NF-κB信号通路在原花青素拮抗砷诱导的BEAS-2B细胞炎性损伤中的作用研究

发布时间：2018-06-13 14:29

本文选题：砷 + 肺　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:原花青素(PC)具有较强抗炎性损伤作用,探讨葡萄籽原花青素(GSPE)拮抗砷诱导的肺脏炎性损伤机制及NF-κB信号通路在其中的作用,为砷中毒防治提供参考。方法:本研究以人支气管上皮细胞(Human bronchial epithelial cells,BEAS-2B)细胞为对象,根据As_2O_3(0、5、10和20μmol/L)、GSPE(0、25和50mg/L)和BAY 11-7082(NF-κB抑制剂,抑制刺激物所诱导的IκB-α磷酸化,0和5μmol/L)的剂量分为48组。采用MTT法测量细胞的存活率,用流式细胞术测定细胞的凋亡率;依据ELISA法测定BEAS-2B细胞匀浆中IL-1β、IL-6、IL-10、TNF-α和CRP的表达水平;q RT-PCR测定BEAS-2B细胞匀浆中IκB-α、IKKα、IKKβ、NF-κBp65、NF-κBp50的m RNA水平;Western-Blot测定BEAS-2B细胞匀浆中P-IκB-α、IκB-α、IKKα/β、NF-κBp65、NF-κBp50的蛋白表达水平。多组之间的比较采用方差分析,两两之间的比较使用Bonferroni法。结果:1.随着As_2O_3的染毒剂量增加,暴露砷24h时BEAS-2B的存活率下降(100%至65.26%,P均0.05),凋亡率升高(1.7%至30.4%,P均0.05);于48h时呈现出类似的趋势。在施加BAY 11-7082后,细胞存活率升高,凋亡率下降;当使用GSPE拮抗砷时,细胞存活率升高(65.26%至83.38%,P均0.05),凋亡率下降(30.4%至13%,P均0.05)。2.与空白对照组相比,砷染毒于BEAS-2B24h时使促炎因子(IL-1β、IL-6、TNF-α和CRP),ROS和LPO的表达水平增加(P均0.05),抑炎因子(IL-10)的表达水平减少(P0.05);且随着砷的染毒剂量增加,促炎因子,ROS和LPO的表达水平升高(P均0.05),抑炎因子下降(P0.05)。相较于砷染毒组,在施加BAY 11-7082之后发现,促炎因子,ROS和LPO的表达量明显下降(P均0.05),而抑炎因子上升(P0.05);使用GSPE干预后,促炎因子,ROS和LPO的表达水平下降(P均0.05),而抑炎因子的表达量增加(P0.05)。3.qRT-PCR结果表明,相较于空白对照组,砷促进IKKα、IKKβ、NF-κBp65、NF-κBp50 m RNA的水平升高(P均0.05),降低IκB-αm RNA的水平(P0.05)。与砷染毒组相比较,在施加BAY 11-7082或GSPE后发现,IKKα、IKKβ、NF-κBp65、NF-κBp50 m RNA的水平降低(P均0.05),IκB-αm RNA的水平增加(P0.05)。4.Western-Blot结果显示,同空白对照组相比较,砷可以增加P-IκB-α、IKKα/β、NF-κBp65、NF-κBp50与β-actin的比值(P均0.05),减少IκB-α与β-actin的比值(P0.05)。相较于砷染毒组,在施加BAY 11-7082或GSPE后,P-IκB-α、IKKα/β、NF-κBp65、NF-κBp50与β-actin的比值(P均0.05)减少,IκB-α与β-actin的比值增加(P0.05)。结论:砷暴露会增加BEAS-2B的凋亡率,降低存活率,可能是激活了NF-κB信号通路,诱导促炎性因子的表达水平增加,降低抑炎因子的表达量,导致炎性损伤;而GSPE可以抑制砷所致的NF-κB信号通路的激活,减少促炎性因子的表达,增加了抑炎因子的表达,表明了GSPE可以通过抑制砷所致NF-κB信号通路的激活,从而调节下游炎性因子的表达量,拮抗了砷所致的炎性损伤作用。
[Abstract]:Objective: proanthocyanidin (PC) has a strong anti-inflammatory injury effect. To explore the mechanism of GSPE antagonizing arsenic induced pulmonary inflammatory injury and the role of NF- 魏 B signaling pathway in preventing and treating arsenic poisoning. Methods: human bronchial epithelial cells BEAS-2B) cells were divided into 48 groups according to the doses of As2O3 / L (10 and 20 渭 mol / L) and BAY11-7082 (NF- 魏 B inhibitor). Cell viability was measured by MTT assay and apoptosis rate was measured by flow cytometry. The expression of IL-10 TNF- 伪 and CRP in BEAS-2B cell homogenate was determined by Elisa. The protein expression of P-I 魏 B- 伪 -taik 魏 Bp65, NF- 魏 Bp50 in BEAS-2B cell homogenate was determined by Western-Blot. The expression level of P-I 魏 B- 伪 -taik- 伪 -kapp65 NF- 魏 Bp50 protein in BEAS-2B cell homogenate was determined by Western Blot, and the expression of I 魏 B- 伪 / 尾 -NF- 魏 Bp65 NF- 魏 Bp50 protein in BEAS-2B cell homogenate was determined by Western-Blot. The analysis of variance was used for the comparison between the two groups and the Bonferroni method was used for the comparison between the two groups. The result is 1: 1. The survival rate of BEAS-2B decreased by 100% to 65.26% and the apoptosis rate increased by 1.7% to 30.4% after exposure to arsenic for 