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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 study the effects of fine particulate matter (PM2.5) on the injury of EA.hy926 human umbilical vein endothelial cells and the protective effect of curcumin. Methods EA.hy926 cells exposed to PM2.5 in Guangzhou urban area were collected and divided into blank group, model group A (PM2.5, 20μg · m L -1), model group B (PM2.5, 200μg · M L -1), model group C (PM 2.5, 400 μg · m L -1). After the preparation of the model, the dose of 200μg · m L ~ (-1) of PM2.5 could significantly affect the survival rate, apoptosis, inflammation and oxidative stress of EA.hy926 cells. For this drug intervention, the dosage of PM2.5 200μg · m L -1. The following experiments were divided into blank group, model group (200μg · m L ~ (-1) PM2.5), low, medium and high doses of three experimental groups (PM2.5 + low dose curcumin group, PM2.5 + Group and PM2.5 + high dose curcumin group), the cells were pretreated with 5, 10, 20μmol·L -1 curcumin for 1 h, and then treated with 200 μg · m L -1 PM2.5 The cells were exposed to Anthrapyrazolone (SP600125) (10μmol·L -1) for 24 hours. The cells were treated with 200μg · m L -1 PM2.5 for 24 hours. The cell viability was measured by MTT colorimetric assay and the apoptosis was detected by flow cytometry. The protein expression of p-JNK, Bax and Bcl-2 was detected by Western blotting, and the expression of Bcl-2 protein was detected by enzyme-linked immunosorbent assay (IL-6) and tumor necrosis factor-α (TNF-α) were measured. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. Results Compared with the blank group of indicators, the model B, C group were significantly changed, indicating successful modeling. Compared with the model group (83.27 ± 1.41)%, the survival rates of EA.hy926 cells in experimental group and control group were (88.37 ± 1.46)%, (91.49 ± 1.34)% and (88.35 ± 1.06)%, respectively (P <0.05). Compared with the model group, the expression of p-JNK protein in experimental group and control group was (0.41 ± 0.03)%, %, (0.39 ± 0.04)% and (0.10 ± 0.01)%, respectively (P <0.05). Compared with model group, the ratio of Bax / Bcl-2 protein was 4.29 (16.34 ± 1.29)% and (9.44 ± 0.98)%, respectively, compared with the model group (22.35 ± 1.21)% ) And (14.45 ± 1.12)%, respectively (P <0.05). Compared with the model group, IL-6 was (523.67 ± 14.36) ng · L -1, (465.31 ± 14.29), (375.52 ± 14.55) and (480.44 ± 16.12) ng · L -1, respectively (P <0.05), and the levels of TNF-α in the model group were ( 387.34 ± 8.42 ng · L -1. The levels of TNF-αin the middle and high dosage groups were (329.45 ± 7.57), (240.46 ± 7.34), (352.53 ± 8.25) ng (P <0.05). Compared with the model group, the SOD activity was (11.46 ± 0.35) μmol·L -1. The SOD activity in the experimental group and the control group (13.87 ± 0.31), (16.43 ± 0.32) and (14.45 ± 0.36) μmol·L -1, respectively. The content of MDA in the model group was (4.15 ± 0.12) μmol·L -1, The contents of MDA in experimental group and control group were (3.21 ± 0.22), (2.38 ± 0.23) and (3.49 ± 0.26) μmol·L -1 respectively (P <0.05) (1.14 ± 0.06) kU · L -1. Compared with the model group, the LDH activities of the two experimental groups and the control group were (0.94 ± 0.05), (0.73 ± 0.05) and (0.84 ± 0.06) kU · L ~ (-1), respectively (P <0.05). Conclusion Curcumin can reduce the injury of EA.hy926 cells by inhibiting the JNK pathway.