Study On Apoptosis And The Related Mechanism Induced By Microcystins-LR In The Human Bronchial Epithelial Cells

ObjectiveMicrocystins (MCs) are a group of cyclic hepatotoxic peptides produced bycyanobacteria, and represent a serious threat to aquatic organisms, wild life andhumans. MCs are extremely stable environmental pollutants, and are not easy to beeffectively removed by the conventional measures of water treatment. Toxins releasedinto the water from the broken algal cells can be taken through skin contact,inhalation, haemodialysis and ingestion (the oral route). There were reports about thatthe cyanobacterial toxins may enter the body through inhaling spindrift produced inthe process of water entertainment, which can cause the respiratory disease.A stable research model of Human Bronchial Epithelial Cells (16HBE) treatedwith Microcystins-LR (MC-LR) was established in this experiment. The effects ofMC-LR on apoptosis, expression of apoptosis-related proteins andmitochondrial-related molecular were observed in order to study on apoptosis and therelated mechanism induced by microcystins-LR in16HBE cells.Methods1. Cell viability assay: Cell viability was assessed by MTT staining as describedpreviously. The EC50value was defined as the concentration of drug that inhibits50%cell growth compared with the control.2. Reactive oxygen and Mitochondrial Membrane Potential assay: The indexeswere detected with DCFH-DA and JC-1respectively.3. Apoptosis assay: The apoptosis rates were measured by Annexin V-FITC andPI combined with flow cytometry.4. Cell viability assay by pretreatment with Z-VAD-FMK: Cell viability wasassessed by MTT and the concentration of Z-VAD-FMK was chosed in thesubsequent experiment.5. The expression of Caspase-3, Caspase-9, Cyt-c, Bcl-2and Bax were detected with western blot.Results1. The cells viability decreased with the increase of MC-LR concentration. Thecells viability change had statistically significant (P<0.05) in1,10,20,30and40μg/ml MC-LR group compared with the control group (0μg/ml).2. When the treatment time was constant, the amount of ROS of each groupincreased with the increase of MC-LR concentration, compared with the controlgroup(0μg/ml MC-LR). When the MC-LR concentration was5μg/ml or10μg/ml, thefluorescence intensity values were increased with the increase of treatment time andhad significant difference (P <0.05).3. When the16HBE cells were exposed to10μg/ml MC-LR for24h or48h, thecollapse of MMP were caused in16HBE cells and was found significant difference (P<0.05).4.16HBE cells were treated with MC-LR (2.5,5,10μg/ml) for24h, cellapoptosis rates were6.03%,17.90%,26.10%respectively. After exposed48h, cellapoptosis rate were19.40%,23.80%,32.23%respectively.5. The cells viability decreased after treated with120μM and140μMZ-VAD-FMK and the viability change had significant difference (P<0.05) comparedwith the control group (0μM). When the MC-LR concentration was5μg/ml and10μg/ml, respectively, the viability was decreased by pretreatment with10μMZ-VAD-FMK compared with no pretreatment group (0μM Z-VAD-FMK).6. Western blot results showed that after exposed to MC-LR for24h, the relativeexpression of Caspase3, Caspase-9, Cyt-c, Bax in16HBE cells increased and theexpression of Bcl-2decreased compared with the control group, and the differencewas statistically significant (P <0.05). After exposed to MC-LR for48h, the trend ofthe relative expression of the target proteins was consistent to the relative expressionof treated for24h. In addition, when16HBE cells were exposed to the the sameconcentration of MC-LR, the relative expressions of Caspase-3, Caspase-9, Cyt-c,Bax increased with the increase of the exposure time, the expression of Bcl-2decreased with the increase of the exposure time, and had time-dose effect.7. The apoptosis rate was inhibited in the pretreatment with Z-VAD-FMK and was found significant difference (P <0.05) compared with non-Z-VAD-FMK group.the relative expressions of Caspase-3and Caspase-9were inhibited in thepretreatment with Z-VAD-FMK and was found significant difference (P<0.05)compared with non-Z-VAD-FMK group.Conclusion1. MC-LR induced the increase of the rate of apoptosis in the16HBE cells.2. MC-LR induced apoptosis through mitochondrial Caspase-dependent pathwayin the16HBE cells.