Objective To evaluate the role of autophagy in inflammatory response in the lungs of silicosis rats. Methods The specific pathogen-free healthy male Wistar rats were randomly divided into four groups with 10 rats in each group. The rats in the control group were given an equal volume of 0.9% sodium chloride solution; all rats in the silicon dioxide (SiO2 ) group, the SiO2 + 3-methyladenine (3-MA) group, and the SiO2 +rapamycin (RAPA) group were given a mass concentration of 0.05 mg/L of SiO2 suspension (1.0 mL/rat) to establish a rat model of silicosis using non-exposed tracheal instillation method. Two days before SiO2 exposure, the rats in SiO2 +3-MA group were intraperitoneally injected with 3-MA at a dose of 2.5 mg/kg body weight. The rats in the SiO2 +RAPA group were intraperitoneally injected with a dose of RAPA 1.0 mg/kg body weight, once a day, and once every other day after modeling, for a total of 10 injections. The pathological changes in the lung tissue of the rats in each group were evaluated using hematoxylin-eosin and Masson staining. The relative expression levels of transforming growth factor-β (TGF-β) , interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the lung tissue of the rats in each group were detected using enzyme-linked immunosorbent assay. The relative expression levels of microtubule-associated protein 1 light chain 3 (LC3) and yeast autophagy-related gene 6 (Beclin1) in lung tissue of rats in each group were detected by Western blotting. Results The pathological observation of rat lung tissue showed that scattered inflammatory nodules and interstitial inflammatory cells were found in the lung tissue of the rats in the SiO2 group. In the SiO2 +3-MA group, the inflammation in the lung tissue was more severe and the alveolar cavity had viscous secretions. The rats in the SiO2 +RAPA group had less inflammation and smaller inflammatory nodules than the SiO2 group. Compared with the control group, the TNF-α, IL-1β, TGF-β, Beclin1 protein relative expression levels and LC3 Ⅱ/Ⅰ ratio in the lung tissue were increased in the SiO2 group (all P<0.05). Compared with the SiO2 group, the relative expression levels of TGF-β and TNF-α in the lung tissue of the rats increased (all P<0.05), and the ratio of LC3 Ⅱ/Ⅰ and Beclin1 protein relative expression decreased in the SiO2+3-MA group (all P<0.05). Compared with the SiO2+ 3-MA group, the relative expression levels of TNF- α, IL-1β and TGF- β in the lung tissue of the SiO2 +RAPA group were decreased (all P<0.05), and the LC3Ⅱ/Ⅰ ratio and the relative expression level of Beclin1 protein were increased (all P<0.05). Conclusion Autophagy occurs when inflammatory reaction occurs in the lungs of silicosis model rats; autophagy has inhibitory effect on pulmonary inflammation.

Objective To investigate bone mineral density-related factors to decrease the prevalence of osteoporosis. Methods This croas-sectional survey enrolled 46 219 adults less than 60 years old. Anthropometry, blood pressure, serum lipid, glucose, electrolytes, uric acid and homocysteine were detected. Bone mineral density (BMD) of distal forearm was measured by using peripheral dual energy-ray detector (MestriscanTM). Our data were analyzed by Pearson's correlation analysis and stepwise multiple regression analysis. Results All BMD-related factors except age showed significant difference between male and female. BMD of female subjects was lower than that of male participants (0.50±0. 15 vs 0. 54 ±0. 15,t = 22. 38 ,P < 0. 05 ). The prevalence of osteoporosis in female was higher ( 29. 51% vs 26. 48%, χ2 =47.90,P <0. 05). BMD increased with age before 40-year old, and then decreased more rapidly in female after 50-year old. Conclusion BMD of male is higher. Cigarette smoking, waist-hip ratio, systolic blood pressure, serum sodium, total cholesterol, and ALP were negatively correlated with BMD.

