Study On The Mechanisms Of Dioscin Alleviating Crystalline Silica-Induced Pulmonary Inflammation And Fibrosis Through Regulating Alveolar Macrophage Autophagy

Objective:Occupational silica exposure can lead to silicosis,characterizing by chronic inflammation,abnormal tissue repair and irreversible pulmonary fibrosis.Due to the lack of understanding of silicosis,effective clinical treatment is still unclear.Therefore,it is urgent to find specific molecular targets and economic effective drugs.Alveolar macrophages play important roles in the pathogenesis and development of silicosis.Our previous studies have elucidated that dioscin,an effective natural monomer,plays an active role in the treatment of silicosis by regulating the innate immune response.However,the specific molecular mechanism remains to be explored.The purpose of this study is to explore the possible mechanism of dioscin regulating macrophages in the process of silica-induced pulmonary fibrosis,which provids basis for the treatment of silicosis.Methods:The experimental silicosis mice model was established by intratracheal instillation of crystalline silica?CS?.Realtime-PCR and immunoblotting analysis were used to detect the expression of autophagy-related genes and proteins in lung tissues of experimental silicosis mice exposed to CS at early?7 days?and late?56 days?stage.The effect of autophagy on the alveolar macrophages apoptosis was validated in vitro by flow cytometry and TUNEL analysis through establishing alveolar macrophage model exposed to CS and giving autophagy activator or autophagy inhibitors.Possible biological effects of dioscin were predicted by bioinformatics and LC3-GFP-mRFP transfected macrophages were constructed to observe the dynamic regulation of dioscin on autophagy flux.In CS exposure models in vivo and in vitro,immunofluorescence,immunoblotting and apoptotic detection were used to further confirm the autophagy activation and anti-apoptotic effect of dioscin;the effect of dioscin on autophagy and apoptosis was correlated through the combined administration of dioscin and autophagy inhibitor.The above experiments were conducted to demonstrate the effect of dioscin in regulating autophagy on the CS-induced alveolar macrophages apoptosis.Atg5-silenced alveolar macrophage line?MH-S?and conditional Atg5-knockout mice(Atg5^flox/floxDppa3^Cre/+)were used to establish experimental silicosis model,and dioscin was administered in vivo and in vitro.Dioscin'effect on mitochondria was detected by immunofluorescence and immunohistochemistry.the effects of dioscin on mitochondrial ROS content was detected by mitoSOX^TM staining;the effects of dioscin on mitochondrial membrane potential through regulating autophagy were detected by JC-1,TMRE and Rhodamine123 staining;the effects of dioscin on mitochondrial function through regulating autophagy were measured by ATP assay;cytochrome c and caspase staining.3/7 activity was used to measure the effect of dioscin on mitochondrial apoptotic pathway activation by regulating autophagy.The above experiments were conducted to illustrate the effect of dioscin in regulating autophagy on CS-induced mitochondrial damage.ELISA and realtime-PCR analysis were used to detect cytokines in MH-S cells,alveolar macrophages and BALF at transcriptional and secretory levels;Realtime-PCR and immunoblotting were used to detect MMPs in MH-S cells and lung tissues at gene and protein levels.The effects of dioscin on inflammation were observed by immunohistochemical staining and HE staining.Collagen deposition was detected by realtime-PCR and sirius red staining staining to measure the effect of dioscin-induced autophagy on silicosis fibrosis.The above experiments were conducted to illustrate the effects of dioscin in regulating autophagy on CS-induced inflammation and fibrosis.Result:1.Autophagy increased in lung tissue of experimental silicosis miceImmunoblotting and realtime-PCR were used to detect the expression of autophagy-related proteins and genes in lung tissues at early and late stages of silicosis.The results showed that the expression of LC3II,ATG5,BECN1 and P62 protein in lung tissue of mice in the experimental group increased at both early and late stages of silica exposure?P<0.05?;the expression of autophagy-related genes increased at two time points,and the changes of P62 genes in the early stage and Atg5,Ulk1 and P62 in late stages of CS exposure were statistically significant.2.Promoting autophagy can reduce CS-induced apoptosis of alveolar macrophagesFlow cytometry and TUNEL analysis were used to detect the apoptosis of MH-S cells exposed to CS,respectively or in combination with autophagy activator,autophagy inhibitors and dioscin.The results showed that inhibition of autophagy could increase the apoptotic level of MH-S cells stimulated by CS and promote autophagy could reduce the apoptotic level of MH-S cells stimulated by CS?P<0.05?.3.Dioscin reduces apoptosis by promoting alveolar macrophage autophagy The results of apoptotic experiment showed that dioscin could reduce the apoptotic level of cells,but when it was given with autophagy inhibitors,dioscin could not reduce the apoptotic level.Laser confocal microscopy showed that dioscin could promote the autophagic flux of MH-S cells;Immunoblotting showed that compared with the experimental group,the expression of LC3II and BECN1 protein in MH-S cells increased,while the expression of P62 protein decreased in a dose-effect relationship?P<0.05?;the phosphorylation level of AKT/mTOR pathway in MH-S cells significantly decreased in the experimental group,and it had a dose-effect relationship?P<0.05?.Dose-effect relationship?P<0.05?.Immunoblotting and immunofluorescence analysis were used to detect the changes autophagy of lung tissue and alveolar macrophages in silicosis model mice treated with dioscin.Compared with the experimental group,the expression of LC3II and BECN1 protein in lung tissue and alveolar macrophages increased,while the expression of P62 protein decreased in the experimental group,regardless of in early or late stage of CS stimulation?P<0.05?;fluorescence expression of LC3II protein increased and P62 