ASMase/Cer信号通路在SiO2致NIH3T3纤维化的作用及rAC的干预效应

职业与健康 ›› 2026, Vol. 42 ›› Issue (20): 2777-2782.

ASMase/Cer信号通路在SiO₂致NIH3T3纤维化的作用及rAC的干预效应

易吉平¹, 杨秀鸿¹, 王有根², 曾明³()

¹ 湖南省职业病防治院（南华大学附属职业病防治院），湖南长沙 410021
² 中国铁路南昌局集团有限公司南昌疾病预防控制所，江西南昌 330002
³ 中南大学湘雅公共卫生学院，湖南长沙 410013

收稿日期:2025-08-05 修回日期:2025-11-13 出版日期:2026-10-15 发布日期:2026-09-14
通信作者: 曾明，E-mail：zengming@csu.edu.cn
作者简介:易吉平，女，主管医师，主要从事毒理学研究工作。
基金资助:
湖南省自然科学基金项目(2025JJ80779);湖南省卫生健康委科研计划项目(D202312046214)

Role of the ASMase/Cer signaling pathway in SiO₂ inducing NIH3T3 cell fibrosis and the intervention effect of rAC

YI Jiping¹, YANG Xiuhong¹, WANG Yougen², ZENG Ming³()

¹ Hunan Prevention and Treatment Institute for Occupational Diseases（The Affiliated Prevention and Treatment Institute for Occupational Diseases， University of South China），Changsha， Hunan 410021， China
² Nanchang Disease Prevention and Control Institute of China Railway Nanchang Group Co.， Ltd.，Nanchang， Jiangxi 330002， China
³ Xiangya School of Public Health， Central South University，Changsha， Hunan 410013， China

Received:2025-08-05 Revised:2025-11-13 Online:2026-10-15 Published:2026-09-14
Contact: ZENG Ming，E-mail：zengming@csu.edu.cn

摘要/Abstract

摘要：

目的了解酸性鞘磷脂酶/神经酰胺（acid sphingomyelinase/ceramide，ASMase/Cer）信号通路在SiO₂致小鼠胚肺成纤维细胞（embryonic fibroblast，NIH3T3）纤维化中的作用及重组酸性神经酰胺酶（recombinant acid ceramidase，rAC）的干预效应，为矽肺纤维化的防治提供新的线索。方法采用200 μg/mL SiO₂粉尘刺激肺泡巨噬细胞（primary alveolar macrophage，PAM），12 h后收集细胞培养上清液，作用于NIH3T3细胞建立矽尘诱导肺纤维化体外细胞模型。将NIH3T3细胞分为3组，分别为对照组、SiO₂刺激组和rAC干预组。各组细胞分别处理6、12、18、24、36和48 h。测定PAM中转化生长因子β1（transforming growth factor-β1，TGF-β1）、丙二醛（malondialdehyde，MDA）、活性氧（reactive oxygen species，ROS）的含量以及NIH3T3细胞中羟脯氨酸（hydroxyproline，HYP）含量和Ⅲ型胶原（type III collagen，ColⅢ）蛋白表达情况以及ASMase、Cer的含量。结果经200 μg/mL SiO₂粉尘刺激PAM细胞12 h后，细胞内ROS、MDA和上清液TGF-β1的含量较对照组上升[（51.605±0.769）vs （37.194±1.017）flu-value/mgpro、（1.250±0.170） vs （0.694±0.098）nmol/mgprot、（2.023±0.013） vs （1.932±0.011）ng/mL]，差异均有统计学意义（均P<0.05）；与对照组相比，SiO₂刺激组NIH3T3细胞 HYP 含量于染毒后12~48 h、Col Ⅲ蛋白表达量和ASMase活性于染毒后24~48 h以及Cer含量于染毒后24~36 h均有增加，且差异均有统计学意义（均P<0.05）。rAC预处理后，PAM细胞内ROS、MDA和上清液TGF-β1含量较SiO₂刺激组下降[（42.830±0.826） vs （51.605±0.769）flu-value/mgpro、（0.903±0.098） vs（1.250±0.170）nmol/mgprot、（1.976±0.012） vs （2.023±0.013）ng/mL]，差异均有统计学意义（均P<0.05）；与SiO₂刺激组相比，rAC干预组NIH3T3细胞内HYP和Col Ⅲ蛋白表达含量以及ASMase活性于染毒后24~48 h明显降低（P<0.05），Cer含量于染毒后18~48 h下降，差异均有统计学意义（均P<0.05）。结论在SiO₂粉尘诱导的肺纤维化体外细胞模型中，ASMase/Cer信号通路被激活，外源性给予rAC干预能有效抑制该通路的激活，并发挥抗纤维化的作用。提示靶向ASMase/Cer通路的rAC疗法是治疗矽肺纤维化的潜在新策略。

