康慧敏, 李柔, 王汉钦, 郑云帆, 陈适. 不同浓度鸦胆子苦醇对小鼠矽肺纤维化的影响[J]. 环境与职业医学, 2024, 41(5): 539-545. DOI: 10.11836/JEOM23378
引用本文: 康慧敏, 李柔, 王汉钦, 郑云帆, 陈适. 不同浓度鸦胆子苦醇对小鼠矽肺纤维化的影响[J]. 环境与职业医学, 2024, 41(5): 539-545. DOI: 10.11836/JEOM23378
KANG Huimin, LI Rou, WANG Hanqin, ZHENG Yunfan, CHEN Shi. Effects of different concentrations of brusatol on silicosis fibrosis in mice[J]. Journal of Environmental and Occupational Medicine, 2024, 41(5): 539-545. DOI: 10.11836/JEOM23378
Citation: KANG Huimin, LI Rou, WANG Hanqin, ZHENG Yunfan, CHEN Shi. Effects of different concentrations of brusatol on silicosis fibrosis in mice[J]. Journal of Environmental and Occupational Medicine, 2024, 41(5): 539-545. DOI: 10.11836/JEOM23378

不同浓度鸦胆子苦醇对小鼠矽肺纤维化的影响

Effects of different concentrations of brusatol on silicosis fibrosis in mice

  • 摘要: 背景

    矽肺是一种肺部弥漫性纤维化疾病,由长期暴露于游离二氧化硅(SiO2)粉尘引起,发病机制复杂,缺乏有效的治疗。鸦胆子苦醇(Bru)具有多种生物活性,其在矽肺纤维化中的作用尚不明确。

    目的

    探究不同浓度Bru对SiO2诱导小鼠矽肺纤维化的影响。

    方法

    将30只雄性C57BL/6J小鼠随机分为对照组、矽尘组、Bru低剂量(1 mg·kg−1)组、Bru中剂量(2 mg·kg−1)组、Bru高剂量(4 mg·kg−1)组,每组6只;除对照组外,其余各组均采用一次性非气管暴露法滴注50 μL、60 mg·mL−1 SiO2悬浊液建立矽肺小鼠模型,对照组滴注等量生理盐水;Bru组于染尘的同时腹腔注射Bru,连续注射5 d,随后隔天注射,染尘28 d后处死小鼠,收集肺组织。测定小鼠肺系数;采用苏木精-伊红(HE)染色和Masson染色观察小鼠肺组织的病理变化;Western blot法检测小鼠肺组织中凋亡蛋白Cleaved-caspase 3、纤维化相关蛋白α-平滑肌肌动蛋白(α-SMA)、I型胶原蛋白(Col-I)、自噬相关蛋白Beclin1、微管相关蛋白1轻链3(LC3)、Sequestosome 1(p62/SQSTM1)蛋白、Kelch样环氧氯丙烷相关蛋白1(Keap1),核因子E2相关因子2(Nrf2)的表达水平;实时荧光定量逆转录PCR(RT-qPCR)检测小鼠肺组织中Caspase 3α-SMACol-ImRNA水平。

    结果

    与对照组相比,矽尘组小鼠的肺系数明显升高(P<0.01);肺组织中肺泡壁受损,出现炎性细胞浸润、纤维结节和胶原纤维沉积;Cleaved-caspase 3、α-SMA和Col-I的蛋白表达及转录水平均明显上调(P<0.01);Beclin1、LC3-Ⅱ/I、p62、Nrf2表达水平增加(P<0.05,P<0.01),而Keap1表达水平下降(P<0.05)。与矽尘组相比,Bru低、中剂量组小鼠肺系数降低(P<0.05);肺组织的病理损伤及胶原沉积明显改善;Cleaved-caspase 3、α-SMA和Col-I的蛋白表达及转录水平下降(P<0.05,P<0.01),Beclin1、LC3-Ⅱ/I、p62、Nrf2表达水平也均明显下降(P<0.05,P<0.01),中剂量组Keap1水平上升(P<0.05)。与矽尘组相比,Bru高剂量组肺系数、病理损伤、Cleaved-caspase 3、α-SMA和Col-I的蛋白表达及转录水平差异无统计学意义(P>0.05);Beclin1、LC3-Ⅱ/I及Nrf2表达水平降低(P<0.01),p62蛋白表达水平差异无统计学意义(P>0.05),Keap1蛋白水平上升(P<0.01)。

