| Objective: Silicosis is a kind of pneumoconiosis caused by long-term inhalation of free silica dust,which is manifested by pulmonary progressive fibrosis,and its fibrotic lesions impair lung function which eventually leads to respiratory failure.Research on the pathogenesis of silicosis and finding effective treatments are still the focus of current work.This study is intended to explore the role of endoplasmic reticulum stress in the fibrosis process of silicosis,and the possible mechanism of IRE1α-TXNDC5 pathway in the process of fibroblast activation and lung fibrosis,expecting to provide potential targets and credible basis for the treatment of silicosis.Methods: In this study,an experimental silicosis mouse model was established by intratracheal instillation with crystalline silica suspension.Western blot was used to detect the protein expression of endoplasmic reticulum stress-related pathways in the lung tissue of mice exposed to silica for 56 days;The endoplasmic reticulum stress inhibitor TUDCA was administered in vivo,and western blot was used to verify the expression of endoplasmic reticulum stress-related pathway proteins in mouse lung tissue;ELISA was used to detect the effect of inhibiting endoplasmic reticulum stress response on the secretion of pro-inflammatory cytokines in bronchoalveolar lavage fluid(BALF);The HE staining method was used to detect the inflammatory pathological changes in mouse lung tissue.Besides,western blot was used to detect the effect of inhibiting endoplasmic reticulum stress on the protein levels of Fn and Col-1 in mouse lung tissue;and the Masson staining was used to evaluate the collagen deposition in the lungs.Western blot and Realtime-PCR methods were used to detect the protein and gene expression of TXNDC5 in the lungs of mice exposed to silica dust for 56 days;Immunohistochemistry and immunofluorescence methods were used to detect the expression and localization of TXNDC5 in lung tissue;the endoplasmic reticulum stress inhibitor TUDCA was administered to mice in vivo,and the western blot was used to verify the effect of inhibiting endoplasmic reticulum stress on the expression of TXNDC5 protein in mouse lung tissue.The mouse fibroblast cell line NIH-3T3 was used for in vitro experiments.Western blot was used to detect the endoplasmic reticulum stress response and TXNDC5 expression in NIH-3T3 fibroblasts activated by TGFβ1;immunofluorescence was used to detect the localization and expression of TXNDC5 in activated fibroblasts.After transfection of sh RNA,western blot and immunofluorescence methods were used to verify the effect of silencing Txndc5 on the process of fibroblasts producing extracellular matrix;a wound healing assay was used to detect its effect on cell migration,which confirmed the effect of silencing Txndc5 on fibroblast activation.The effect of IRE1α-XBP-1 pathway on TXNDC5 was verified by administration of fibroblast cell line IRE1α inhibitor 4μ8C and transfection of si RNA to knock down XBP-1 in vitro.Western blot and immunofluorescence methods were used to detect the effect of inhibiting IRE1α-XBP-1 pathway on TXNDC5 and extracellular matrix.IRE1α inhibitor 4μ8C was administrated to silica dust-exposed mice in vivo.Through monitoring the lung function of mice,the improvement effect of pharmacologically inhibiting IRE1α function on the lung function of mice was observed;HE staining was used to detect the effect of inhibiting IRE1α function on the inflammatory pathological changes in mouse lung tissue;ELISA was used to detect the effect of pro-inflammatory cytokine secretion in BALF after inhibiting IRE1α function;Western blot and immunohistochemistry were used to detect the expression of Fn and Col-1 in mouse lung tissue to verify the effect of inhibiting IRE1α function on extracellular matrix in lung tissue;Realtime PCR was used to detect the effect of inhibiting IRE1α function on fibrosis-related genes;Masson staining was performed to analyze the effect of inhibiting IRE1α function on collagen deposition in lung tissue;the hydroxyproline content in the lung tissue of each group of mice was measured to detect the effect of inhibiting IRE1α function on the degree of pulmonary fibrosis caused by silica dust.Results:1.Crystalline silica exposure causes endoplasmic reticulum stress in the lung tissue of mice.Western blot was used to detect the protein expression of endoplasmic reticulum stress-related pathways in the lung tissue of mice exposed to crystalline silica for 56 days.The results showed that compared with the control group,the protein levels of IRE1α,ATF6,PERK and Bi P in the lung tissue of the silicosis model mice were significantly up-regulated(p<0.05)after exposure to crystalline silica for 56 days,which showed that endoplasmic reticulum stress occurs in the silicosis model.2.The endoplasmic reticulum stress inhibitor TUDCA can effectively inhibit the endoplasmic reticulum stress response in mouse lung tissue.Western blot was used to detect the expression of endoplasmic reticulum stressrelated pathway proteins in the lung tissue of crystalline silica-exposed model mice after the inhibitor TUDCA was administered.The results showed that compared with the model group,the protein expressions of IRE1α,ATF6,PERK and Bi P were significantly decreased after exposure to crystalline silica at day 7 and day 56(p <0.05).3.Inhibition of endoplasmic reticulum stress can reduce inflammatory and fibrotic changes in the lungs of silicosis model mice.The ELISA assay results showed that in the silicosis model,the secretion of