关键词: 二氧化硅, 肺纤维化, 酸性鞘磷脂酶, 神经酰胺, 信号通路, 重组酸性神经酰胺酶

Abstract:

Objective To investigate the role of the acid sphingomyelinase/ceramide（ASMase/Cer） signaling pathway in silica-induced fibrosis of mouse embryonic lung fibroblasts（NIH3T3） and the intervention effects of recombinant acid ceramidase（rAC），providing new insights for the prevention and treatment of silicosis fibrosis. Methods Primary alveolar macrophages（PAM） were stimulated with 200 μg/mL SiO₂ dust for 12 hours，and the cell culture supernatant was collected and applied to NIH3T3 cells to establish an in vitro cell model of silica-induced pulmonary cell fibrosis. NIH3T3 cells were divided into three groups：control group，SiO₂ stimulation group，rAC intervention group. Each group was treated for 6，12，18，24，36，and 48 hours. The levels of transforming growth factor-β1（TGF-β1），malondialdehyde（MDA），and reactive oxygen species（ROS） in the PAM，as well as the levels of ASMase，Cer，hydroxyproline（HYP），and type III collagen（Col III） expression in NIH3T3 cells were measured. Results Compared with the control group，after stimulation with 200 μg/mL SiO₂ for 12 h，the levels of MDA and ROS in PAM cells，as well as TGF-β1 in the supernatant were increased[（51.605±0.769） vs（37.194±1.017）flu-value/mgpro，（1.250±0.170） vs（0.694±0.098）nmol/mgprot，（2.023±0.013） vs （1.932±0.011）ng/mL]，and the differences were statistically significant（all P<0.05）；In the NIH3T3 cells，HYP content was elevated after exposing to SiO₂ 12-48 h，Col III protein expression and ASMase activity were increased at 24-48 h，and Cer content was raised at 24-36 h，and the differences were statistically significant（all P<0.05）. Compared to the SiO₂ stimulation，the levels of MDA，ROS in PAM cells and TGF-β1 in the supernatant reduced after pretreatment with rAC[（42.830±0.826） vs （51.605±0.769）flu-value/mgpro，（0.903±0.098） vs （1.250±0.170）nmol/mgprot，（1.976±0.012） vs （2.023±0.013）ng/mL]，the differences were statistically significant（all P<0.05）；In NIH3T3 cells，rAC intervention led to significant reductions in HYP content，Col III protein expression，and ASMase activity at 24-48 h，as well as a decrease in Cer content at 18-48 h，and the differences were statistically significant（all P<0.05）. Conclusion In the in-vitro cell model of SiO₂ dust-induced pulmonary fibrosis，the ASMase/Cer signaling pathway is activated. Exogenous rAC can effectively suppress this activation and exert anti-fibrotic effects，suggesting that rAC therapy targeting the ASMase/Cer axis represents a promising new strategy for treating silicotic fibrosis.

