Regulation Of Ac-SDKP On Apoptosis Of Alveolar Type?Epithelial Cells Mediated By Endoplasmic Reticulum Stress During Development Of Silicosis

Silicosis is a chronic diffuse interstitial fibronodular lung disease caused by the inhalation of dust containing free crystalline silica.Silicosis has become one of the major occupational health and safety problems in China.Studying the mechanism of silicosis and exploring effective therapeutic targets has been a difficult and hot issue in the field of occupational disease research all over the world.The alveolar type II epithelial cells are considered to be the"defensive cells"of the alveoli,and the functional status of alveolar type II epithelial cell is one of the determinants of the pathological changes of lung injury.However,in the process of silicosis fibrosis,the role and mechanism of SiO₂ stimulation on alveolar type II epithelial cells still need further study.N-acetyl-seryl-aspartyl-lysyl-proline?Ac-SDKP?is a kind of endogenous tetrapeptide which plays a physiological role at basal concentrations,preventing organ collagen accumulation.Our previous studies showed that Ac-SDKP could inhibit fibroblast proliferation and myofibroblast differentiation and thus suppress the formation and development of silicosis.This study aims to explore the effect and mechanism of SiO₂ on the apoptosis of alveolar type II epithelial cells,and to further investigate whether Ac-SDKP can inhibit the apoptosis of alveolar type II epithelial cells induced by SiO₂and then play the role of anti-silicosis,reveal the molecular mechanism of its anti-silicosis effects in depth.The study was divided into three parts:Part one The effect and mechanism of SiO₂ on the apoptosis of human alveolar type II epithelial cellsObjective:To observe and analyze the effect and mechanism of SiO₂ on the apoptosis of human alveolar type II epithelial cells by using human alveolar type II epithelial cell line A549 as the research object.Methods:1 The human type II alveolar epithelial cell line A549 was divided into control group and SiO₂?50?g/cm²?stimulation group.2 The ultrastructural changes and apoptosis of A549 cells between the normal control group and the SiO₂ stimulation group were observed using a transmission electron microscope?TEM?.3 Protein samples extracted from the normal control group and SiO₂stimulation group were separated by two-dimensional gel electrophoresis,and then were stained by Coomassie brilliant blue to show the protein spots.The protein spots on the gel were analyzed by ImageMaster 6 image analysis,screening of differentially expressed proteins.Matrix mass spectrometry?Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF-MS?was used to obtain peptide mass fingerprinting and protein analysis.4 The expression of GRP78,phospho-PERK,phospho-eIF2?,CHOP and Caspase-12 in A549 cells was detected by Western blot.Results:1.Observation of ultrastructure by TEM showed that A549 cells in the control group had microvilli on the cell membrane,abundant cytoplasm with no vacuoles,integrated organelle,homogeneous chromatin and distinct nucleolus.However,in Si O₂-treated A549 cells,the endoplasmic reticula were dilated,and typical apoptotic characteristics were observed,including cytoplasmic vacuolization,nuclear shrinkage,and highly condensed chromatin,which had moved around the nuclear membrane.2.Eleven differential protein points were screened by proteomics technology.Compared with the control group,5 protein spots were up-regulated and 6 protein spots were down regulated in SiO₂ stimulation group.The expression of GRP78,a marker protein of endoplasmic reticulum stress,was up-regulated.3.Western blot results showed that GRP78 protein levels were significantly increased in SiO₂-treated cells from 3 h?P<0.01?.CHOP and Caspase-12 protein levels were increased in SiO₂-treated cells at 6 h and the levels continued to increase over the following 48 h?P<0.01?.Part two Effects and mechanism of Ac-SDKP on apoptosis of A549 cells induced by Si O₂ via regulating endoplasmic reticulum stressObjective:Taking human alveolar type II epithelial cell line A549 as the research object.To observe the regulatory effect of Ac-SDKP on SiO₂ induced endoplasmic reticulum stress in human alveolar type II epithelial cell line A549,and to explore the mechanism of Ac-SDKP inhibiting the apoptosis of alveolar type II epithelial cells induced by SiO₂.Methods:1 The human type II alveolar epithelial cell line A549 was divided into four groups:1)controls;2)SiO₂?50?g/cm²?stimulation group;3)Ac-SDKP(10^-8mol/L)treatment group;and 4)4-PBA?1mmol/L?treatment group.2 The effects of Ac-SDKP and 4-PBA on the ultrastructure of A549 cells stimulated by SiO₂ were observed by transmission electron microscopy.3.The effect of Ac-SDKP and 4-PBA on the apoptosis of A549 cells stimulated by SiO₂ was detected by Flow Cytometry Annexin V-FITC/PI staining.4 The effects of Ac-SDKP and 4-PBA on the expression of GRP78,CHOP and Caspase-12 in A549 cells were assessed by Immunocytochemistry.5 The effects of Ac-SDKP and 4-PBA on the expression of GRP78,phospho-PERK,phospho-eIF2?,CHOP and Caspase-12 protein in A549 cells stimulated by SiO₂ were detected by Western blot.Results:1.Observation of ultrastructure by TEM showed that showed that pretreatment of Ac-SDKP and 4-PBA significantly attenuated endoplasmic reticulum dilation and improved cytoplasmic vacuolation,nuclear shrinkage,chromatin condensation and margination.2.The result of flow cytometry Annexin V-FITC/PI staining showed that compared with the control group,the percentage of early apoptosis and proportion of late apoptotic/necrotic cells a was higher in SiO₂-treated cells compared to control cells.Pretreatment with Ac-SDKP and 4-PBA significantly reduced Si O₂ induced A549 cell apoptosis.The percentage of early apoptosis in Ac-SDKP treatment group and 4-PBA treatment