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核因子-κB反义寡核苷酸对博莱霉素诱导的小鼠肺纤维化发生及其中白细胞介素-4表达的影响:小鼠实验

[Effects of antisense oligonucleotide to nuclear factor-kappaB on the development of bleomycin-induced pulmonary fibrosis and IL-4 expression therein: experiment with mice].

作者信息

Zhang Xiao-ye, Zhou Yan, Liu Wei-qing, Zhao Li, Li Sheng-qi

机构信息

Department of Respiratory Medicine, Shengjing Hospital of China Medical University, Shenyang 110004, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2007 Jan 16;87(3):195-9.

Abstract

OBJECTIVE

To investigate the expression of nuclear factor-kappaB (NF-kappaB) in the cells in the bronchoalveolar lavage fluid (BALF) of idiopathic pulmonary fibrosis (IPF) and its effects on IL-4 expression therein, and to investigate the therapeutic role of antisense oligonucleotide (ASON) to NF-kappaB on IPF.

METHODS

C57BL/6 mice were randomly divided into 4 groups: bleomycin (BLM) group (n = 35, injected with BLM through caudal vein), control group [n = 20, injected with normal saline (NS) via caudal vein], ASON group (n = 35, injected with ASON to p65, a subunit of NF-kappaB, at the dose of 900 microg), and SON group (n = 35, injected with sense oligonucleotide to p65 subunit). Six hours after intravenous injection, the BLM, ASON, and SON groups were treated with BLM-A5 (5 mg/kg dissolved in 20 microl NS) by intratracheal installation, and the control group was treated with NS (20 microl). 0.5, 1, 3, 7, 14, and 28 days following intratracheal instillation of BLM or 0.5, 1, 14 days following intratracheal instillation of NS, 5 mice of every group were sacrificed and bronchoalveolar lavage was performed. The BALF was collected and assayed with ELISA for IL-4. Immunohistochemistry (IHC) and microscope image analysis were completed to detect the expression of p65 and IL-4 in the bronchoalveolar lavage cells. Another 5 mice from each group were sacrificed 28 days after intratracheal instillation with their total right lungs taken out to undergo pathohistological examination. The content of hydroxyproline in the left lung was detected by high performance liquid chromatography and ELISA.

RESULTS

(1) Twenty-eight days after intratracheal instillation, the BLM and the SON groups showed consolidation of the lung parenchyma with loss of the alveolar architecture and increased cellularity, while the ASON and control groups showed no significant pulmonary consolidation or fibrosis. (2) Twenty-eight days after intratracheal instillation, the hydroxyproline content of the BLM group was 876.8 +/- 91.1 nmol/lung, significantly higher than that of the control group (347.6 +/- 53.9 nmol/lung, t = -9.833, P < 0.001); the hydroxyproline content of the ASON group was 505.6 +/- 34.8 nmol/lung, significantly lower than that of the BLM group (t = -9.862, P < 0.001); however, the hydroxyproline content of the SON group was 775.2 +/- 68.9 nmol/lung, not significantly different from that of the BLM group (t = 2.118, P = 0.102). (3) One day after the intratracheal instillation of BLM, the value of average integral optical density of p65 in the bronchoalveolar lavage cells of the BLM, SON, and ASON groups were 275 +/- 13, 233 +/- 60, 233 +/- 60, and 126 +/- 34 respectively, all significantly higher that of the control group (38 +/- 18, t = 27.350, 8.039, and 6.107, P < 0.001, = 0.001, and = 0.004), that of the ASON group being significantly lower than those of the BLM and SON groups (t = 7.664 and -3.407, P = 0.002 and 0.027). (4) IHC showed that 1 day after the intratracheal instillation, the value of average integral optical density of IL-4 of the BLM, SON, and ASON groups were 134 +/- 16, 128 +/- 2, and 80 +/- 9 respectively, all significantly higher than that of the control group (33 +/- 12, t = 10.346, -5.927, and 5.313, P < 0.001, = 0.004, = 0.006), that of the ASON group being significantly lower than those of the BLM and SON groups (t = 6.967 and -3.591, P = 0.002 and 0.023). (5) ELISA showed that 1 day after the intratracheal instillation the IL-4 level BLM, SON, and ASON groups were (20.8 +/- 7.2) ng/L, (21.4 +/- 8.0) ng/L, and (9.7 +/- 1.4) ng/L respectively, all significantly higher than that of the control group [(1.6 +/- 3.6) ng/L, t = 6.494, 4.143, and 4.331, P = 0.003, 0.014, and 0.012], that of the ASON group being significantly lower than those of the BLM and SON groups (t = -3.553 and -3.577, P = 0.024 and 0.023) (6) Correlation analysis showed that 1 day after intratracheal instillation the expression of p65 was positively correlated with IL-4 expression in the bronchoalveolar lavage cells in the treatment group (r = 0.890, P < 0.05) and the ASON group (r = 0.909, P < 0.05).

