Observe The Mitochondrial Metabolism During The Fibroblast Transdifferentiation Induced By SiO₂ Dust

Background and objectiveSilicosis is an extensive fibrosis of lung tissue caused by long-term inhalation of free silica(SiO2)dust.Its occurrence and development is a complex process with the participation of a variety of cells,organelles and molecules participate together,and the transdifferentiation of lung fibroblasts to myofibroblasts is an important link.It is generally believed that energy needs to be consumed in the process of cell transdifferentiation to meet the cell synthesis and secretion of related proteins,and the production of energy requires the participation of mitochondrial metabolism.Previous studies have proved that in lung diseases such as idiopathic pulmonary fibrosis(IPF),chronic obstructive pulmonary disease(COPD),lung cancer and other lung diseases,there are changes and effects of mitochondrial morphology and function,but its status and role in the process of silicosis fibrosis is still unclear.Therefore,through the establishment of SiO2 dust-induced lung fibroblast transdifferentiation model in vitro,using laser confocal microscope,Western blot,real-time fluorescence quantitative PCR and other technologies,the mitochondrial function changes during transdifferentiation process are observed,and its potential relevance is explored,which provides new ideas and reference basis for further understanding the regulation mechanism of mitochondria during the pathogenesis of silicosis and further clarifying the pathogenesis of silicosis.Materials and Methods1.MaterialsHuman embryonic lung fibroblasts(MRC-5)and human monocytes(THP-1)were purchased from the Cell Bank of the Chinese Academy of Sciences.2.MethodsPropylene glycol monomethyl ether acetate(PMA)was used to induce human monocytes(THP-1 cells)to differentiate into macrophages,and CCK-8 experiments were used to determine the concentration of SiO2.qPCR was used to detect the mRNA expression levels of transdifferentiation markers FN1,COL1,and ?-SMA and so on after different concentrations of TGF-? stimulated human embryo lung fibroblasts(MRC-5 cells),and then to determine the toxoic concentration of TGF-?.Co-culture models of THP-1 cells induced macrophages and MRC-5 cells were constructed using the Transwell chamber.The experiments were divided into a blank control group,a SiO2 exposure group,and a TGF-? exposure group.The morphological changes of the cells were observed under a light microscope respectively at 0h,24h,48h and 72h after exposure.And the expression of mRNA and protein of transdifferentiation markers before and after the experiment were detected by qPCR,Western blot,and immunohistochemistry.A simplified model was established by directly stimulating MRC-5 cells with TGF-? to determine the concentration of stimulation.The control group and the stimulation group were exposed to the virus for 0h,24h,48h and 72h respectively,and the changes of cell morphology were observed under optical microscope.qPCR and Western blot were used to detect the expression levels of mRNA and protein such as transdifferentiation markers FN1,COL1,?-SMA before and after the experiment.For the above experimental groups,the changes of mitochondrial membrane potential were detected by rhodamine 123 kit,The levels of mitochondrial reactive oxygen species(ROS)were detected by Mito SOX Red,mitochondrial ATP levels were detected by ATP kit,western blot was used to detect the expression of mitochondrial marker proteins and autophagy related proteins,qPCR was used to detect mRNA levels of mitochondrial marker proteins such as VDAC,TOM40,COX4,etc.3.Statistical analysisSPSS21.0 was used for statistical analysis.When the measurement data conforms to the normal distribution after normality test,the results are expressed as mean ±standard deviation(X±S).The mean comparison between two independent samples was performed by two independent samples t test.One-way analysis of variance was used for comparison of mean differences between multiple groups.LSD-t test was used for pairwise comparison between groups when variances were equal,and Dunnett-t test was used for variances.P-value<0.05 was applied to declare statistical significance.Results1.Establishment and observation of SiO2 dust induced lung fibroblast transdifferentiation modelIn the co-culture model,150 ?g/ml SiO2 exposure concentration and 5 ng/ml TGF-? exposure concentration were selected for experiments.After exposure for 0h,the morphology of cells was the same among the blank control group,SiO2 exposure group and TGF-? exposure group,and the expression difference of mRNA and protein such as transdifferentiation markers FN1,COL1 and ?-SMA had no statistical significance.After 24h,48hand 72h of treatment,compared with the blank control group,the SiO2 exposure group and the TGF-? exposure group all had changes in cell morphology.The mRNA level and protein expression of FN1,COL1,COL3,CTGF,SERPINE1,VCAN,?-SMA increased with time,while the protein expressions of FN1,COL1,?-SMA increased at 24h,increased most obviously at 48h and decreased at 72h,with statistical significance(P<0.05).In the TGF-? direct stimulation experiment,compared with the control group,the morphology of MRC-5 cells stimulated by TGF-? did not change significantly after 0h,24h,48h,72 h.After 24h,48h and 72h of stimulation,the mRNA expressions of FN1,COL1,COL3,CTGF,SERPINE1,VCAN,?-SMA and the protein expressions of FN1,COL1,?-SMA are all increased with time,with statistically significant difference(P<0.05).2.Basic function of mitochondria and expression level of related proteins during fibroblast transdifferentiationIn the co-culture model,compared with the blank control group,the SiO2 exposure group and the TGF-? exposure group have increased mitochondrial membrane potential,increased ROS level,decreased ATP level,VDAC mRNA and protein expression increased,TOM40 and COX4 expression decreased only in 72h,and LC3?/LC3? ratio increased,with statistically significant difference(P<0.05).In the TGF-? direct stimulation experiment,compared with the control group,the change trend of membrane potential,ROS,ATP and expression level of related molecules in the stimulation group is the same as that in the co-culture model,and the difference is also statistically significant(P<0.05).ConclusionsSiO2 dust and TGF-? promoted fibroblast transdifferentiation in vitro.Mitochondrial membrane potential increased,ROS level increased,ATP level decreased,and some mitochondrial marker protein homeostasis changed.It is suggested that the mechanism of mitochondrial metabolism is involved in the process of fibroblast transdifferentiation.