The Regulatory Role And Mechanism Of HIF-1α In The Development Of Silicosis

ObjectiveHIF-1α has been reported to be important in diseases such as liver fibrosis and myocardial fibrosis,but its role in silicosis is still unclear.In this study,we established a mouse silicosis model combined with an NIH-3T3 cell transdifferentiation model to investigate the effect and molecular mechanism of HIF-1α in silicosis.This experiment provides new targets and theories for investigating silicosis treatments.Materials and Methods1 MaterialNIH-3T3 mouse lung fibroblasts were purchased from the Cell Bank of the Chinese Academy of Sciences.Male,4-week-old C57BL/6N mice(15～20 g)were purchased from Beijing Viton Lihua Laboratory Animal Technology Co.2 Methods2.1 Construction of NIH-3T3 cell transdifferentiation model in vitro and observation of HIF-1α expression changes and its action mechanismNIH-3T3 cells were treated with different concentrations of TGF-β1 to establish a transdifferentiation model.The protein expression levels of α-SMA,COLIA1 and HIF-1α were detected by Western blot technique,and the expression levels of α-SMA,COLIA1 and HIF-1α mRNA were detected by RT-qPCR.NIH-3T3 cells were treated with 7.5 ng/mL of TGF-β1,and the protein expression levels of α-SMA,COLIA1,and HIF-1α were detected at different times using Western blot technique,and the expression levels of α-SMA,COLIA1,and HIF-1α mRNA were detected by RT-qPCR.DMOG,KC7F2,SIS3 and HIF-1α/NC-siRNA were used to intervene in the transdifferentiation model,respectively.The expression levels of α-SMA,COLIA1,HIF-1α,SMAD3 and PSMAD3 proteins were detected using Western blot technique.α-SMA,COLIA1 and HIF-1α mRNA expression levels were detected by RT-qPCR.2.2 Detection of changes in HIF-1α expression in a mouse silicosis model and observation of intervention effectsForty C57BL/6N mice were randomly divided into a saline control group,a silicosis model group,a KC7F2 intervention group,and an intervention control group.The silicosis model was established by non-exposed tracheal drip injection of 100 μL SiO2 dust suspension(50 mg/mL)(saline control group was dripped with an equal amount of saline),KC7F2 group mice were injected intraperitoneally with KC7F2 solution(0.75 mg/kg)at a frequency of once every three days,and the intervention control group mice were injected with an equal amount of saline.The extent of lung tissue injury and fibrosis was assessed using HE and Masson staining,and the expression of COLIA1 and HIF-1α protein in mice lung tissue was detected using immunohistochemistry.The expression of α-SMA,COLIA1,HIF-1α,and SMAD3 were detected by RT-qPCR or Western blot.3 Statistical analysisThe experimental data were statistically analyzed by SPSS 25.0,and the measurement data were expressed by mean ± standard deviation.The difference between the two groups of samples was compared by students t test,the diversity difference was compared by one-way ANOVA,and the pairwise comparison was compared by LSD-t test.Inspection level α=0.05。Result1 Establishment of NIH-3T3 cell transdifferentiation model and investigation of the mechanism of HIF-1α action1.1 Effect of different concentrations of TGF-β1 treatment on NIH-3T3 transdifferentiation modelNIH-3T3 cells were treated with 0,2.5,5,7.5,and 10 ng/mL TGF-β1 for 24 h,αSMA protein,and mRNA expression levels and pSMAD3/SMAD3 ratio were significantly higher at 5 ng/mL,COLIA1 protein and mRNA expression levels and HIF-1α protein expression levels were significantly higher at 7.5 ng/mL,and HIF-1αmRNA expression levels were not changed.1.2 Effect of different time TGF-β1 treatment on NIH-3T3 transdifferentiation model7.5 ng/mL TGF-β1 treated NIH-3T3 cells 0,2,4,6,8,10,12,24,48 h,COLIA1,α-SMA protein,and mRNA expression continued to increase,HIF-1α protein expression levels and pSMAD3/SMAD3 ratio increased first and then decreased,reaching a maximum at 4 h.1.3 Effect of intervention with HIF-1α on NIH-3T3 transdifferentiation modelCompared with the Control group,COLIA1,α-SMA protein,mRNA expression levels,and pSMAD3/SMAD3 ratio were increased in the DMOG group;COLIA1,αSMA protein,and mRNA expression levels and pSMAD3/SMAD3 ratio were decreased in the KC7F2 and HIF-1α-siRNA groups.1.4 Effect of intervention with HIF-1α and pSMAD3 on NIH-3T3 transdifferentiation modelCompared with the Control group,COLIA1,α-SMA protein,mRNA expression levels,and pSMAD3/SMAD3 ratio was increased in the DMOG group.Compared with DMOG,COLIA1,α-SMA expression levels,and pSMAD3/SMAD3 ratio was decreased in the DMOG+SIS3 group.2 Mouse silicosis model establishment and the effect of KC7F2 intervention2.1 Effect of KC7F2 intervention on HIF-1α expression on histopathological changes in the lungs of silicosis micePulmonary tissues of mice in the Silica group showed disruption and thickening of alveolar walls,different degrees of inflammatory aggregation,increased secretion of extracellular matrix,and cellular nodules in severe lesions.Compared with the Silica group,the pulmonary tissues of the KC7F2 group showed reduced inflammatory aggregation and infiltration.2.2 Effects of KC7F2 intervention on HIF-1α expression on mRNA and protein expression levels in lung tissue of silicosis miceCompared with the Saline group,α-SMA,COLIA1,HIF-1α protein,mRNA expression levels,and pSMAD3/SMAD3 ratio were significantly higher in the Silica group.Compared with the Silica group,α-SMA,COLIA1,HIF-1α protein,mRNA expression levels,and pSMAD3/SMAD3 ratio were decreased in the KC7F2 group.ConclusionHIF-1α promotes phosphorylation of SMAD3 and transdifferentiation of NIH3T3 cells into myofibroblasts.Inhibition of HIF-1α expression alleviated lung injury and fibrosis development in mice with silicosis.