瘦素/ERK信号在云锡矿粉诱导大鼠II型肺泡上皮细胞转化中的作用 Translated title: Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust

Abstract

背景与目的

目前，云南个旧锡矿有大量矿工从事开采工作，这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关，大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡II型上皮细胞（immortalized rat alveolar cells type II, RLE-6TN）恶性转化过程中，瘦素（leptin）及其介导的细胞外调节蛋白激酶（extracellular regulated protein kinase, ERK）信号通路所起的作用。

方法

采用200 μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代，建立毒染细胞模型，命名为R ₂₀₀细胞，正常培养组命名为R细胞，通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）抑制剂（U0126）对R ₂₀₀细胞的最佳作用浓度。自第20代起，将R组、R ₂₀₀组细胞分别与leptin及MEK抑制剂U0126共培养，对各组细胞的形态改变进行观察，并利用苏木素-伊红（hematoxylin-eosin, HE）染色技术鉴别第40代细胞的形态学差异，通过刀豆凝集素A（concanavalin A, ConA）及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。

结果

ng/mL时，其促增殖效应最为显著，30 μmol/L U0126可抑制毒染细胞R ₂₀₀增殖，与对照组相比具有统计学差异（P<0.05）。自第25代起，leptin诱导的R ₂₀₀组（R ₂₀₀L组）细胞形态发生变化，至第30代出现恶性转化，至第40代时恶性转化特征明显；而R ₂₀₀组细胞及U0126诱导的R ₂₀₀组（R ₂₀₀LU组）细胞则在第40代时才出现恶性转化特征。R ₂₀₀L组细胞凝集速度较R ₂₀₀LU组快，其余各组细胞P30出现凝集，且随ConA浓度增加，细胞凝集速度加快。R ₂₀₀L组细胞自P40可见克隆形成，克隆形成率为2.25‰±0.5‰，R ₂₀₀LU组及R ₂₀₀组未见克隆集落。R ₂₀₀L组细胞pERK表达增强；加入U0126阻断后，R ₂₀₀L组细胞pERK磷酸化水平降低。

结论

Leptin可以促进云锡矿粉毒染肺上皮细胞的恶性转化，ERK信号通路可能是其促进云锡矿粉引发的肺泡II型上皮细胞转化的重要途径。

Translated abstract

Background and objective

Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust.

Methods

Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R ₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R ₂₀₀ cells was determined using the MTT method. Starting from the 20 ^th generation, the cells in the R group were co-cultured with leptin, while the cells in the R ₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method.

Results

Both the cells in the R group and R ₂₀₀ group express leptin receptor OB-R. Compared to the R ₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R ₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25 ^th generation, the cell morphology of the leptin-induced R ₂₀₀ group (R ₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30 ^th generation. The characteristics of malignant transformation became evident by the 40 ^th generation in the R ₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R ₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R ₂₀₀ (R ₂₀₀LU) group. Additionally, the cells in the R ₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R ₂₀₀LU group and R ₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R ₂₀₀L group. However, when the cells in the R ₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased.

Conclusion

Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.