group were9.01%and 9.52%,respectively.The proportion of late apoptotic/necrotic cells was 3.09%and 3.75%in Ac-SDKP treatment group and 4-PBA treatment group,respectively.3.Immunocytochemical staining results showed that the cells positive for GRP78,Caspase-12 and CHOP protein significantly increased after SiO₂stimulation of 48h.GRP78 was mainly expressed in the cytoplasm,and CHOP and Caspase-12 protein were expressed in the cytoplasm and nucleus.Furthermore,pretreatment of Ac-SDKP/4-PBA significantly decreased the number of cells positive for GRP78,Caspase-12 and CHOP protein compared with the SiO₂ stimulation group.4.Western blot results showed that expression of GRP78,phospho-PERK,phospho-eIF2?,CHOP and Caspase-12 protein level in Si O₂ stimulation group was significantly higher than that in control group.Ac-SDKP and 4-PBA pretreatment significantly reduced Si O₂ induced GRP78,phospho-PERK,phospho-eIF2?,CHOP and Caspase-12 protein expression.Part three Regulation of Ac-SDKP on apoptosis of alveolar type II epithelial cells mediated by endoplasmic reticulum stress in rats with silicosisObjective:Taking rat model of silicosis as the research object.To observe the role of endoplasmic reticulum stress in type II alveolar epithelial cells during the development of silicotic fibrosis,and to explore the anti-firotic effect of Ac-SDKP on rat exposed to silica via regulating endoplasmic reticulum stress in type II alveolar epithelial cells.Methods:1 The establishment of rat silicosis model:using the HOPE-MED8050exposure control apparatus to construct the rat silicosis model.The animals were randomly divided into the control group and the silicosis 4 weeks,8weeks,12 weeks,16 weeks,24 weeks and 32 weeks,groups.Ten rats in each group.After exposed to dust for the corresponding time periods,animals in silicosis 4 weeks,8 weeks,12 weeks,16 weeks groups were euthanized.Animals in silicosis 24 weeks and 32 weeks were then reared under normal conditions to simulate the development of silicosis in patients after removal of the dust.2 The pathological changes of lung tissue at different time points were observed by H.E staining and Masson trichromatic staining.The expression and location of SP-D,GRP78,CHOP,Caspase-12,phospho-PERK,phospho-eIF2?and type I collagen were detected by immunohistochemistry and Western blot.3 According to the results of the rat silicosis model,the animals were randomly divided into 4 groups at 16 weeks after exposure to dust:1)control for 32w group,2)Silicosis for 32w group,3)Ac-SDKP treatment group,and 4)4-PBA treatment group.4 H.E staining and Van Gieson staining were used to observe the effects of Ac-SDKP and 4-PBA on the pathological changes of lung tissue in rats.Western blot method was used to detect the protein expression of SP-D,GRP78,CHOP,Caspase-12,phospho-PERK,phospho-eIF2?and type I collagen5 The effects of Ac-SDKP and 4-PBA on the ultrastructure of alveolar type II epithelial cells in silicosis rats were observed by transmission electron microscopy.6 The co-expression of SP-D/GRP78 and SP-D/Caspase-12 in lung tissue of rats was detected by immunofluorescence staining.Results:1.The results of H.E and Masson trichromatic staining showed the completely normal parenchymal structures in the control group and no obvious infiltration of inflammatory cells.Macrophages were visible in the lumen of the alveoli 4 weeks after silica inhalation.Multiple cellular nodules,composed of macrophages,were present in the lung tissue of rats after 8weeks of silica inhalation.The silica nodules became larger as the duration of silica exposure increased,and multiple fused nodules were present in the lungs after 16 weeks of silica inhalation.Cell fibrous nodules formed after 24 weeks of silica exposure.The nodule area reached nearly 50%after 32 weeks of silica exposure.2.Immunohistochemical staining showed that SP-D,GRP78,CHOP and Caspase-12 positive alveolar type II epithelial cells in lung tissues were significantly higher than those in control group at 4 and 8 weeks after exposure to dust.After 12 weeks of exposure,SP-D positive cells began to decrease.The number of GRP78 positive cells continued to increase until 24weeks of dust exposure,and the number of GRP78 positive cells was not significantly changed after 24 weeks.The expression of CHOP and Caspase-12 protein increased continuously with the duration of dust exposure.The quantitative results of Western blot protein were in accordance with the results of immunohistochemical staining.In addition,the quantitative results of Western blot protein also showed that the expression of phosphor-PERK and phosphor-eIF2?increased continuously with the prolongation of the silica exposure time.3.H.E and Van Gieson staining results showed that the areas of the silicon nodules in the lung tissue of Ac-SDKP and 4-PBA treatment group were significantly reduced compared with the silicosis model group,and the fibrosis extent in the lung tissue was significantly reduced.4.Observation of ultrastructure by TEM showed that Ac-SDKP and4-PBA significantly reduced the degree of endoplasmic reticulum expansion induced by SiO₂ in alveolar type II epithelial cells,and the features of apoptosis?such as cytoplasmic vacuolation,nuclear condensation,chromatin condensation and margination?were alleviated.5.Western blot results showed that the anti-fibrosis treatment of Ac-SDKP and 4-PBA alleviated the protein expression of SP-D,GRP78,CHOP,Caspase-12,phosphor-PERK,phosphor-eIF2?and type I collagen in lung tissue of rats.Conclusions:1.Endoplasmic reticulum stress is involved in the apoptosis of alveolar type II epithelial cells induced by long-term SiO₂ dust stimulation in vivo and in vitro2.Ac-SDKP effectively suppresses the apoptosis of AECs induced by SiO₂ by attenuating endoplasmic reticulum stress triggered Caspase-12 and PERK/eIF2?/CHOP signaling pathway activation.