CONCLUSION

The expression of NF-kappaB is significantly increased and augments the expression of IL-4 indirectly in the BALF cells during the process of BLM-induced lung fibrosis. ASON significantly inhibits the NF-kappaB activation and the IL-4 expression, and may be useful in gene therapy for pulmonary fibrosis.

摘要

目的

探讨核因子-κB(NF-κB)在特发性肺纤维化(IPF)患者支气管肺泡灌洗液(BALF)细胞中的表达及其对其中白细胞介素-4(IL-4)表达的影响,并研究NF-κB反义寡核苷酸(ASON)对IPF的治疗作用。

方法

将C57BL/6小鼠随机分为4组:博来霉素(BLM)组(n = 35,经尾静脉注射BLM)、对照组(n = 20,经尾静脉注射生理盐水(NS))、ASON组(n = 35,以900μg的剂量向NF-κB的一个亚基p65注射ASON)和正义寡核苷酸(SON)组(n = 35,向p65亚基注射正义寡核苷酸)。静脉注射6小时后,BLM组、ASON组和SON组经气管内注入BLM-A5(5mg/kg溶于20μl NS),对照组经气管内注入NS(20μl)。在气管内注入BLM后0.5、1、3、7、14和28天,或在气管内注入NS后0.5、1、14天,每组处死5只小鼠并进行支气管肺泡灌洗。收集BALF并用ELISA法检测IL-4。完成免疫组织化学(IHC)和显微镜图像分析以检测支气管肺泡灌洗细胞中p65和IL-4的表达。每组另取5只小鼠在气管内注入28天后处死,取出其右肺全肺进行病理组织学检查。通过高效液相色谱法和ELISA法检测左肺中羟脯氨酸的含量。

结果

(1)气管内注入28天后,BLM组和SON组显示肺实质实变,肺泡结构丧失,细胞增多,而ASON组和对照组未显示明显的肺实变或纤维化。(2)气管内注入28天后,BLM组的羟脯氨酸含量为876.8±91.1nmol/肺,显著高于对照组(347.6±53.9nmol/肺,t = -9.833,P < 0.001);ASON组的羟脯氨酸含量为505.6±34.8nmol/肺,显著低于BLM组(t = -9.862,P < 0.001);然而,SON组的羟脯氨酸含量为775.2±68.9nmol/肺,与BLM组无显著差异(t = 2.118,P = 0.102)。(3)气管内注入BLM 1天后,BLM组、SON组和ASON组支气管肺泡灌洗细胞中p65的平均积分光密度值分别为275±13、233±60、233±60和126±34,均显著高于对照组(38±18,t = 27.350、8.039和6.107,P < 0.001、= 0.001和= 0.004),ASON组显著低于BLM组和SON组(t = 7.664和-3.407,P = 0.002和0.027)。(4)IHC显示,气管内注入1天后,BLM组、SON组和ASON组IL-4的平均积分光密度值分别为134±16、128±2和80±9,均显著高于对照组(33±12,t = 10.346、-5.927和5.313,P < 0.001、= 0.004和= 0.006),ASON组显著低于BLM组和SON组(t = 6.967和-3.591,P = 0.002和0.023)。(5)ELISA显示,气管内注入1天后,BLM组、SON组和ASON组的IL-4水平分别为(20.8±7.2)ng/L、(21.4±8.0)ng/L和(9.7±1.4)ng/L,均显著高于对照组[(1.6±3.6)ng/L,t = 6.494、4.143和4.331,P = 0.003、0.014和0.012],ASON组显著低于BLM组和SON组(t = -3.553和-3.577,P = 0.024和0.023)(6)相关性分析显示,气管内注入1天后,治疗组(r = 0.890,P < 0.05)和ASON组(r = 0.909,P < 0.05)支气管肺泡灌洗细胞中p65的表达与IL-4的表达呈正相关。

结论

在BLM诱导的肺纤维化过程中,NF-κB的表达显著增加,并间接增强了BALF细胞中IL-4的表达。ASON显著抑制NF-κB的激活和IL-4的表达,可能对肺纤维化的基因治疗有用。

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