| BackgroundThe most common inflammatory disease of the airways is asthma. The incidence of asthma is increasing throughout the world, imposing enormous burdens on health care. The prevalence of asthma in developed countries is approximately10%in adults and even higher in children, whereas in developing countries, the prevalence is lower but increasing rapidly.Asthma is a disease of airway dysfunction that inflammatory response played an important role in the pathogenesis of this disease. In addition to biochemical stimuli, physical forces also play a critical role in regulating the structure, function, and metabolism of the lung. In asthma patients, the lung suffered more stretch than that during normal respiration, and the hyperstretch aggravated the airway obstruction in asthma by inducing myogenic and pulmonary inflammatory responses. Hyperstretch can induce the inflammatory responses in asthma, but the mechanism remains unclear. The bronchial epithelial cells (BEpiCs) are the first point of contact for aeroallergens and have recently become an interesting target for many studies to investigate their involvement in the modulation of inflammatory responses. BEpiCs are not only considered to be a physical barrier, but also to actively contribute to airway inflammation. They can synthesize inflammatory cytokines, which may affect the recruitment and activation of immune cells to the local tissue and also create a microenvironment where these activated immune cells can take part in the inflammatory processes.Poly(ADP-ribose) polymerase-1(PARP-1), an abundant nuclear enzyme present in eukaryotes, accounts for nearly90%of total cellular PARP activity. Poly(ADP-ribose) polymerase-1(PARP-1) is a nuclear enzyme that can regulate a variety of inflammatory cytokines expression. In the nucleus, PARP-1can be activated by DNA breaks and participates in a variety of cellular processes by catalyzing the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP-ribose to form long branches of ADP-ribose polymers on target proteins, including transcription factors and itself. However, excessive activation of PARP-1results in the intracellular depletion of NAD+and ATP, thereby causing a cellular energy crisis and cell death. PARP-1activation has been associated with the pathogenesis of numerous diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. Moreover, PARP-1has been suggested to regulate the expression of a variety of key inflammatory genes, such as vascular cell adhesion molecule-1(VCAM-1), which is regulated by nuclear factor-KB (NF-κB). PARP-1has been proved to be closely associated with the pathogenesis of asthma. It has also demonstrated that PARP-1is activated by allergen challenge in an animal model of asthma, and PARP-1inhibition prevents infiltration of inflammatory cells into the lungs after allergen exposure. PARP-1deficiency inhibited IL-5expression through calpain-dependent degradation of STAT-6in allergen induced asthma. However, the effect of mechanical stretch on PARP-1during asthma is still unclear.NF-κB is an important transcription factor for the amplification loop of inflammatory reactions; its activation and nucleus translocation can combine with the fixed sequence of many gene promoters to start the transcription of plural inflammatory mediators which plays a very important role in the development of an inflammatory disease. Increasing evidence showed that NF-κB played an essential role in the expression of inflammatory mediators in asthma. Previous study has found that PARP-1inhibition by inhibitor3-AB can inhibit NF-κB activation and IL-8expression induced by H2O2.ObjectiveIn the present study, we aimed to investigate the role and mechanism of PARP-1in mechanical stretch-induced inflammation in human bronchial epithelial cells (HBEpiCs).MethodsHBEpiCs were simulated by mechanical stretch in our experiment. Cells under static were used as the control. PARP-1expression was interfered by small interfering RNA (siRNA).The expression of inflammatory cytokine related gene and protein of stretch-preconditioned and static cells were detected separately by quantitative reverse transcription polymerase chain reaction (RT-PCR), western bloting, enzyme linked immunosorbent assay (ELISA), small interference RNA (siRNA) and immunofluorescence. Oxidative stress was evaluated by DHE staining. DNA damage was assessed by comet assay.Results1. IL-8and vascular cell adhesion molecule-1(VCAM-1) expression were regulated by hyperstretch in a time-dependent manner.HBEpiCs were stimulated by hyperstretch for various times. Hyperstretch induced a time-dependent regulation of IL-8and VCAM-1expression. Compared with cells under static condition,24-h hyperstretch could significantly increase the mRNA and protein expression of VCAM-1, and the ELISA also showed that IL-8secretion was upregulated by hyperstretch stimulation (P<0.05).2. Hyperstretch could increase PARP-1expression and activity by inducing superoxide production and DNA damage.Compared with the static cells, PARP-1expression was significantly increased by hyperstretch, and PARP-1activity was also upregulated (P<0.05).There was almost no superoxide production in cells under static, while hyperstretch