    结论

    低、中剂量Bru可能通过Keap1-Nrf2通路调控自噬,改善自噬降解障碍,降低矽肺小鼠的肺系数,减轻矽肺小鼠肺组织中的细胞凋亡,延缓矽肺纤维化的进展。

     

    Abstract: Background

    Silicosis is a diffuse fibrosis of the lungs caused by long-term inhalation of free silicon dioxide (SiO2). It has a complex pathogenesis and lacks effective treatment. Brusatol (Bru) has a variety of biological activities, and its role in silicosis fibrosis is unclear yet.

    Objective

    To investigate the effects of different concentrations of Bru on SiO2-induced silicosis fibrosis in mice.

    Methods

    Thirty male C57BL/6J mice were randomly divided into five groups: a control group, a silica group, and three Bru intervention groups with low, medium, and high doses (1, 2, and 4 mg·kg−1), with 6 mice in each group. Except the control group, the remaining groups were established as SiO2-induced silicosis mouse models by using a single tracheal infusion of 50 μL 60 mg·mL−1 SiO2 suspension. The control group was dosed with equal amount of saline. The Bru intervention groups were injected intraperitoneally with Bru for 5 consecutive days and then injected every other day. After 28 d of exposure, the mice were executed and lung tissues were collected. The lung coefficient of the mice was measured, and the pathological changes of the lung tissues were observed after hematoxylin-eosin (HE) and Masson staining. The levels of apoptotic protein Cleaved-caspase 3, fibrosis-related protein α-smooth muscle actin (α-SMA), type I collagen (Col-I), autophagy-associated protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), Sequestosome 1 (p62/SQSTM1), Kelch like ECH-associated protein-1 (Keap1), and nuclear factor erythroid 2 related factor 2 (Nrf2) were detected by Western blot. The mRNA levels of Caspase 3, α-SMA, and Col-I were measured by realtime fluorescence-based quantitative PCR.

    Results

    Compared with the control group, the lung coefficient of mice in the silica group was significantly increased (P < 0.01); the lung tissues of the silicosis mice showed damaged alveolar walls, along with infiltration of inflammatory cells, fibrous nodules, and collagen deposition; furthermore, the protein and mRNA levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly increased (P < 0.01); the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were increased, while that of Keap1 was decreased (P < 0.05). The interventions with low and medium doses of Bru reduced lung coefficient (P < 0.05) and protected against pathological damage and collagen deposition in the lung tissues of the silicosis mice; the protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I were significantly decreased in the low and medium dose groups (P < 0.05, P < 0.01), the expression levels of Beclin1, LC3-II/I, p62, and Nrf2 were also decreased (P < 0.05, P < 0.01), and the expression level of Keap1 was increased in the medium dose group (P < 0.05). However, compared with the silica group, the differences in lung coefficient, pathological damage, and protein and mRNA expression levels of Cleaved-caspase 3, α-SMA, and Col-I in the Bru high dose group were not statistically significant (P > 0.05). In addition, the high dose of Bru decreased Beclin1, LC3-II/I, and Nrf2 expression levels (P < 0.01), did not change p62 protein expression level (P > 0.05), while increased Keap1 protein level (P < 0.01).

    Conclusion

    Low and medium doses of Bru might regulate autophagy through the Keap1-Nrf2 pathway, ameliorate autophagic degradation impairment, reduce pulmonary coefficient, attenuate apoptosis, and delay the progression of fibrosis in SiO2-induced silicosis mice.

     

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