| Objective1: To examine whether cigarette smoke extract (CSE) induced human airway smooth muscle cells (HASMCs) death occurs through apoptosis or not;2: Observe the changes in the expression of Bcl-2 family genes (Bcl-2, Bax and Bad) in human airway smooth muscle cells to know if mitochondrial pathway is involved in CSE-induced apoptosis;3:Observe the changes in the expression of TNFR super family genes (Fas and FasL) in human airway smooth muscle cells to know if death receptor pathway is involved in CSE-induced apoptosis;4: To measure the level of reactive oxygen species (ROS) in human airway smooth muscle cells under the incubation of CSE;5: To test if CSE-induced airway smooth muscle cell apoptosis was mediated by ROS, which could regulate the NF-κB expression.Methods and MaterialsHASMCs were isolated and cultured, which were divided into four does groups (2.5%CSE, 5%CSE, 10%CSE and 20%CSE) and three time groups (24hours, 48hours and 72hours), respectively.We analysis of cell viability and proliferation by MTT assay and Immunocytochemical staining (PCNA). The apoptosis was detected by Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry.The level of ROS was determined by NBT assay and fluorescent probe of DCFH-DA.The expression of Bcl-2, Bax, Bad, Fas, FasL and NF-κB mRNA was tested by Realtime-PCR. The Bax and Bad protein was detected using immunohistochemical SABC method.Results1. In the presence of 2.5% CSE, cell viability was increased to 103.3% at 24h. After 24-hour or longer incubation, CSE at the concentration of 5% or more, cell viability kept on reducing. Viability of cells exposed to CSE remained decline at concentration of 5%, 10% and 20% to 92%, 89.4% and 77.8%, respectively, and viability of cells in 2.5% CSE at 48h and 72h were also decreased to 93.1% and 91.2%. HASMCs in control group presented a (84.3±7.2)% positive staining with PCNA. After incubated with 2.5%, 5%, 10% and 20% CSE for 48h, PCNA immunoreactivity was significantly decreased in HASMCs.2. HASMCs interfered with 2.5% CSE for 24h have little changes compared with control. After the HASMCs were incubated with 5 % CSE for 24h, the early apoptotic cells could be observed, and after the cells being treated with 10 % CSE for 24h, some late apoptotic cells could be observed. The apoptotic bodies could be seen clearly. When the concentration of CSE was increased to 20%, both the apoptotic cells and the necrosis cells increased seriously. On the contrary, the untreated cells did not show these apoptotic characteristics. Despite the necrosis cells raised with the increased does of CSE, most of the death cells are apoptotic cells, and with increased doses of CSE or increased duration of CSE, the number of HASMCs exhibiting AO staining (normal cells or early apoptotic cells) decreased and exhibiting EB staining (late apoptotic cells) increased. HASMCs did not respond to serum restitution if they were exposed to CSE 2.5% and 5% solution for 24h. Except for this, CSE treatment of cells resulted in accumulation of the cells in the G1 phase of the cell cycle in time- and dose-dependent manner.3. Real-time PCR analysis showed that CSE consistently decreased the level of Bcl-2 expression and increased the level of Bax expression compared to the control. The expression of Bad mRNA was top at the 48h. Then it went to lower, but the expression level at 72h still more than 24h group. The level of Fas mRNA expression in CSE-induced HASMCs was significantly increased to 2.27±0.13, 4.62±0.22, 5.35±0.31 and 6.14±0.28 after treated with 2.5%, 5%, 10% and 20% CSE for 48h compared with control group. FasL was expressed at much lower levels compared with the expression of Fas, and there was no significant change in FasL mRNA expression upon different concentration CSE treatment at 24h, 48h and 72h.4. The protein level of Bcl-2 and Bad was decreased and increased according to the concentration and incubation time of CSE, respectively.5. HASMCs exposed to CSE increased yields of nitroblue formazan in accordance with CSE doses. When HASMCs were exposed to 2.5% CSE, nitroblue formazan of 0.0171±0.009 was detected. The addition of CSE concentration to 20% increased production of nitroblue formazan to 0.228±0.018. Compared with control, intracellular ROS levels were dramatically increased in CSE-treated groups, and it was concentration-dependent manner in average fluorescence values of DCFH-DA in the cells. Mild exposure to cigarette smoke (2.5%CSE for 24h) induced NF-κB activation, and except for that expression of NF-κB keep in decrease in dose- and time-dependent manner, which is according to CSE reduced cell viability by MTT assay. Both p65 and p50 subunit were obey the above regularity.ConclusionAt low CSE concentrations within 2.5%CSE for 24h, HASMCs proliferation is induced, and at high does or longer than 24h, CSE exposure resulted in a time- and does-dependent loss of cell viability in HASMCs. According to the results of flow cytometric analysis, it is documented that cell cycle stage of HASMCs induced by CSE was arrested in G1 phase. At high concentration or longer incubation time, cell death was induced, both apoptosis and necrosis occurred, but the most of the death cells were apoptotic cells. Bax, Bad up-regulation and Bcl-2 down-regulation are involved in the modulation of HASMCs apoptosis through the disruption of the proapoptotic/antiapoptotic balance in mitochondrial apoptosis pathway. The expression of Fas, but not FasL, mRNA increases with the treatment of CSE. Our study demonstrates that oxidative stress as represented by the rapid and accelerating induction of intracellular and extracellular ROS increased documenting along with the gradient increase of CSE, and also showes that although both lower and higher concentration of CSE increased generation of ROS, lower concentration of CSE up-modulated NF-κB activity but higher one down-modulated the expression level of NF-κB, and the trend of NF-κB expression was according to the proliferation and apoptosis of the cells.These suggest: 1. CSE disturbance the balance between apoptosis and proliferation in HASMCs contributes to the pathogenesis of COPD. 2. Both mitochondrial and death receptor pathways of apoptosis were activated through CSE-mediated transcription in HASMCs, and it seems that the mitochondrial pathway had more effection in the way of apoptosis. 3. ROS have diphasic action on affect the expression of NF-κB on cigarette smoke induced proliferation/apoptosis unbalance of HASMCs. Accumulation of ROS by oxidant stress can affect gene transcription through its effects on redox-sensitive transcription factor NF-κB, which maybe the major trigger of apoptosis, in the process of CSE treatment. Therefore, ROS and NF-κB may play an important role in the pathogenesis of COPD.
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