欧前胡素衍生物对COPD肺泡Ⅱ型细胞活性及耐药蛋白的影响

doi:10.11958/20240581

摘要/Abstract

摘要：

目的探究欧前胡素衍生物（IMP-1）对慢性阻塞性肺疾病（COPD）肺泡Ⅱ（ATⅡ）型细胞活性及耐药蛋白的影响。方法 ATⅡ型细胞分为空白组（常规培养基进行培养的生长良好的ATⅡ型细胞）、香烟烟雾提取物（CSE）组（加入2%CSE诱导培养）、IMP-1组（采用10 μmol/L的IMP-1干预经CSE诱导的ATⅡ型细胞）。采用四甲基偶氮唑盐（MTT）法测定ATⅡ型细胞24 h、48 h、72 h增殖率，Hoechst 33342染色法检测ATⅡ型细胞凋亡率，流式细胞分析仪分析各周期细胞占比，软琼脂克隆形成实验检测ATⅡ型细胞克隆数量，Western blot法检测多耐药基因1（MDR1）及多药耐药相关蛋白1（MRP1）蛋白表达水平。结果与空白组比较，CSE组细胞凋亡率、ATⅡ型细胞周期G₀/G₁期占比升高，在24 h、48 h、72 h细胞的增殖率和S、G₂/M期占比降低（P<0.05）；与CSE组比较，IMP-1组细胞增殖率和S、G₂/M期占比及细胞克隆数量升高，ATⅡ型细胞周期G₀/G₁期占比和MDR1、MRP1蛋白降低（P<0.05）。结论 IMP-1可能通过下调CSE诱导的ATⅡ型细胞的MDR1、MRP1蛋白表达，从而抑制细胞的异常活化，增加其凋亡。

关键词: 肺疾病，慢性阻塞性, 欧前胡素衍生物, 肺泡Ⅱ型上皮细胞, 香烟烟雾提取物, 多耐药基因1, 多药耐药相关蛋白1

Abstract:

Objective To explore effects of a derivative of imperatorin (IMP-1) on the activity and resistance proteins of alveolar type Ⅱ (ATⅡ) cells in chronic obstructive pulmonary disease (COPD). Methods AT Ⅱ cells were divided into the blank group (well growing AT Ⅱ cells cultured in conventional medium), the cigarette smoke extract (CSE) group (induced culture with 2% CSE added) and the IMP-1 group (intervention with 10 μ mol/L of IMP-1 on CSE induced AT Ⅱ cells). Proliferation rates of AT Ⅱ cells at 24, 48 and 72 hours were measured using MTT method. Apoptosis of ATⅡ cells was detected by Hoechst 33342 staining. The proportion of cells in each cycle was analyzed by flow cytometry. The number of ATⅡ cell clones was detected by soft AGAR clone formation assay. Multidrug resistance gene 1(MDR1) and multidrug resistance associated protein 1 (MRP1) were detected by Western blot assay. Results Compared with the blank group, the apoptosis rate and the proportion of G₀/G₁ phase of ATⅡ cell cycle were increased in the CSE group, and the proliferation rate and the proportion of S, G₂/M phase were decreased at 24 h, 48 h and 72 h (P<0.05). Compared with the CSE group, the cell proliferation rate, the proportion of S and G₂/M phases and the number of cell clones were increased in the IMP-1 group, and the proportion of G₀/G₁ phase in ATⅡ cell cycle and the protein levels of MDR1 and MRP1 were decreased in the IMP-1 group (P<0.05). Conclusion IMP-1 may downregulate the expression of MDR1 and MRP1 proteins induced by CSE in AT Ⅱ cells, inhibiting the progression of AT Ⅱ cells from S phase to G2 phase and from G2 phase to M phase, thereby inhibiting abnormal cell activation and increasing apoptosis.

Key words: pulmonary disease, chronic obstructive, imperatorin derivatives, alveolar type Ⅱ epithelial cells, cigarette smoke extract, multidrug resistance gene 1, multidrug resistance associated protein 1

中图分类号:

R563.9

图/表 7

表1 各组ATⅡ型细胞增殖率和凋亡率比较（n=6，%，$\bar{x} \pm s$）

Tab.1 Comparison of AT Ⅱ cell proliferation rate between the three groups

组别	增殖率			凋亡率
组别	24 h	48 h	72 h	凋亡率
空白组	100.00±0.00	100.00±0.00	100.00±0.00	9.33±1.02
CSE组	35.45±1.08^a	41.46±1.21^a	50.33±1.35^a	85.57±2.34^a
IMP-1组	53.89±1.66^ab	69.76±1.84^ab	82.14±2.05^ab	40.26±1.59^ab
F	5 050.439^＊＊	3 195.432^＊＊	1 893.672^＊＊	2 925.154^＊＊

图1 荧光显微镜下各组ATⅡ型细胞凋亡情况（Hoechst 33342染色，×200）

Fig.1 Apoptosis of AT Ⅱ cell in each group under fluorescence microscopy (Hoechst 33342 staining，×200)

图2 各组ATⅡ型细胞周期占比比较

Fig.2 Comparison of the number of ATⅡ cell cycle cells between the three groups

图3 各组ATⅡ型细胞克隆数量比较

Fig.3 Comparison of the number of AT Ⅱ cell clones between the three groups

表2 各组ATⅡ型细胞周期占比及克隆数目比较（n=6，$\bar{x} \pm s$）

Tab.2 Comparison of the number of ATⅡ cell cycle and the number of clones between the three groups

组别	G₀/G₁/%	S/%	G₂/M/%	克隆数目/个
空白组	8.31±0.33	46.10±5.56	45.59±5.49	148.33±3.36
CSE组	53.85±5.85^a	22.62±0.83^a	23.53±0.78^a	78.57±1.15^a
IMP-1组	21.78±2.49^ab	39.56±3.10^ab	38.66±3.65^ab	115.13±2.61^ab
F	242.836^＊＊	64.096^＊＊	52.028^＊＊	48.120^＊＊

图4 各组ATⅡ型细胞中MDR1、MRP1蛋白比较

Fig.4 Comparison of MDR1 and MRP1 proteins in AT Ⅱ cells between the three groups

表3 各组ATⅡ型细胞中MDR1、MRP1蛋白比较（n=6，$\bar{x} \pm s$）

Tab.3 Comparison of MDR1 and MRP1 proteins in AT Ⅱ cells between the three groups

组别	MDR1	MRP1
空白组	1.02±0.04	1.10±0.02
CSE组	2.35±0.15^ab	2.51±0.13^a
IMP-1组	1.60±0.09^ab	1.57±0.07^ab
F	229.663^＊＊	450.337^＊＊

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