Effect of mTOR Signaling Pathway Inhibition on Bleomycin-induced Pulmonary Fibrosis
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摘要:
目的 探讨mTOR信号通路对博来霉素诱导肺纤维化的影响。 方法 6～8周龄健康雄性C57BL/6小鼠30只，喂养1周，分为对照组（NC组，n = 5）、博来霉素组（BLM组，n = 5），雷帕霉素+博来霉素组（Rapa+BLM组，n = 5），在7 d、28 d各采用颈椎脱臼法处死小鼠5只，取肺组织。HE染色观察肺组织炎症浸润，Masson's 染色观察肺组织的纤维化严重程度。Western blot、qPCR检测CollagenⅠ、Collagen Ⅲ和α-SMA的表达水平，观察肺组织纤维化程度。Western blot检测各组细胞中mTOR、P70S6K和其磷酸化水平的表达。 结果 与NC组相比，BLM组肺组织肺泡间隔增厚，有明显的炎性改变和胶原沉积，CollagenⅠ、Collagen Ⅲ、α-SMA蛋白表达均显著升高（P < 0.01），CollagenⅠmRNA、Collagen ⅢmRNA、α-SMAmRNA表达增加（P < 0.05），p-mTOR、p-p70S6K表达升高（P < 0.05）；与BLM组比较，Rapa+BLM组肺组织结构改善，炎症及胶原沉积减轻，Collagen Ⅰ、CollagenⅢ、α-SMA蛋白表达有下降趋势（P > 0.05），CollagenⅠmRNA有下调趋势（P > 0.05），Collagen ⅢmRNA、α-SMAmRNA下降（P < 0.05）。 结论 在博来霉素诱导的肺纤维化中观察到mTOR异常活化；抑制mTOR信号通路的活化能有效缓解肺纤维化的形成。 Abstract:Objective To investigate the effect of mTOR signaling pathway on bleomycin-induced pulmonary fibrosis. Methods 30 healthy male C57BL/6 mice aged 6～8 weeks were fed for 1 week and divided into control group （NC group, n = 5）, bleomycin group （BLM group, n = 5）, and rapamycin + bleomycin group （Rapa+BLM group, n = 5）. Mice were euthanized by cervical dislocation at 7 and 28 days, and lung tissues were collected. HE staining was used to observe inflammatory infiltration in lung tissue, and Masson's staining was used to assess the severity of lung fibrosis. Western blot and qPCR were used to detect the expression levels of collagen I, collagen III and α-SMA to evaluate the degree of lung fibrosis. Western blot was used to detect the expression of mTOR, P70S6K and their phosphorylation levels in each group. Results Compared with the NC group, the BLM group showed thickened alveolar septa, obvious inflammatory changes, and collagen deposition.The protein expression of Collagen I, Collagen III, and α-SMA were significantly increased （P < 0.01）, with increased mRNA expression of Collagen I, Collagen III, and α-SMA （P < 0.05）, and elevated p-mTOR and p-p70S6K expression （P < 0.05）. Compared with the BLM group, the Rapa+BLM group showed improved lung tissue structure, reduced inflammation and collagen deposition, a downward trend in Collagen I, Collagen III, and α-SMA protein expression （P > 0.05）, a downward trend in Collagen I mRNA （P > 0.05）, and decreased Collagen III and α-SMA mRNA expression （P < 0.05）. Conclusion Abnormal mTOR activation was observed in bleomycin-induced pulmonary fibrosis; inhibiting mTOR signaling pathway activation can effectively alleviate the formation of pulmonary fibrosis. -
Key words:
- Pulmonary fibrosis /
- Bleomycin /
- Rapamycin
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图 1 肺组织染色结果
A：肺组织HE染色（ bar 100 μm）：NC组肺泡结构正常，未见明显胶原沉积；BLM组小鼠肺组织结构破坏，肺泡间隔不规则的增厚、肺泡腔变小，可见炎性细胞浸润肺泡腔及间质；B：肺组织Masson’ s染色（bar 100 μm）：NC组未见明显胶原沉积；BLM组可见不规则增厚的肺泡间隔中胶原沉积，胶原蓝染明显；BLM+Rapa组胶原染堆积有所减少。
Figure 1. Lung tissue staining
图 2 Western blot法检测BLM诱导小鼠肺纤维化中mTOR/P70S6K通路蛋白表达的影响
A：p-mTOR和p-p70S6K Western blot 结果；B：p-mTOR和p-p70s6k表达情况；与NC组比较，* P < 0.05；** P < 0.01。
Figure 2. Effect of mTOR/P70S6K pathway protein expression in BLM-induced lung fibrosis in mice detected by Western blot
图 3 Western blot法检检查抑制m-TOR后对博来霉素诱导小鼠体内Collagen Ⅰ、CollagenⅢ、α-SMA蛋白表达
A：Collagen Ⅰ、CollagenⅢ、α-SMA Western blot 结果；B：Collagen Ⅰ、CollagenⅢ、α-SMA 表达情况；与NC组比较，BLM组：* P < 0.05；*** P < 0.001。
Figure 3. Western blot analysis of Collagen I，Collagen III，and α-SMA protein expression in mouse tissues after inhibition of m-TOR induced by bleomycin
图 4 RT-qPCR检测检查抑制m-TOR后对博来霉素诱导小鼠体内CollagenⅠmRNA、Collagen ⅢmRNA、α-SMAmRNA的表达
A：7 d和28 d Collagen Ⅰ RT-qPCR检测结果；B：7 d和28 d CollagenⅢ RT-qPCR检测结果；C：7 d和28 d α-SMA RT-qPCR检测结果；7 d：与NC组比较，BLM组：*P < 0.05；***P < 0.001；与BLM组比较，**P< 0.01；28 d：与NC组比较，BLM组：#P < 0.05；###P < 0.001；与BLM组比较，#P < 0.05；##P < 0.01。
Figure 4. RT-qPCR detection of Collagen I mRNA，Collagen III mRNA，and α-SMA mRNA expression in mouse models after inhibiting m-TOR with bleomycin induction
表 1 引物序列
Table 1. Primer Sequence
名称 引物序列（5′→3′） Collagen-I-F F: CAAAGACGGGAGGGCGAGT Collagen-I-R R: CCATAGGACATCTGGGAAGCAA Collagen-III-F F: TGCCCACAGCCTTCTACACC Collagen-III-R R: GATAGCCACCCATTCCTCCC α-SMA-F F: GTAGCACCAGAAGAGCACCC α-SMA-R R: GCACAGCCTGAATAGCAACAT actin-F F: CGTGCGTGACATCAAAGAGAAG actin-R R: CCAAGAAGGAAGGCTGGAAAA 
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