The Role Of Lipid Metabolism Reprogramming And Its Key Gene PGC1α In The Pathogenesis Of Silicosis

Objective To study the mechanism of lipid metabolic reprogramming regulating the occurrence and development of silicosis fibrosis.In addition,overexpress and down its key gene,the peroxisome proliferator-activated receptor-γcoactivator 1-α（PGC1α）and its agonist 2-（4-tert-butylphenyl）benzimidazole（ZLN005）were used to intervene in lipid metabolic reprogramming,and the effects and mechanisms of regulating lipid metabolic pathways on silicosis fibrosis were observed.Methods 1 A dataset of silicosis rats was retrieved from GEO database.Silicosis rat and mouse models were constructed by injecting SiO₂ suspension into trachea at one time.NR8383 rat alveolar macrophages,alveolar macrophages in the lavage fluid of silicosis patients and primary fibroblasts were selected as cell culture materials in vitro.2 The experimental protocol was as follows:1)Differential genes in silicosis rats were screened by bioinformatics analysis,and verified on silicosis rat models and NR8383 cells to observe whether there is lipid metabolic reprogramming in the progression of silicosis.2)In order to observe the effect of PGC1αon lipid metabolic reprogramming,NR8383 cells were stimulated with SiO₂ and treated with si-PGC1α,overexpressed PGC1αplasmid,and ZLN005,respectively.3)The supernatant from the above NR8383 cells of each group was used to stimulate rat primary fibroblasts,and the silicosis mouse model was constructed by a one-time infusion of SiO₂ suspension into the trachea,and the silicosis mice were treated with ZLN005 to observe the effect of PGC1αon silicosis fibrosis by regulating lipid metabolism reprogramming.3 Differential genes were screened and enriched with R packages.HE staining was used to observe the pathological changes of lung tissue in rats and mice.The collagen deposition in lung tissue was observed by VG staining.Expressions of PGC1α,CD36,COL（40）and TNF-αin lung tissues were observed by immunofluorescence staining.The expressions of COL（40）,Eca,FN,IL-6,IL-1β,PGC1α,CD36,LXR,ABCA1,SDHA,NDUFS1 and COXⅣin animal tissues and cells were detected by western blotting.The expressions of Abcg1,Ccl5,IL-1β,SOD2,Cybb,Il18,Cyba,Cd14,Cxcl2,Ccl3,Cxcl1,Ccl2 and CD36 in rat lung were detected by real-time quantitative PCR.Lipid levels in animal lung tissues and cells were detected by oil red O and BODIPY 493/503 staining.The contents of TC,FC and CE and the ratio of CE to TC in serum of animals and cells were detected by total cholesterol and free cholesterol kits.Lipid metabolomics detects markers of differential lipid metabolism in animal tissues,animal serum,and cells.The changes of mitochondrial complex（40）,ⅡandⅣin NR8383 were detected by O2K.Fluorescent probes Mito SOX^TM,DHE and DCFH-DA were used to detect mitochondrial ROS levels,intracellular reactive oxygen species and hydrogen peroxide content,respectively.The level of TGF-β1 in NR8383 cell supernatant was detected by ELISA.Computed tomography and whole body plethysmography were used to detect fibrosis and lung function in mice.Results 1 The differentially expressed genes in silicosis rats were screened and analyzed by bioinformatics methods,and verified on silicosis rat models and SiO₂-stimulated NR8383cells:1)426 differential genes were identified in SiO₂-stimulated rats,which enriched lipid and atherosclerotic pathways.2)Compared with the control group,the m RNA levels of Abcg1,IL-1β,SOD2,Cyba,Cd14,Cxcl2,Ccl3,Cxcl1,Ccl2 and CD36 in lung tissues of silicosis model group were increased,while Ccl5,Cybb and Il18 were decreased.3)Silencing CD36 in NR8383 reduced the enhancement of SiO₂-induced BODIPY 493/503fluorescence staining.4)In the lung tissue of silicosis rats,compared with the control group,the nodules in the silicosis model group gradually increased,fibrosis increased in a time-dependent manner,the contents of TC,FC and CE in serum were significantly increased,the expressions of COL（40）,CD36 and Fas were up-regulated,while the expressions of Eca,PGC1α,LXR and P-ACC/ACC were down-regulated.5)Lipid metabolomics screening differentiated metabolites in lung tissue and plasma of silicosis rats showed that they were mainly enriched in metabolic pathways such as glycerophospholipid metabolism.6)There are lipid metabolism disorders in alveolar macrophages of silicosis patients.2 In vitro intervention of PGC1αexpression in NR8383 cells:1)SiO₂ stimulation decreased the expression of ABCA1,LXR,PGC1α,SDHA and NDUFS1 proteins,increased the expression of IL-1β,IL-6,CD36 proteins,increased levels of intracellular TC,FC and CE,and enhanced fluorescence staining of BODIPY 493/503.Mitochondrial respiratory chain was damaged,ROS was enhanced and membrane potential decreased.Lipid metabolomics analysis of their differences in lipid metabolites mainly enrichment of glycerol phospholipid metabolism and other metabolic pathways.In addition,the stimulation of SiO₂ led to the increase of TGF-β1 secreted by macrophages.2)Overexpression of PGC1αinhibited the increase of lipid content stimulated by SiO₂,restored the activities of complexes（40）andⅡin the mitochondrial respiratory chain,and the secretion of TGF-β1 tended to decrease.3)Silencing PGC1αaggravated the increase of lipid content induced by SiO₂ stimulation,reduced the activity of complex（40）in the mitochondrial respiratory chain,and increased the secretion of TGF-β1.4)ZLN005 inhibited the increase of lipid content stimulated by SiO₂,and restored the activities of complexes（40）andⅡin mitochondrial respiratory chain.The differential lipid metabolites of ZLN005 were analyzed by lipid metabolomics,and it was found that it mainly enriched glycerophospholipid metabolic pathways.3 Primary fibroblasts were stimulated with the supernatant of NR8383 cells respectively,and ZLN005intervention was given in silicosis mouse model:1)The expressions of COL（40）and FN were increased and Eca was decreased due to SiO₂ stimulation.2)Overexpression of PGC1αdecreased the increase of COL（40）and FN expression and the decrease of Eca expression induced by SiO₂.3)Silencing of PGC1αexacerbated the increase of COL（40）and FN expression and the decrease of Eca expression induced by SiO₂.4)Compared with the control group,the lipid metabolism was disturbed and fibrosis was enhanced in silicosis model group.5)Compared with the silicosis model group,ZLN005 decreased the lipid content in the lung tissue of silicosis mice,increased the expressions of PGC1α,Eca and LXR,decreased the expressions of COL（40）,FN,TNF-α,IL-6 and CD36,and inhibited the increase of TC,FC and CE in serum of silicosis mice.Conclusions 1 The process of silicosis fibrosis is accompanied by abnormal activation of lipid metabolism and reprogramming of lipid metabolism.2 Overexpression of PGC1αand ZLN005 can improve SiO₂-induced lipid metabolism disorder,and silenced PGC1αcan aggravate SiO₂-induced lipid metabolism disorder.3 By targeting PGC1α,ZLN005 can inhibit SiO₂-induced lipid metabolism disorder and regulate lipid metabolic reprogramming,thereby inhibiting the progression of silicosis fibrosis.Figure 94;Table 7;Reference 322...