protein decreased in alveolar macrophages of mice in the experimental group.Flow cytometry and TUNEL were used to detect primary alveolar macrophages.The results showed that dioscin could reduce the apoptotic induced by CS at two time points?P<0.05?.4.Dioscin can promote mitophagy of alveolar macrophages.Immunoblotting results showed that dioscin could promote the expression of autophagy-related proteins LC3II and BECN1 in MH-S cells and reduce the expression of P62 protein.Meanwhile,dioscin could promote the expression of mitophagy-related proteins PINK1and Parkin,while the effect of dioscin could be reversed by Atg5 silence.Immunofluorescence results showed that the expression of autophagy marker LC3?red?or coincidence points?yellow?of autophagy and mitochondrial marker in the experimental group was higher than that in the experimental group.5.Dioscin helps maintain mitochondrial function through promoting macrophage autophagyFlow cytometry and fluorescence microscopy were used to detect the expression of mitochondrial ROS in alveolar macrophages.The results showed that dioscin could alleviate the overexpression of mitochondrial ROS induced by CS,but this effect disappeared in Atg5-silenced cells?P<0.05?.JC-1 stainstaining was used to test MMP.We observed that dioscin maintained mitochondrial membrane potential in an autophagy-dependent manner and reduced the mitochondrial membrane potential caused by silica?P<0.05?.TMRE and Rhodamine 123 staining were used to detect mitochondrial permeability.The results showed that dioscin could reduce the increase of mitochondrial membrane permeability caused by silica through autophagy.The mitochondrial function was measured by measuring the content of ATP in cells.Dioscin could enhance mitochondrial function,and Atg5 silence could reverse the effect of dioscin?P<0.05?.We obtained macrophages from alveolar lavage fluid of Atg5^flox/floxDppa3^Cre/+mice silicosis model,and examined the effect of dioscin on mitochondrial function in vivo.Similar to the results of in vitro assay,dioscin can reduce the high expression of mitochondrial ROS,decrease of mitochondrial membrane potential,increase of mitochondrial permeability and the decrease of mitochondrial function in alveolar macrophages induced by CS,but the effects of dioscin was reversed in autophagic deficient Atg5^flox/floxDppa3^Cre/+mice alveolar macrophages?P<0.05?.6.Dioscin inhibits the activation of mitochondrial apoptosis pathway through promoting macrophage autophagyCytochrome c,a key protein in mitochondrial apoptosis,was detected by immunofluorescence assay.We observed that CS could increase the fluorescence intensity of cytochrome c and widely distribute in cytoplasm.Dioscin could restrict the distribution of cytochrome c.Atg5 silence could reverse the effect of dioscin.The results of caspase3/7 activity assay showed that dioscin could reduce the increase of caspase 3/7 activity induced by CS,and Atg5 silence could reverse the effect of dioscin?P<0.05?.7.Dioscin decreases alveolar macrophage cytokine secretion through promoting autophagyELISA was used to detect the expression of macrophage cytokines in the supernatant of MH-S cells.The results showed that dioscin could significantly reduce the levels of IL-1?,IL-6 and MCP-1 secreted by MH-S cells stimulated by CS.Both Atg5 silence and BECN1silence could reverse the effect of dioscin?P<0.05?.Realtime-PCR and ELISA were used to detect the expression of specific cytokines in alveolar macrophages of silicosis model mice treated with dioscin at transcriptional and translation levels.The results showed that dioscin could significantly reduce the expression of Il-1?,Il-6 and MCP-1 genes and secretion of factors in alveolar macrophages with statistical significance.However,the secretion levels of IL-1?,IL-6 and MCP-1 in the alveolar lavage fluid of Atg5^flox/floxDppa3^Cre/+mice didn't decrease due to the administration of dioscin,while the secretion levels of IL-1?,IL-6 and MCP-1 in CS group were higher than those in Atg5^flox/floxDppa3^+/+mice?P<0.05?.8.Dioscin decreases MMPs secretion in macrophages through promoting autophagyImmunoblotting analysis was used to detect the expression of MMPs in MH-S cells.The results showed that dioscin could significantly reduce the levels of MMP9 and MMP12secreted by MH-S cells stimulated by CS,and Atg5 silence could reverse the effect of dioscin?P<0.05?.The expression of MMPs in lung tissue of silicosis model mice treated with dioscin was detected by Realtime-PCR and immunoblotting at gene and protein levels.The results showed that dioscin could significantly reduce the transcription and protein expression of MMP9 and MMP12 genes with statistical significance.However,the transcription levels of Mmp9 and Mmp12 genes in lung tissues of Atg5^flox/floxDppa3^Cre/+mice with autophagy deficiency did not decrease due to dioscin administration,while the transcription levels of Mmp9 and Mmp12 genes in model group were higher than those in Atg5^flox/floxDppa^3+/+mice?P<0.05?.9.Dioscin delays the inflammation and fibrosis process of silicosisThe aggregation of macrophages in tissues was detected by immunohistochemistry.We observed that dioscin could significantly reduce the increase of macrophage marker F4/80caused by CS.HE staining was used to detect inflammatory cell infiltration and inflammatory damage.We observed that dioscin could significantly alleviate lung tissue damage caused by CS,but dioscin could not alleviate lung inflammation induced by CS in Atg5^flox/floxDppa3^Cre/+mice.Realtime-PCR and Sirius red staining were used to detect collagen deposition.The results showed that dioscin could significantly alleviate the increased transcription of col1a1 and col3a1 genes induced by CS?P<0.05?and the expression of collagen1and collagen3.Conclusion:Dioscin increases the removal of damaged mitochondria and reduces activation of mitochondria-dependent apoptotic pathways through promoting autophagy to protect alveolar macrophages from CS-induced apoptosis,thus reduces mitochondrial ROS production and excessive secretion of macrophage inflammatory factor,further alleviates CS-caused lung inflammation and fibrosis.