Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure / 中南大学学报(医学版)

OBJECTIVES@#The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.@*METHODS@#The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting.@*RESULTS@#After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group.@*CONCLUSIONS@#Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Regulatory effect of Ac-SDKP on phosphorylated heat shock protein 27/SNAI1 pathway in silicotic rats / 中华劳动卫生职业病杂志

Objective: To study the effect of anti-fibrotic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on phosphorylated heat shock protein 27 (P-HSP27) and zinc finger family transcriptional repressor 1 (SNAI1) expression to explore the anti-silicosis fibrosis effect of Ac-SDKP. Methods: In December 2014, the rat silicosis animal model was prepared by one-time bronchial infusion of silicon dioxide (SiO(2)) dust. 80 SPF healthy adult Wistar rats were selected, and the rats were divided into 8 groups according to the random number table method, 10 in each group. Model control group for 4 weeks (feeding for 4 weeks) , model control group for 8 weeks (feeding for 8 weeks) : bronchial perfusion with normal saline 1.0 ml per animal. Silicosis model group for 4 weeks (feeding for 4 weeks) and silicosis model group for 8 weeks (feeding for 8 weeks) : bronchial perfusion of 50 mg/ml SiO(2) suspension 1.0 ml per animal. Ac-SDKP administration group for 4 weeks (feeding for 4 weeks) , Ac-SDKP administration group for 8 weeks (feeding for 8 weeks) : Ac-SDKP 800 μg·kg(-1)·d(-1) was administered by intraperitoneal pump. Ac-SDKP preventive treatment group: 48 h after Ac-SDKP 800 μg·kg(-1)·d(-1) administration, bronchial perfusion of SiO(2) suspension 1.0 ml per animal, raised for 8 weeks. Ac-SDKP anti-fibrosis treatment group: after bronchial perfusion of 1.0 ml of SiO(2) suspension for 4 weeks, Ac-SDKP 800 μg·kg(-1)·d(-1) was administered for 4 weeks. Western blotting was used to detect the expression of P-HSP27, SNAI1, α-smooth muscle actin (α-SMA) , and collage typeⅠ and Ⅲ in each group. The expression of P-HSP27 and SNAI1 was detected by immunohistochemistry, and the co-localized expression of P-HSP27 and α-SMA was detected by laser confocal microscopy. Results: Compared with the model control group, the expressions of P-HSP27, SNAI1, α-SMA, and collage typeⅠ and Ⅲ in the silicosis fibrosis area of the rats in the silicosis model group were enhanced, and the differences were statistically significant (P<0.05) . After Ac-SDKP intervention, compared with silicosis model group for 8 weeks, the expressions of P-HSP27, SNAI1 α-SMA, and collage typeⅠ and Ⅲ in the Ac-SDKP preventive and anti-fibrosis treatment groups were significantly decreased, and the differences were statistically significant (P<0.05) . However, the expressions of P-HSP27 SNAI1, and collage typeⅠ and Ⅲ between the Ac-SDKP administration group and the model control group did not change significantly, and the differences were not statistically significant (P>0.05) . Laser confocal results showed that the positive cells expressing P-HSP27 and α-SMA in the lung tissue of the silicosis model group were more than those in the model control group. Compared with the silicosis model group, the Ac-SDKP prevention and anti-fibrosis treatment groups expressing the positive cells of P-HSP27 and α-SMA decreased. Compared with the model control group for 8 weeks, there were some double-positive cells expressing P-HSP27 and α-SMA in the nodules of the silicosis model group for 8 weeks. Conclusion: Ac-SDKP may play an anti-silicic fibrosis effect by regulating the P-HSP27/SNAI1 pathway.

Interventional effect of asiaticosdide on rats exposed to silica dust / 中华劳动卫生职业病杂志

Objective: To investigate the effect of asiaticoside for fibrosis in lung tissues of rats exposed to silica and to explore its possible mechanism. Methods: 144 SD male rats were randomly divided into control group, model group, positive drug control group, asiaticoside high-dose group, medium-dose group and low-dose group, each group included 24 rats. Rats in the control group were perfused with 1.0 ml of normal saline, and the other groups were given 1.0 ml 50 mg/ml SiO(2) suspension. Gavage of herbal was given from the next day after model establishment, once a day. Rats in the positive drug control group were administration with 30 mg/kg tetrandrine and rats in the low-dose group, medium-dose group and high-dose group were given 20 mg/kg, 40 mg/kg and 60 mg/kg asiaticoside for fibrosis respectively. Rats in the control group and the model group were given 0.9% normal saline. The rats were sacrificed in on the 14th, 28th and 56th day after intragastric administration and collect the lung tissues to detect the content of hydroxyproline, TGF-β(1) and IL-18, observe the pathological changes of the lung tissues by HE and Masson staining and determine the expressions of Col-I, a-SMA, TGF-β in lung tissues by Western Blot. Results: On the 14th day, 28th day and 56th day after model establishment, the lung tissues of rats in the model group showed obvious inflammatory response and accumulation of collagen fibers, and the degree of inflammation and fibrosis increased with time. The intervention of asiaticoside could effectively inhibit the pathological changes of lung tissues. The contents of hydroxyproline, IL-18 and TGF-β1 in lung tissues of model group were higher than those in the control group (P<0.05) , while the level of hydroxyproline, IL-18 and TGF-β1 in asiaticoside groups were significantly decreased, and the difference was statistically signicant (P<0.05) . Compared with the control group, the expression levels of Col-I, TGF-β1and α-SMA in lung tissue of model group were increased (P<0.05) , while the expression level of Col-I, TGF-β1 and α-SMA were decreased after the intervention of asiaticoside, and the difference was statistically signicant (P<0.05) . Conclusion: Asiaticoside can inhibit the increase of Col-I, TGF-β1 and α-SMA content in the SiO(2)-induced lung tissues of rats, reduce the release of TGF-β1 and IL-18 inflammatory factors in lung tissue, and then inhibit the synthesis and deposition of extracellular matrix in rat lung tissue, and improve silicosis fibrosis.