ABSTRACT
Background@#Obstructive sleep apnea (OSA) is associated with pulmonary fibrosis. Chronic intermittent hypoxia (CIH) is considered to be a surrogate of OSA. However, its exact role in pulmonary fibrosis remains uncertain. Therefore, we investigated the mechanism underlying CIH-induced pulmonary fibrosis and the role of the anti-fibrotic agent in bleomycin (BLE) induced lung injury. @*Methods@#Mice were divided into eight groups: the normoxia (NOR), CIH, NOR plus BLE, CIH plus BLE, NOR plus pirfenidone (PF), CIH plus PF, NOR plus BLE and PF, and CIH plus BLE and PF groups. BLE was administered intratracheally on day 14 following CIH or NOR exposure. Subsequently, the mice were exposed to CIH or NOR for an additional 4 weeks. PF was administered orally on day 5 after BLE instillation once daily for 3 weeks. @*Results@#In the BLE-treated groups, CIH-induced more collagen deposition in lung tissues than NOR, and significantly increased hydroxyproline and transforming growth factor-β expression. The CIH and BLE-treated groups showed increased lung inflammation compared to NOR or CIH groups. Following CIH with BLE treatment, nuclear factor-κB (NF-κB) protein expression was significantly increased, whereas nuclear factor-erythroid-related factor 2 (Nrf2) and heme oxygenase-1 protein levels were decreased. After PF treatment, NF-κB and Kelch-like ECH-associated protein 1 expression were suppressed, and Nrf2 expression was increased. @*Conclusion@#CIH accelerated lung fibrosis in BLE-induced lung injury in mice, potentially by regulating the NF-κB/Nrf2 signaling pathway. Our results implicate PF as a potential therapeutic agent for treating pulmonary fibrosis in individuals with OSA and idiopathic pulmonary fibrosis.
ABSTRACT
Background@#Obstructive sleep apnea (OSA) is associated with pulmonary fibrosis. Chronic intermittent hypoxia (CIH) is considered to be a surrogate of OSA. However, its exact role in pulmonary fibrosis remains uncertain. Therefore, we investigated the mechanism underlying CIH-induced pulmonary fibrosis and the role of the anti-fibrotic agent in bleomycin (BLE) induced lung injury. @*Methods@#Mice were divided into eight groups: the normoxia (NOR), CIH, NOR plus BLE, CIH plus BLE, NOR plus pirfenidone (PF), CIH plus PF, NOR plus BLE and PF, and CIH plus BLE and PF groups. BLE was administered intratracheally on day 14 following CIH or NOR exposure. Subsequently, the mice were exposed to CIH or NOR for an additional 4 weeks. PF was administered orally on day 5 after BLE instillation once daily for 3 weeks. @*Results@#In the BLE-treated groups, CIH-induced more collagen deposition in lung tissues than NOR, and significantly increased hydroxyproline and transforming growth factor-β expression. The CIH and BLE-treated groups showed increased lung inflammation compared to NOR or CIH groups. Following CIH with BLE treatment, nuclear factor-κB (NF-κB) protein expression was significantly increased, whereas nuclear factor-erythroid-related factor 2 (Nrf2) and heme oxygenase-1 protein levels were decreased. After PF treatment, NF-κB and Kelch-like ECH-associated protein 1 expression were suppressed, and Nrf2 expression was increased. @*Conclusion@#CIH accelerated lung fibrosis in BLE-induced lung injury in mice, potentially by regulating the NF-κB/Nrf2 signaling pathway. Our results implicate PF as a potential therapeutic agent for treating pulmonary fibrosis in individuals with OSA and idiopathic pulmonary fibrosis.
