Effects Of Lipoic Acid Synthase Gene On Silicosis By Regulating NRF2 Signaling And Its Mechanism

Objectives To investigate the effects of lipoic acid synthase（LIAS）on silicosis fibrosis by the nuclear factor E2 related factor 2（NRF2）signaling pathway and to evaluate the effect of lipoic acid on cellular redox balance.Methods 1 A total of 107 patients with silicosis and 45 healthy controls were randomly selected in this study.The elementary informations from the participants of two groups were investigated,and physical examination and pulmonary function were carried out.PBMCs were isolated from peripheral blood and NRF2 protein expression was detected by immunofluorescence.The dose-response relationship beween LIAS and NRF2 m RNA expression levels and their association with silicosis were analyzed by restricted cubic spline（RCS）and logistic regression.Key DEGs and signaling pathways were identified using RNA-Seq and bioinformatics technology.2 To establish experimental model of silicosis with wildtype and Lias ^H/Hmice,a new antioxidant mouse model which have overexpressed Lias gene compared to wild type.The pathological changes of pulmonary fibrosis and effectsα-lipoic acid on oxidative stress,inflammatory and pulmonary fibrosis biomarkers were examined in the mice after crystalline silica instillation.3THP-1 cells were differentiated into macrophages with 160 nm PMA.Cell viability and ROS levels were detected after treatment using SiO₂and ALA,the expression levels of LIAS and NRF2 were measured by immunohistochemistry and immunofluorescence,and the cell mitochondrial pressure was detected by Seahorse.Results 1 The positive expression rate of NRF2 in PBMCs from patients with stage I silicosis was significantly higher than that of the control group（P<0.001）.Results of RCS showed that there was a linear dose-response relationship between LIAS and NRF2m RNA expression levels.The results of multivariate analysis showed that LIAS and NRF2 were increased the risk of silicosis incidence in stageⅠsilicosis patients and NRF2 was a protective factor in silicosis patients with stage II and III after adjusting for potential confounding factors including age,education level,BMI and smoking.The immunomodulatory abnormalities of silicosis are related to the lymphocyte oxidative state.Omics analysis revealed 1158 differential genes,of which 475 were up-regulated and 683 were down-regulated.PPI analysis revealed NRF2 plays a pivotal role in antioxidant regulation.NRF2-regulated DEGs were associated with glutathione metabolism,transforming growth factor-β,and the extracellular matrix receptor interaction signaling pathway in PBMCs from patients with silicosis.Ten key genes were found from 27 differential genes related to three pathways through PPI network.Smad2,BMP4,mapk3 and transcription factors THBS1 and ITGB3 increased,while the expressions of CD44 and SMAD3 decreased.All differences were statistically significant（P<0.05）.2 Lias ^H/Hmice alleviated pathological alterations in the early stage of pulmonary fibrosis induced by silica.Oxidative stress was significantly increased in mice after silica instillation.The antioxidant defense was strengthened through NRF2 and LIAS in Lias ^H/Hmice.The inflammatory factor and chemokines levels decreased after inhibition of oxidative stress by LIAS.The activity of NF-κB was inhibited in Lias ^H/Hmice after silica instillation.The protein expression levels of TGF-β1,Vimentin andα-SMA in Lias ^H/H+SiO₂group were lower than that in WT+SiO₂group.All differences were statistically significant（P<0.05）.3 Compared with the control group,the cell viability was decreased in SiO₂exposed groups and increased in 100μM ALA group after intervention for 24 h and 48 h.The activity of THP-1-derived macrophages was increased and the production of ROS was decreased after 50μM and 100μM ALA intervened THP-1-derived macrophages treated by 50μg/ml SiO₂for 24 h and 48 h.Compared with the control group,the expression levels of LIAS and NRF2 and the cell oxygen consumption were increased in THP-1-derived macrophages in 50μg/ml SiO₂exposed group.Compared with SiO₂exposed group,the expression of LIAS,Nrf2 and OCR were decreased after the ALA intervention group for 24 h.All differences were statistically significant（P<0.05）.Conclusions 1 LIAS/NRF2 pathway is involved in the pathophysiological process of silicotic fibrosis by regulating the oxidative stress network.2 The overexpression of lipoic acid synthase gene in mice retarded the development of silica-induced pulmonary fibrosis through regulating Nrf2,NF-κB pathway and epithelial mesenchymal transformation.3 Lipoic acid can protect macrophages mitochondria,reduce oxidative stress induced by SiO₂and improve cell viability.Figure 31;Table 22;Reference 312...