Cigarette smoking extract induce cell death in BEAS-2B bronchial epithelia cells
To evaluate the effect of cigarette smoking extract on BEAS-2B bronchial epithelia cells. BEAS-2B cells were exposure to various dosage of CSE. As shown in Fig. 1. CSE treatment significantly reduce cell viability in 5% CSE and 10% at 24 hours treatment. And the IC50 were approximately 5% CSE. The dose lower than 2.5% CSE show slight toxicity, while the dose grater that 2.5%. These results show that CSE treatment remarkably increase bronchial cell injury in dose greater than 5% CSE.
Tiotropium/olodaterol reverse CSE-induced cell death in BEAS-2B bronchial epithelia cells
To evaluate the effect of tiotropium/olodaterol on CSE-induced cell death. We pre-treat BD for 4 hours followed by 5% CSE treatment for 24 hours and the cell viability were determined by MTT assay. As shown in Fig. 2, pre-treatment with BD were significantly increase in cell viability compared with 5% CSE treatment alone. This result show that pre-treatment with BD have protective effect against cell injury caused by CSE treatment.
Tiotropium/olodaterol reverse CSE-induced mitochondria membrane potential disruption in BEAS-2B bronchial epithelia cells
It is established that changes in mitochondrial membrane potential (Δψm) are integral to the cell life/death transition. To evaluated whether CSE affected the mitochondrial membrane potential (Δψm) and the effect of Tiotropium/olodaterol on CSE-mediated MMP change. As shown in Fig. 3, treated cells were stained with the fluorescent cationic dye JC-1. Loss of Δψm is an indicator of mitochondrial damage during cell death. After cells were treated with 5% CSE for 24 h, red fluorescence was detected in BEAS-2B cells, suggesting a reduction in Δψm caused by 5% CSE. Furthermore, pre-treatment with Tiotropium/olodaterol decrease the red fluorescence expression. BEAS-2B cells were pretreated 4 h with Tiotropium/Olodactoro and then treated with 5% CSE for 24 h showed significantly retard mitochondrial dysfunction.
Tiotropium/olodaterol inhibit CSE-induced autophagy in BEAS-2B bronchial epithelia cells
Previous studies indicated that autophagy is an important pathway of programmed cell death as well as COPD pathogenesis [4, 5]. We evaluated whether 5% CSE induce autophagy and the effect of pretreatment with Tiotropium/olodaterol. We firstly evaluated autophagy by acridine orange staining (AO) in FACS analysis. For autophagolysosomes, the protonated form of AO accumulates and aggregates, which are characterized by yellow-orange fluorescence (FL3). Staining of normal cells with AO, a weak base, which is characterized by green fluorescence (FL1). As shown in Fig. 4A, CSE treatment resulted in the appearance of yellow-orange (FL-3) fluorescence after 24 h. Another autophagy marker, named the conversion of LC3-I (18 kD) to LC3-II (16 kD), were analysed by western blot.. As shown in Fig. 4B, CSE treatment for 24 h, the ratio of LC-II/LC3-I as well as LC3-II level were increased in BEAS-2B bronchial epithelial cells. Pre-treatment with Tiotropium/olodaterol for 4 h, AVO and the conversion of LC3-I to LC3-II slightly decrease in compared with 5% CSE treatment only.
Notably, CSE treatment resulted in the appearance of yellow-orange (FL-3) fluorescence were also found in 6 h (Fig. 5A). Furthermore, CSE treatment for 6 h, the ratio of LC-II/LC3-I as well as LC3-II level were increased in BEAS-2B bronchial epithelial cells (Fig. 5B). Pre-treatment with Tiotropium/olodaterol for 4 h, AVO and the conversion of LC3-I to LC3-II significantly decrease in compared with 5% CSE treatment only. These finding suggest that Tiotropium/olodaterol have inhibitory effect on autophagy in short-term of CSE treatment.
Tiotropium/olodaterol induce ERK and JNK activation in BEAS-2B bronchial epithelia cells
ERK and JNK activation were involved in cell survival and cell death by regulating autophagy [21, 22]. To further understand the molecular mechanisms of action of Tiotropium/olodaterol on the underlying mechanism. We examined the effect of Tiotropium/olodaterol on ERK and JNK. As shown in Fig. 6A and 6B, pre-treatment with Tiotropium/olodaterol significantly increase ERK activation as well as JNK, following by CSE treatment for 3 h and 6r, respectively. These findings suggest that Tiotropium/olodaterol inhibit autophagy via inducing ERK and JNK activation.