Study On The Effect Of STING On Fibroblasts Fibrosis Mediated By Macrophage Supernatant Exposed To Silica

Objective: Silicosis is an occupational lung disease caused by inhalation of silica and manifests as silica nodules and diffuse interstitial fibrosis formation.Interferon gene stimulating factor(STING)has been found to have the ability to induce innate immune responses in a variety of tissue cells,but the specific mechanism of action of STING in the supernatant-mediated fibroblasts of silica exposed macrophages needs to be further explored.In this study,we firstly examined whether silica exposed macrophage supernatant could promote the expression of STING in fibroblasts,and secondly investigated the effect of silica exposed macrophage supernatant on fibroblast fibrosis after silencing STING in fibroblasts,to provide a theoretical basis and experimental foundation for elucidating the role of STING signaling pathway in the fibroblast fibrogenic effect mediated by silica exposed macrophage supernatant.Methods: In vitro experiments were performed using MH-S alveolar macrophages and MLg mouse lung fibroblasts.The treatment group was silica suspension stimulated MH-S cells for 18 h and the culture fluid was collected and used as conditioned culture supernatant,while the control group only added an equal volume of saline.Western Blot and Realtime-PCR were applied to detect the protein and gene expression of STING in MLg cells by silica exposure of MH-S supernatant.STING-silenced MLg cell lines were constructed by lentiviral transfection of sh-RNA and divided into the following four groups: control group(Saline-CM+sh-NC),silica group(CS-CM+sh-NC),STING-silenced control group(Saline-CM+sh-STING)and STING-silica group(CS-CM+sh-STING).The effects of STING silencing on the STING signaling pathway and extracellular matrix production process in fibroblasts were verified at the protein and gene levels by Western Blot and Realtime-PCR;the expression of α-SMA,Col-Ⅰ and Fn in STING-silenced MLg cells was detected by immunofluorescence;the effect of silica exposure to MH The above experiments together investigated the mechanism of STING signaling pathway on the effect of silica exposed MH-S supernatant on fibroblast fibrosis.Results: 1.Supernatant from silica exposed macrophages promotes STING expression in fibroblasts.Western Blot and Realtime-PCR results showed that protein and gene expression of STING was elevated in silica exposed MH-S cells after 6h and 18 h of conditioned culture supernatant-mediated MLg cells compared to control(P<0.05).2.STING silencing reduced levels of fibroblast fibrosis-associated protein expression mediated by silica exposed macrophage supernatant and inhibited the migration ability of fibroblasts.STING-silenced MLg cell lines were established and the expression of STING signalling pathway and extracellular matrix proteins were detected by Western Blot and Realtime-PCR.Compared with the control group,silica exposure to MH-S supernatant increased the expression of STING signaling pathway-related proteins and genes in MLg cells,and the expression levels of α-SMA,Col-Ⅰ and Fn were up-regulated(P<0.05).After silencing of STING,the expression levels of STING signaling pathway-related proteins and genes in MLg cells mediated by silica exposure to MH-S supernatant were reduced,and the expression levels ofα-SMA,Col-Ⅰ and Fn were significantly inhibited(P<0.05).Immunofluorescence results showed that the percentage of cells expressing α-SMA,Col-Ⅰ or Fn positivity was significantly reduced after silencing STING in MLg cells mediated by silica exposure to MH-S supernatant.The results of the cell scratch assay showed that the migration ability of fibroblasts mediated by silica exposure to MH-S supernatant was significantly enhanced,whereas the migration ability of fibroblasts was significantly inhibited after silencing of STING.Conclusion:1.Silica exposed macrophage supernatants upregulate STING expression in fibroblasts.2.Silencing STING in fibroblasts reduces the expression of proteins associated with fibroblast fibrogenic effects mediated by silica exposed macrophage supernatants and inhibits fibroblast migration capacity.