ABSTRACT
PURPOSE: In order to elucidate the mechanisms mediating cyclosporine A (CsA)-induced renal tubular cell injury, we examined the effects of ceramide, second messenger derived from sphingolipid breakdown, and phospholipase A2 (PLA2), enzyme responsible for release of arachidonic acid, on CsA-induced apoptosis of cultured LLC-PK1 renal tubular cell line. METHODS: The apoptosis was evaluated by flow cytometric analysis and DNA gel electrophoresis. The activities of cytosolic (c)- and secretory (s)-PLA2 were measured by ELISA methods and Western blotting of cPLA2 was also investigated. RESULTS: The exposure to CsA (10microgram/mL) significantly increased the percentage of cells displaying annexin-V binding from 5.1+/-2.0% in control to 24.7+/-6.5% (P<0.05), indicating apoptosis. The addition of ceramide (10 micromol/mL) significantly inhibited the increase of apoptosis induced by CsA (24.7+/-6.5% vs. 14.7+/-6.0). The treatment with PLA2 (5 U/mL) also decreased CsA (5microgram/mL)-induced apoptosis (20.4+/-5.7% vs. 13.6+/-5.2, P<0.05). But there was no dose-dependent further protectective effect of ceramide or PLA2. Concerning the changes of PLA2 activities, cPLA2 activity after CsA exposure (10microgram/mL) was increased from 3.92+/-2.01 nmol/min/mL in control to 9.81+/-3.07 (P<0.05), and the addition of ceramide (5 micro mol/mL) significantly inhibited the increase of cPLA2 activity after CsA exposure (4.64+/-1.52). The Western blotting of cPLA2 (110 kD) also showed similar results. Meanwhile there was no significant changes of sPLA2 activitiy which was markedly low. CONCLUSION: The apoptosis of renal tubular epithelial cell following CsA expsoure appears to be mediated by membrane phospholipid breakdown via sphingomyelinase and PLA2. The mechanism(s) mediating the protective effect of ceramide and PLA2 on CsA-induced apoptosis should be further elucidated.
Subject(s)
Animals , Apoptosis , Arachidonic Acid , Blotting, Western , Cell Line , Cyclosporine , Cytosol , DNA , Electrophoresis , Enzyme-Linked Immunosorbent Assay , Epithelial Cells , LLC-PK1 Cells , Membranes , Negotiating , Phospholipases A2 , Phospholipases , Second Messenger Systems , Sphingomyelin Phosphodiesterase , SwineABSTRACT
PURPOSE: One of the major limiting factors in the use of FK506 (Tacrolimus) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of FK506 tubulotoxicity, we examined mechanisms of cellular injury induced by FK506 in cultured LLC-PK1 renal tubular cell line. METHODS: The 3H-thymidine uptake and flow cytometric analysis of apoptosis following FK506 treatment were evlauated. The changes of the Fas and Bcl-2 protein in FK506-induced renal tubular cell injury were also investigated by Western blotting. RESULTS: FK506 treatment significantly decreased 3H-thymidine uptake (M+/-S.D., 3591.6+/-274.3 cpm/well vs 2217.6+/-79.7 at 1 microgram/mL, p<0.05), in a dose dependent manner upto 50microgram/mL, indicating that DNA damage is a sensitive indicator of FK506- induced nephrotoxicity. The addition of FK506 (50 microgram/mL) for 96 hours also induced a significant increase in the percentage of cells displaying annexin-V binding from 3.9+/-2.2% in control cells to 34.9+/-8.5% in treated cells (P<0.05), indicating early apoptotic cellwith an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5microgram/mL. death. This finding was supported by electrophoretic analysis of the DNA extracted from FK506-treatedcells, where a series of bands correspondingto integer multiples of 180 to 200 base pairs was visualized. The treatment with FK506 at a concentration higher than 1 microgram/mL for 96 hours was seen to cause a significant 3.7 (mean) fold elevation in the expression of the 45 kD Fas protein by Western blot analysis. The exposure to FK506 at dose of 5microgram/mL for 96 hours was also associated with an increase of Bcl-2 protein levels up to 6.2 fold (mean) on Western blot analysis, and the dose-dependent further increase of Bcl-2 protein was observed at a dose above 5 microgram/mL. CONCLUSION: FK506 is directly toxic to renal tubular cells with inhibiting DNA synthesis and inducing cell death in the form of apoptosis. The changes of Fas antigen system and Bcl-2 protein may play roles in mediating FK506-induced apoptosis of renal tubular cells.