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

Abstract

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

CLC Number:

R135.2

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.

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References 22

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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

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

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

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

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

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

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