24 h, and a similar trend was observed at 48 h. After application of BAY11-7082, the cell survival rate increased and the apoptosis rate decreased, and the cell survival rate increased by 65.26% to 83.38%, and the apoptosis rate decreased by 30.4% to 0.05%, respectively, when GSPE was used to antagonize arsenic. Compared with the control group, arsenic exposed to BEAS-2B for 24 hours increased the expression levels of IL-6TNF- 伪, CRPG-Ros and LPO, and decreased the expression level of anti-inflammatory factor IL-10 (P0.05N), and with the increase of arsenic exposure dose, The expression levels of Ros and LPO were both increased (P 0.05), and those of anti-inflammatory factor were decreased (P 0.05). Compared with arsenic exposure group, after applying BAY11-7082, it was found that the expression of Ros and LPO decreased significantly, while the anti-inflammatory factor increased P0.050.After GSPE treatment, the expression of Ros and LPO decreased significantly. The expression level of Ros and LPO decreased (P 0.05), while the expression of anti-inflammatory factor increased (P 0.05). The results of qRT-PCR showed that compared with the control group, arsenic increased the level of NF- 魏 Bp65, NF- 魏 Bp50 mRNA and decreased the level of I 魏 B- 伪 mRNA. Compared with the arsenic exposed group, it was found that the level of Ike 伪 -IKK 尾 -NF- 魏 Bp65, NF- 魏 Bp50 mRNA decreased, the level of I 魏 B- 伪 mRNA increased by P0.05. 4. Western-Blot results showed that compared with the control group, arsenic could increase the ratio of P-I B- 伪 -kek 伪 / 尾 -NF- 魏 Bp65 to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 伪 to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 伪 and 尾 -actin to 尾 -actin (P 0.05), and decrease the ratio of I 魏 B- 伪 and 尾 -actin to 尾 -actin (P 0.05), compared with the control group (P 0.05. 4) Western-Blot showed that arsenic could increase the ratio of P-I B- 伪 to 尾 -kek 伪 / 尾 -kappa Bp65, and decrease the ratio of I 魏 B- 伪 to 尾 -actin. Compared with arsenic-exposed group, the ratio of Ike 伪 / 尾 -NF- 魏 Bp65, NF- 魏 Bp50 to 尾 -actin was decreased after BAY11-7082 or GSPE (P 0.05) and the ratio of I 魏 B- 伪 to 尾 -actin increased by P0.05. Conclusion: arsenic exposure can increase the apoptosis rate of BEAS-2B and decrease the survival rate. It may be that arsenic exposure activates NF- 魏 B signaling pathway, induces the increase of pro-inflammatory factor expression, decreases the expression of anti-inflammatory factor, and leads to inflammatory injury. GSPE can inhibit the activation of arsenic induced NF- 魏 B signaling pathway, reduce the expression of pro-inflammatory factors, and increase the expression of anti-inflammatory factor, suggesting that GSPE can inhibit the activation of arsenic induced NF- 魏 B signaling pathway. Thus regulate the expression of downstream inflammatory factors and antagonize the inflammatory injury induced by arsenic.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R595

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