Key words: Silica, Pulmonary fibrosis, Acid sphingomyelinases, Ceramide, Signaling pathway, recombinant acid ceramidase

中图分类号:

R135.2

易吉平, 杨秀鸿, 王有根, 曾明. ASMase/Cer信号通路在SiO₂致NIH3T3纤维化的作用及rAC的干预效应. [J]职业与健康, 2026, 42(20): 2777-2782.

YI Jiping, YANG Xiuhong, WANG Yougen, ZENG Ming. Role of the ASMase/Cer signaling pathway in SiO₂ inducing NIH3T3 cell fibrosis and the intervention effect of rAC. [J]OCCUPATION AND HEALTH, 2026, 42(20): 2777-2782.

图/表 8

表1 SiO2对小鼠PAM细胞存活率和TGF-β1含量的影响（n=3，$\bar{x}±s$）

SiO₂剂量（μg/mL)	细胞存活率（%)	TGF-β1含量（ng/mL)
400	36.328±0.082^a	1.121±0.008^a
200	72.949±1.294^a	1.098±0.002^a
100	73.242±3.981^a	1.086±0.002^a
50	79.395±0.707^a	1.076±0.016^a
25	82.617±1.071^a	1.077±0.001^a
0	100	1.051±0.005

图1 200 μg/mL SiO2染毒PAM不同时间对TGF-β1的影响注：PAM—原代肺泡巨噬细胞；TGF-β1—转化生长因子-β1；a与0 h相比，P<0.05。

表2 SiO2对PAM细胞内ROS、MDA和TGF-β1含量影响及rAC的干预效应（n=3，$\bar{x}±s$）

组别	ROS（flu-value/mgpro）	MDA（nmol/mgprot）	TGF-β1（ng/mL）
SiO₂刺激组	51.605±0.769^a	1.250±0.170^a	2.023±0.013^a
rAC干预组	42.830±0.826^ab	0.903±0.098^ab	1.976±0.012^ab
对照组	37.194±1.017	0.694±0.098	1.932±0.011
F值	63.816	9.800	34.535
P值	<0.001	0.013	<0.001

图2 染毒SiO2对NIH3T3细胞内HYP含量的影响及rAC的干预效应注：HYP—羟脯氨酸；rAC—重组酸性神经酰胺酶；与对照组相比，aP<0.05；与SiO2刺激组相比，bP<0.05。

图3 SiO2对NIH3T3细胞内Col Ⅲ蛋白的影响及rAC的干预作用注：A—细胞条带图；B—SiO2对NIH3T3细胞内Col Ⅲ蛋白表达情况的影响；rAC—重组酸性神经酰胺酶；NIH3T3—胚肺成纤维细胞；与正常对照组比较，aP<0.05；与SiO2刺激组比较，bP<0.05。

图4 SiO2对NIH3T3细胞内ASMase活性变化的影响及rAC的干预效应注：NIH3T3—胚肺成纤维细胞；ASMase—酸性鞘磷脂酶；rAC—重组酸性神经酰胺酶；与对照组相比，aP<0.05；与SiO2刺激组相比，bP<0.05

图5 SiO2对NIH3T3细胞内Cer含量的影响及rAC的干预效应注：Cer—神经酰胺；rAC—重组酸性神经酰胺酶；与对照组相比，aP<0.05；与SiO2刺激组相比，bP<0.05。

表3 NIH3T3细胞内ASMase/Cer信号通路与HYP及Col Ⅲ表达相关性分析（r值）

指标	ASMase	Cer	HYP	Col Ⅲ
ASMase	1.000
Cer	0.660^b	1.000
HYP	0.855^b	0.544^a	1.000
Col Ⅲ	0.395	0.814^b	0.335	1.000

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ASMase/Cer信号通路在SiO₂致NIH3T3纤维化的作用及rAC的干预效应

Role of the ASMase/Cer signaling pathway in SiO₂ inducing NIH3T3 cell fibrosis and the intervention effect of rAC

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