OBJECTIVE: To investigate the effect of toluene diisocyanate(TDI) on the activation of autophagy and expression of inflammatory cytokines interleukin(IL)-4 and IL-6 in normal human bronchial epithelial cells(16 HBE). METHODS: i) We prepared TDI-human serum albumin(HSA) and determined the mass concentration of TDI in TDI-HSA. ii) The cells were treated with TDI-HSA and HSA at concentrations of 0.00-400.00 mg/L for 12 hours. CCK-8 assay was used to determinate the cell viability, and TDI-HSA and HSA doses were selected for subsequent experiments. iii) The cells were treated with TDI-HSA and HSA at doses of 0.00-120.00 mg/L for 12 hours, and the levels of reactive oxygen species(ROS) in the cells were detected by flow cytometry. The levels of IL-4 and IL-6 in the cell supernatant were measured by enzyme-linked immunosorbent assay. iv) The cells were treated with TDI-HSA at doses of 0.00-120.00 mg/L for 12 hours, and the autophagy activity was observed under transmission electron microscope. Western blot was utilized to detect the expression of Beclin1, microtubule-associated protein 1 light chain(LC3β) and P62. RESULTS: i) The mass concentrations of TDI in 40.00, 80.00 and 120.00 mg/L TDI-HSA groups were 0.44, 0.89 and 1.33 mg/L respectively. ii) The results of CCK-8 showed that TDI-HSA and HSA at doses below 120.00 mg/L did not affect cell viability, and 0.00-120.00 mg/L was selected as the TDI-HSA and HSA treatment doses for subsequent experiments. iii) The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells in the TDI-HSA group at 40.00, 80.00, and 120.00 mg/L were higher than that in HSA group at the same dose(P<0.01). The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells increased with the increase of TDI-HSA doses(P<0.01). iv) Transmission electron microscopy showed that the number of autophagic lysosomes in 16 HBE cells increased significantly, and the number of mitochondrial vacuoles increased in 40.00, 80.00, 120.00 mg/L TDI-HSA group compared with 0.00 mg/L group. With the increase of TDI-HSA dose, the relative expression of Beclin1 protein and LC3β-Ⅱ/Ⅰ ratio in 16 HBE cell supernatant increased(P<0.05), and the relative expression of P62 protein decreased(P<0.05). CONCLUSION: TDI-HSA induces increased expression of ROS and inflammatory factors and induces autophagy activation in 16 HBE cells. Autophagy may be an important factor for the development of airway inflammation in TDI-induced occupational asthma.

Objective: To screen the changes of microRNA (miRNA) expression profiles in lung tissues of early silicosis rats, and provide a basis for functional analysis of differential microRNA. Methods: SPF Wistar male rats were randomly divided into a negative control group and SiO₂-exposed groups, with 30 rats in each group. The model of silicosis in rats was established by intratracheal instillation of 1 ml SiO₂ suspension, and the control rats were treated with 1mL in the same way to sterilize normal saline. The lung tissues of two group were collected at the 1, 7, 14, 21, 28 d after SiO₂-exposed. Three of the rat lung tissues were used for pathological observation, and the other three were used to screen differentially expressed miRNAs in lung tissue by miRNA microarray technology. miRNA chip screening and RT-qPCR were used to verify the expression levels of miRNA-423-5p and miRNA-26a-5p in the two groups. miRNA-423-5p and miRNA-26a-5p are predicted by target genes and analyzed by GO (gene ontology) enrichment analysis and KEGG (kyoto encyclopedia of genes and genomes) pathway analysis. Results: In the control group, the inflammatory response of lung tissue 21 and 28 days was significantly reduced compared with 1, 7 and 14 days, and the inflammatory cells infiltrated in the lung tissue of the SiO₂-exposed rats. The rats in the control group had a small amount of collagen at 21 and 28 days, but a large amount of collagen fiber deposition began to appear in the lung tissue of rats exposed to SiO₂ after 21 days. Compared with the control group, the expression levels of micro RNA-423-5p was significantly up-regulated and the expression of microRNA-26a-5p was significantly down-regulated in the SiO₂-exposed rats lung tissues dust at different time points (P<0.05) . Conclusion The up-regulation of miRNA-423-5p and the down-regulation of miRNA-26a-5p in lung tissues of early silicotic rats may be related to the occurrence and development of early silicosis.

Objective: To investigate the role of microRNA-29b-3p (miRNA-29b-3p) and miRNA-34c-3p in the process of pulmonary fibrosis, we detected the expression levels of miRNA-29b-3p and miRNA-34c-3p in the lung tissue of rats exposed to silica and A549 cells. Methods: SPF male Wistar rats were randomly divided into 1, 7, 14, 21, 28 d control group and silica (SiO₂) dusting group, with 6 rats in each group. One-time non-exposure method was used to infuse 1ml SiO₂ suspension. The rat SiO₂ dusting group was established in the liquid, and the control rats were intratracheally injected with 1 ml of sterile physiological saline in the same manner. The lung tissues of each group were collected at the corresponding time points after dusting. Three of the rats were taken out for pathological observation, and the other three were used to screen differentially expressed miRNAs in lung tissue by miRNA microarray technology. A549 cells were cultured at the in vitro cell level and divided into control group, SiO₂ stimulation group and TGF-β₁ stimulation group, and cells were collected at 12, 24 and 48 h after treatment. The expression levels of miRNA-29b-3p and miRNA-34c-3p in rat lung tissue and A549 cells were verified by real-time PCR (qRT-PCR), target gene prediction of miRNA-29b-3p and miRNA-34c-3p and perform GO enrichment analysis and KEGG pathway analysis. Results: The weight growth rate of the control group was significantly higher than that of the SiO₂ dusting group. Compared with the control group, the lung mass and lung coefficient of the SiO₂ dusting group were significantly increased (P<0.05). The inflammatory response of the lungs in the control group was significantly reduced at 21 and 28 days, and the inflammatory cells infiltrated in the lung tissue of the SiO2 group. The rats in the control group had a small amount of collagen at 21 and 28 days. A large amount of collagen fiber deposition began to appear in the lung tissue of rats exposed to SiO₂ for 21 days. Compared with the control group, the expression levels of miRNA-29b-3p and miRNA-34c-3p in the SiO₂ dusting group were significantly down-regulated, and there was significant difference compared with the control group (P<0.05). The expression levels of miRNA-29b-3p and miRNA-34c-3p in A549 cells treated with SiO₂ and human recombinant TGF-β1 were significantly lower than those in the control group at 24 h and 48 h, and the difference was statistically significant (P<0.05). Conclusion Down-regulation of miRNA-29b-3p and miR-34c-3p in rat lung tissue A549 cells may be associated with the development of early silicosis and is expected to be an indicator of early silicosis diagnosis and prognosis.