proinflammatory factors IL-1β,TGFβ1 and IL-6 in the BALF decreased after inhibition of endoplasmic reticulum stress;Western blot results showed that inhibition of endoplasmic reticulum stress can reduce the protein expression of Fn and Col-1 in lung tissue of mice after 56 days exposure to crystalline silica(P <0.05);From the results of HE staining and Masson staining,it was observed that after the inhibition of endoplasmic reticulum stress,the inflammatory and fibrotic pathological changes of the lung tissue were reduced.4.TXNDC5 expression is enhanced in lung tissue of silicosis model mice.Western blot and Realtime-PCR method were used to detect the protein and gene expressions of TXNDC5 in the lung tissue of mice exposed to crystalline silica for 56 days.The results showed that the protein and gene expression of TXNDC5 in the lung tissue of the silicosis model group increased significantly(P <0.05).The results of immunohistochemical staining showed that the expression of TXNDC5 in the silicosis area in the lung tissue sections was significantly increased.Immunofluorescence double staining results showed that TXNDC5 was highly expressed in α-SMA-positive fibroblasts,indicating that TXNDC5 was involved in the process of silicosis fibrosis.5.Inhibition of endoplasmic reticulum stress can reduce the increase of TXNDC5 expression caused by crystalline silica exposure.Western blot was used to detect the changes of TXNDC5 expression in the lung tissues of mice exposed to crystalline silica.The results showed that after the endoplasmic reticulum stress was inhibited in vivo,the increased expression of TXNDC5 caused by crystalline silica was significantly inhibited(P <0.05).6.TXNDC5 can regulate the activation of fibroblasts.Western blot was used to detect the expression of endoplasmic reticulum stress receptor proteins IRE1α,ATF6,PERK and TXNDC5 in activated NIH-3T3 fibroblasts,the results showed that the expression of IRE1α,ATF6,PERK and TXNDC5 increased steadily with time.The results of immunofluorescence showed that the up-regulation of TXNDC5 is highly corresponded with the higher expression of α-SMA in activated fibroblasts.After transfection of sh RNA to silence Txndc5,western blot was used to detect the expression of Fn and Col-1 in activated fibroblasts,the results showed that after silencing Txndc5,the expression levels of Fn and Col-1 in activated fibroblasts were significantly suppressed(P <0.05).Immunofluorescence results showed that after silencing Txndc5,the percentage of cells expressing Col-1 or α-SMA was significantly reduced.The results of the wound healing assay showed that the migration ability of fibroblasts activated by TGFβ1 increased significantly,while the ability was significantly inhibited after silencing Txndc5.7.IRE1α-XBP-1 pathway can regulate the expression of TXNDC5 and affect the activation of fibroblasts.Western blot results showed that IRE1α inhibitor 4μ8C can reduce the expression of TXNDC5 in activated fibroblasts(P <0.05).In lung tissues of silicosis model,western blot and Realtime-PCR results showed that 4μ8C can inhibit the protein and gene expression of TXNDC5.After silencing Xbp1 by the transfection of si RNA in vitro,western blot and immunofluorescence results showed that the expression of TXNDC5 in activated fibroblasts decreased significantly(p <0.05).Western blot and immunofluorescence results showed that the use of 4μ8C or si RNA to silence Xbp1 can significantly reduce the expression of Fn and Col-1 in activated fibroblasts.8.Inhibition of IRE1α endonuclease activity can improve the level of lung function in silicosis model mice.The dynamic changes of the lung function of the mice were detected by monitoring and recording the breathing frequency and the minute volume in each group of mice,the results showed that inhibition of IRE1α endonuclease activity by 4μ8C can effectively improve the lung function level of silicosis model mice.9.Inhibition of IRE1α endonuclease activity can reduce the inflammatory and fibrotic changes in lung tissue of silicosis model mice.The ELISA assay results showed that in the silicosis model,the secretion of proinflammatory factors IL-1β,TGFβ1 and IL-6 in the BALF significantly decreased after 4μ8C treatment(p <0.05);Western blot and immunohistochemical staining results showed that inhibition of IRE1α endonuclease activity can reduce the expression of Fn and Col-1 in lung tissues after 56 days exposure to crystalline silica(P <0.05);Realtime PCR results showed that the m RNA level of Col1a1,Col3a1 and Vim in the lung tissue of model mice was significantly reduced after 4μ8C treatment,and the difference was statistically significant(P <0.05);the hydroxyproline content in the lung tissue of each group of mice was measured and the results showed that 4μ8C could effectively reduce the content of hydroxyproline in lung tissue of silicosis model mice.From the results of HE and Masson staining,it was observed that inhibition of IRE1α endonuclease activity alleviated the pathological changes of inflammation and fibrosis in lung tissue.Conclusion: Inhibition of endoplasmic reticulum stress can alleviate lung inflammation and fibrosis in mice caused by silica dust exposure;TXNDC5 mediated fibroblast activation is regulated by the endoplasmic reticulum stress pathway IRE1α-XBP-1;inhibiting the endonuclease function of IRE1α can affect the activation of fibroblasts and the secretion of extracellular matrix by reducing the expression of TXNDC5 protein,thereby alleviating lung inflammation and fibrosis caused by silica dust exposure. |
