(-1)的PM2.5染毒24 h。用噻唑蓝(MTT)比色法测细胞存活率,以流式细胞术测细胞凋亡;用蛋白免疫印迹法检测p-JNK、Bax、Bcl-2蛋白表达,用酶联免疫吸附测定白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)含量,并测定丙二醛(MDA)含量、超氧化物歧化酶(SOD)及乳酸脱氢酶(LDH)活性。结果与空白组的各项指标比较,模型B、C组均有明显改变,说明造模成功。与模型组的(83.27±1.41)%比较,中、高2个剂量实验组及对照组EA.hy926细胞存活率分别为(88.37±1.46)%,(91.49±1.34)%,(88.35±1.06)%显著增加(P<0.05);与模型组的p-JNK蛋白表达(0.82±0.03)%比较,中、高2个剂量实验组及对照组的p-JNK蛋白表达分别为(0.41±0.03)%,(0.39±0.04)%,(0.10±0.01)%明显下调(P<0.05);与模型组的Bax/Bcl-2蛋白比率4.29比较,中、高2个剂量实验组及对照组的分别为1.44,1.10,1.95;与模型组的凋亡率(22.35±1.21)%比较,中、高2个剂量实验组及对照组的凋亡率分别为(16.34±1.29)%,(9.44±0.98)%,(14.45±1.12)%显著降低(P<0.05);与模型组的IL-6为(523.67±14.36)ng·L^(-1)比较,中、高2个剂量实验组及对照组的IL-6分别为(465.31±14.29),(375.52±14.55),(480.44±16.12)ng·L^(-1)显著降低(P<0.05);与模型组的TNF-α含量为(387.34±8.42)ng·L^(-1)比较,中、高2个剂量实验组及对照组的TNF-α含量分别为(329.45±7.57),(240.46±7.34),(352.53±8.25)ng·L^(-1)显著降低(P<0.05);与模型组的SOD活性为(11.46±0.35)μmol·L^(-1)比较,中、高2个剂量实验组及对照组的SOD活性分别为(13.87±0.31),(16.43±0.32),(14.45±0.36)μmol·L^(-1)显著增加;与模型组的MDA含量为(4.15±0.12)μmol·L^(-1)比较,中、高2个剂量实验组及对照组的MDA含量分别为(3.21±0.22),(2.38±0.23),(3.49±0.26)μmol·L^(-1)明显降低(P<0.05);与模型组的LDH活性为(1.14±0.06)k U·L^(-1)比较,中、高2个剂量实验组及对照组的MDA含量分别为LDH活性分别为(0.94±0.05),(0.73±0.05),(0.84±0.06)k U·L^(-1)明显降低(P<0.05)。结论姜黄素可通过抑制JNK通路,减轻PM2.5对EA.hy926细胞的损伤。

Objective To investigate the alleviating effect of curcumin on injury of human umbilical vein endothelial cells EA. hy926 PM2. 5- induced. Methods The samples of fine particulate matter（ PM2. 5） were collected in Guangzhou and made into suspension. Different concentration of PM2. 5 were added in EA. hy926 cells and were randomly divided into blank group,model- A group（ 20 μg · m L-1）,model- B group（ 200 μg·m L-1）,model- C group（ 400 μg·m L-1）. As 200 μg·m L-1PM2. 5 significantly affected the survival rates,apoptosis,inflammation and oxidative stress biomarkers in EA. hy 926 cells,so the concentration of PM2. 5 200 μg·m L-1was sected in this test. And then it were randomly divided into blank group,model group（ 200 μg·m L-1PM2. 5）,test- L,test- M,test- H groups（ 5,10,20 μmol · L-1curcumin for 1 h,and then 200 μg · m L-1PM2. 5 for 24 h）,and control group（ 10μmol·L-1Anthrapyrazolone（ SP600125） for 30 min,and then 200 μg·m L-1PM2. 5 for 24 h）. The survival rate and apoptosis of EA. hy926 cells were measured by MTT ssay,flow cytometry. The expressions of p- JNK,Bax and Bcl- 2 in EA. hy926 cells were measured by Western blot. The contents of interleukin- 6（ IL- 6） and tumor necrosis factor- α（ TNF- α）,malonaldehyde（ MDA） content,superoxide dismutase（ SOD） and lactic dehydrogenase（ LDH） activities in EA. hy 926 cells supernatant were measured by enzyme- linked immuno- sorbent assay and colorimetry,respectively. Results Compared with blank group,the items in model group were all significantly. That means the model was established successfully. Compared to model group on the survival rates with（ 83. 27 ± 1. 41） %,the survival rates in test- M group, test- H group and control group were significantly decreased with（ 88. 37 ± 1. 46） %,（ 91. 49 ± 1. 34） %,（ 88. 35 ± 1. 06） %（ P 〈 0. 05）. Compared to model group on p- JNK expressions with（ 0. 82 ±0. 03） %,the p- JNK expressions in test- M group,test- H group and control group were significantly decreased with（ 0. 41 ± 0. 03） %,（ 0. 39 ± 0. 04） %,（ 0. 10 ± 0. 01） %（ P 〈 0. 05）. Compared to model group on Bax /Bcl- 2 rates with 4. 29,the Bax / Bcl- 2 rates in test- M group,test- H group and control group were significantly decreased with1. 44,1. 10,1. 95. Compared to model group on the apoptosis rate with（ 22. 35 ± 1. 21） %,the apoptosis rate in test-M group,test- H group and control group were significantly decreased with（ 16. 34 ± 1. 29） %,（ 9. 44 ± 0. 98） %,（ 14. 45 ± 1. 12） %（ P 〈 0. 05）. Compared to model group on the IL- 6 with（ 523. 67 ± 14. 36） ng·L-1,the IL- 6 in test- M group,test- H group and control group were significantly decreased with（ 465. 31 ± 14. 29）,（ 375. 52 ±14. 55）,（ 480. 44 ± 16. 12） ng · L-1. Compared to model group on the contents of TNF- α with（ 387. 34 ± 8. 42）ng·L-1,the contents of TNF- α in test- M group,test- H group and control group were significantly decreased with（ 329. 45 ±7. 57）,（ 240. 46 ±7. 34）,（ 352. 53 ±8. 25） ng·L-1（ P 〈0. 05）. Compared to model group on the SOD activitie with（ 11. 46 ± 0. 35） μmol·L-1,the SOD activitie in test- M group,test- H group and control group were significantly increased with（ 13. 87 ± 0. 31）,（ 16. 43 ± 0. 32）,（ 14. 45 ± 0. 36） μmol ·L-1（ P 〈 0. 05）. Compared to model group on the contents of MDA with（ 4. 15 ± 0. 12） μmol·L-1,the contents of MDA in test- M group,test- H group and control group were significantly decreased with（ 3. 21 ± 0. 22）,（ 2. 38 ± 0. 23）,（ 3. 49 ± 0. 26） μmol ·L-1（ P 〈 0. 05）.Compared to model group on the LDH activitie with（ 1. 14 ± 0. 06） k U · L-1,the LDH activitie in test- M group,test- H group and control group were significantly decreased with（ 0. 94 ± 0. 05）,（ 0. 73 ± 0. 05）,（ 0. 84 ± 0. 06）k U·L-1. Conclusion Curcumin alleviates EA. hy926 cells injury PM2. 5- induced via the inhibition of JNK pathway.