OBJECTIVE: To investigate the role of high mobility group protein 1(HMGB1) in toluene diisocyanate(TDI) induced nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3(NLRP3) inflammasome activation in human bronchial epithelial cells(HBECs). METHODS: i) The TDI-human serum albumin(HSA) stimulation experiment: the HBECs in logarithmic growth phase were randomly divided into control group, low-, medium-and high-dose groups that were pretreated with TDI-HSA with the final concentration of 0.00, 40.00, 80.00 and 120.00 mg/L for 12 hours. ii) The HMGB1 expression inhibition experiment: the HBECs in logarithmic growth phase were divided into control group, TDI-HSA group, TDI-HAS+negative-siRNA group, and TDI-HAS+HMGB1-siRNA group. The cells in TDI-HAS+negative-siRNA group and TDI-HAS+HMGB1-siRNA group were infected with HBECs with negative-siRNA lentivirus and HMGB1-siRNA lentivirus, respectively. Cells in these two groups and the TDI-HSA group were treated with 120.00 mg/L of TDI-HSA for 12 hours. The cells in the control group were not treated with TDI-HAS. iii) The expression of HMGB1, NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), pro-caspase-1 and caspase-1 p20 proteins in all groups were detected by Western blot. The number of NLRP3 and caspase-1 inflammasome in TDI-HSA stimulation experiment was observed by immunofluorescence method. RESULTS: i) TDI-HSA stimulation experiment: the relative protein expression of HMGB1 and ASC was higher in HBECs of medium-and high-dose groups than that of control group(all P values were <0.01). The relative protein expression of NLRP3 and casepase-1 p20 and the number of NLRP3-caspase-1 inflammasome were higher in HBECs of 3 dose groups than that of control group(all P values were <0.01). The number of NLRP3-caspase-1 inflammasome in HBECs increased obviously in low-, medium-and high-dose groups as compared to the control group(all P values were <0.05). The number of NLRP3-caspase-1 inflammasome in HBECs increased with the increase of TDI-HSA dose(all P values were <0.01). ii) The HMGB1 expression inhibition experiment: the relative protein expression of HMGB1, NLRP3, ASC, pro caspase-1 and caspase-1 p20 in HBECs were higher in the TDI-HSA group and TDI-HSA + negative-siRNA group than those of the control group(all P values were <0.01). The above indexes of HBECs were lower in the TDI-HAS + HMGB1-siRNA group than those in the TDI-HSA group and TDI-HSA + negative-siRNA group(all P values were <0.01).CONCLUSION: TDI treatment in HBECS can induce the increase of HMGB1 protein expression and activate NLPR3 inflammasome. Inhibition of HMGB1 expression can down-regulate the expression of NLPR3 and its related proteins.

Objective: To observe the repairing effect of adipose mesenchymal stem cells (ADSCs) on lung injury induced by silica in rats. Methods: Primary ADSCs-GFP was obtained from rats. ADSCs-GFP was injected into tail vein of silicosis model rats. The expression of green fluorescence in lungs was observed regularly to determine the homing ability of ADSCs. Primary ADSCs of rats were obtained and randomly divided into control group, exposure group, vehicle group and ADSCs group. Silicosis rat model was established by non-exposed tracheal drip method. 24 hours after silica exposure, rats in ADSCs group were injected with ADSCs of 1×10⁶/kg body weight through tail vein, and the pathological changes of lung tissue were observed and evaluated 28 days after intervention. To explore the early intervention mechanism of ADSCs on pulmonary fibrosis in silicosis model rats, apoptosis-related proteins were detected by immunohistochemistry. Results: 28 days after exposure to silica, rats in the exposure group showed obvious pulmonary fibrosis. Compared with exposure group and vehicle group, ADSCs group showed less pulmonary inflammation, less silica nodules and less collagen deposition area. Immunohistochemical results showed that the expression of Caspase-3 and cytochrome C protein decreased and Bcl-2 protein increased after ADSCs transplantation. Conclusion ADSCs infusion has an obvious intervention effect on postponing early silicosis fibrosis in rats exposed to silica, and its mechanism is related to the regulation of apoptotic process.