Study On The Mechanism Of TUDCA In Crystalline Silica-Induced Pulmonary Injury Through Regulating ER-Phagy

Objective: Silicosis is a systemic disease mainly manifested by pulmonary diffuse fibrillation caused by long-term inhalation of free crystalline silica in production.The development of silicosis is progressive,characterized by chronic inflammation and pulmonary fibrosis.Given that the course of silicosis is slow,the pathogenesis has not been well understood,and the pathological injury of pulmonary fibrosis is irreversible.Our previous studies have shown that autophagy and ER stress play an important role in pulmonary inflammation and fibrosis caused in silicosis,while the relationship between them has not been clarified.Therefore,this project intends to explore the interaction and mechanism of ER-phagy and ER stress on the progression of silicosis,providing new theoretical basis for the prevention and control of silicosis.Methods: In this study,experimental C57BL/6 mice were treated with intratracheal instillation of crystalline silica suspension to establish an animal model of silicosis.The expression of ER stress-related proteins in lung tissues of silicosis model was detected by Western blot.By treating with ER stress inhibitor,the effect of ER stress on autophagyrelated proteins was detected in the same way.Realtime-PCR and Western blot were used to detect the protein transcription and translation of ER-phagy receptor FAM134 B in lung tissues of mice in the different period of silicosis.The effect of ER stress on ERphagy was detected by immunofluorescence.Western blot was used to verify the effect of ER-phagy inhibition on apoptosis in the lung tissue of silicosis model mice.ELISA was used to detect the content of cytokines in the BALF of mice in each group,and HE staining was used to verify the effect of ER-phagy inhibition on pulmonary inflammation caused by silica dust.Masson staining was used to observe the effect of endoplasmic reticulum autophagy inhibition on pulmonary fibrosis induced by crystalline silica.Result: 1.Crystalline silica induces autophagy by activating ER stress in vivo.Western blot was used to detect the expression of ER stress-related proteins in the lung tissues of model mice in the different period of silicosis,and the expression of autophagy related proteins in the lung tissues of mice treated with ER stress inhibitor.We observed increased levels of BIP,PERK,IRE1α and CHOP in lung tissue of mice exposed to silica compared with the control group(P<0.05).ER stress inhibitor could decrease the up-regulation of LC3 II,ATG5,and Beclin1 expression levels induced by silica exposure(P<0.05).2.Silica exposure induces ER-phagy in vivo.Realtime-PCR and Western blot were used to detect the ER-phagy receptor FAM134 B transcription and translation changes in the lung tissues of silica-exposed mice.The results showed that silica exposure up-regulated Retreg1 transcription and FAM134 B translation in lung tissue(P<0.05).The co-localization of the autophagosome membrane marker LC3 and endoplasmic reticulum marker Calnexin was detected by immunofluorescence assay.The results showed that silica exposure up-regulated the fluorescence expression of both LC3 and Calnexin and the co-localization of these two proteins,suggesting that the combination of autophagosome and endoplasmic reticulum increased,while ER stress inhibitor could reverse the effect of silica exposure.3.TUDCA attenuates pulmonary injury in silica-exposed mice by suppressing ER-phagy.Western blot was used to detect the expression of apoptosis-related proteins in lung tissue.The results showed that silica exposure induced the up-regulation of Caspase 3expression,and TUDCA decreased the expression of Caspase 3 in lung tissues of silicaexposed mice by suppressing ER-phagy(P<0.05).The results of ELISA showed that the secretion of TNF-α and MCP-1 in the BALF of silicosis group was increased and the inhibition of ER-phagy could reduce the secretion of these cytokines in the BALF of silica-exposed mice(P<0.05).HE staining was used to detect pulmonary inflammation,and the results showed that TUDCA could significantly alleviate inflammatory cell infiltration and inflammatory damage induced by silica exposure through suppressing ER-phagy.Masson staining was used to detect the symptoms of pulmonary fibrosis.The results showed that suppressing ER-phagy significantly alleviated collagen deposition in lung tissue of silica-exposed mice.Conclusion: 1.Silica exposure activates ER stress and then induces ER-phagy in silicosis mice.2.TUDCA alleviates pulmonary inflammation and fibrosis caused by silica dust by inhibiting ER-phagy,and plays a protective role in lung tissue injury.