Apelin-13对脂多糖诱导大鼠肺微血管内皮细胞炎性因子表达的影响及机制研究

Effects of Apelin-13 on the level of inflammatory factors induced by LPS in rat PMVECs and the mechanisms

目的探讨Apelin-13对脂多糖(LPS)诱导大鼠肺微血管内皮细胞(PMVECs)炎性因子表达的影响,并分析其与活性氧-核因子κB(ROS-NF-κB)信号通路的相关性。方法采用植块法培养大鼠PMVECs。在Apelin-13干预实验中,PMVECs随机分为对照组、模型组和低、中、高剂量实验组。对照组用正常含血清的培养液处理细胞,模型组和实验组均采用10μg·mL-1 LPS处理细胞,实验组分别给予1×10^&-9,1×10^-8和1×10^-7 mol·L^-1 Apelin预处理细胞,2 h后加入终浓度为10μg·mL-1的LPS刺激细胞。用逆转录-聚合酶链反应(RT-PCR)法检测白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)mRNA表达以及蛋白分泌的变化;2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)染色检测PMVECs中ROS的产生;以Western blot法检测NF-κB P65的蛋白表达水平。结果Apelin-13干预实验中,对照组、模型组和低、中、高剂量实验组的IL-6 mRNA相对表达量分别为0.46±0.06,1.06±0.08,0.90±0.08,0.75±0.11和0.62±0.04;MCP-1 mRNA相对表达量分别为0.31±0.06,1.12±0.24,0.91±0.06,0.66±0.09和0.63±0.10;ROS相对生成量分别为(100.00±10.21)%,(442.39±56.41)%,(315.35±46.75)%,(229.51±24.67)%和(216.18±40.59)%;NF-κB p65蛋白的相对表达量分别为(100.00±18.18)%,(376.87±49.39)%,(233.36±52.42)%,(202.63±27.01)%和(183.88±53.09)%。对照组和实验组分别与模型组比较,差异均有统计学意义(P<0.05)。结论Apelin-13通过干预ROS-NF-κB信号通路抑制了LPS诱导的PMVECs中炎性因子的表达。

Objective To investigate the effect of Apelin-13 on inflammatory factors induced by lipopolysaccharide(LPS)in rat pulmonary microvascular endothelial cells(PMVECs),and to analyze its relationship to reactive oxygen species-nuclear factor kappa-B(ROS-NF-κB)signaling pathway.Methods PMVECs were cultured with the explant technique.In the intervention experiment of Apelin-13,PMVECs were randomly divided into 5 groups:control group,model group,expe-rimental-L,-M,-H groups.The control group was treated with normal serum-containing medium,the model and experimental-L,-M,-H group groups were treated with LPS(10μg·mL-1).In addition,the experimental-L,-M,-H groups were also treated with 1×10^-9,1×10^-8 and 1×10^-7 mol·L^-1 Apelin for 2 h before LPS treatment,respectively.Reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)method were used to detect the mRNA expression and protein secretion levels of interleukin 6(IL-6)and monocyte chemotactic protein 1(MCP-1),respectively.ROS was measured by confocal microscopy with DCFH-DA staining.The protein expression of NF-κB P65 was evaluated by Western blot analysis.Results In the Apelin-13 intervention experiment,the levels of IL-6 mRNA in control group,model group and experimental-L,-M,-H groups were 0.46±0.06,1.06±0.08,0.90±0.08,0.75±0.11 and 0.62±0.04,respectively;the levels of MCP-1 mRNA in the above groups were0.31±0.06,1.12±0.24,0.91±0.06,0.66±0.09 and 0.63±0.10,respectively;the relative production of ROS in the above groups were(100.00±10.21)%,(442.39±56.41)%,(315.35±46.75)%,(229.51±24.67)%and(216.18±40.59)%,respectively;the relative protein expression of NF-κB p65 in the above groups were(100.00±18.18)%,(376.87±49.39)%,(233.36±52.42)%,(202.63±27.01)%and(183.88±53.09)%,respectively.There were statistically significant differences between the model group and the control/experimental group(P<0.05).Conclusion Apelin-13 inhibited the expression level of inflammatory factors induced by LPS by interfering the ROS-NF-k B signaling pathway.