stimulation could significantly induce oxidative stress (P<0.05).In the cells under static, there was almost no DNA in the tail, while hyperstretch could increase the DNA content in the cellular tail, which suggested that hyperstretch could induce DNA damage in HBEpiCs.3. Silencing of PARP-1attenuated hyperstretch-induced IL-8and VCAM-1upregulation as well as monocytes adhesion, which were related to the inhibition of nuclear factor-kappa B (NF-κB) translocation.Hyperstretch stimulation markedly increased IL-8secretion and VCAM-1expression as compared with static cells. However, after PARP-1silencing, IL-8secretion and VCAM-1expression were significantly reduced (P<0.05). The negative control of PARP-1siRNA had no significant effect on the expression of inflammatory cytokines.Compared with cells under static condition, stimulation of HBEpiCs with hyperstretch resulted in a significant increase in THP-1cell adherence; while pretreatment of HBEpiCs with PARP-1siRNA before hyperstretch stimulation resulted in a significant decrease in monocyte adhesion.NF-κB p65was mostly cytoplasmic before hyperstretch stimulation, but its localization quickly changed to the nucleus with stimulation. After PARP-1silencing, p65remained primarily cytoplasmic after hyperstretch treatment.Conclusion1. Hyperstretch could induce inflammatory response and superoxide production as well as DNA damage in HBEpiCs.2. PARP-1silencing decreased IL-8and VCAM-1expression, partly through inhibition of NF-κB translocation.3. PARP-1played a fundamental role in hyperstretch-induced inflammation.4. PARP-1silencing could be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma. BackgroundThe most common inflammatory disease of the airways is asthma. The incidence of asthma is increasing throughout the world, imposing enormous burdens on health care. The prevalence of asthma in developed countries is approximately10%in adults and even higher in children, whereas in developing countries, the prevalence is lower but increasing rapidly.Asthma is a disease of airway dysfunction that inflammatory response played an important role in the pathogenesis of this disease. In addition to biochemical stimuli, physical forces also play a critical role in regulating the structure, function, and metabolism of the lung. In asthma patients, the lung suffered more stretch than that during normal respiration, and the hyperstretch aggravated the airway obstruction in asthma by inducing myogenic and pulmonary inflammatory responses. Hyperstretch can induce the inflammatory responses in asthma, but the mechanism remains unclear. The bronchial epithelial cells (BEpiCs) are the first point of contact for aeroallergens and have recently become an interesting target for many studies to investigate their involvement in the modulation of inflammatory responses. BEpiCs are not only considered to be a physical barrier, but also to actively contribute to airway inflammation. They can synthesize inflammatory cytokines, which may affect the recruitment and activation of immune cells to the local tissue and also create a microenvironment where these activated immune cells can take part in the inflammatory processes.Poly(ADP-ribose) polymerase-1(PARP-1), an abundant nuclear enzyme present in eukaryotes, accounts for nearly90%of total cellular PARP activity. Poly(ADP-ribose) polymerase-1(PARP-1) is a nuclear enzyme that can regulate a variety of inflammatory cytokines expression. In the nucleus, PARP-1can be activated by DNA breaks and participates in a variety of cellular processes by catalyzing the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP-ribose to form long branches of ADP-ribose polymers on target proteins, including transcription factors and itself. However, excessive activation of PARP-1results in the intracellular depletion of NAD+and ATP, thereby causing a cellular energy crisis and cell death. PARP-1activation has been associated with the pathogenesis of numerous diseases, including cardiovascular diseases, diabetes, rheumatoid arthritis, endotoxic shock, and stroke. Moreover, PARP-1has been suggested to regulate the expression of a variety of key inflammatory genes, such as vascular cell adhesion molecule-1(VCAM-1), which is regulated by nuclear factor-KB (NF-κB). PARP-1has been proved to be closely associated with the pathogenesis of asthma. It has also demonstrated that PARP-1is activated by allergen challenge in an animal model of asthma, and PARP-1inhibition prevents infiltration of inflammatory cells into the lungs after allergen exposure. PARP-1deficiency inhibited IL-5expression through calpain-dependent degradation of STAT-6in allergen induced asthma. However, the effect of mechanical stretch on PARP-1during asthma is still unclear.NF-κB is an important transcription factor for the amplification loop of inflammatory reactions; its activation and nucleus translocation can combine with the fixed sequence of many gene promoters to start the transcription of plural inflammatory mediators which plays a very important role in the development of an inflammatory disease. Increasing evidence showed that NF-κB played an essential role in the expression of inflammatory mediators in asthma. Previous study has found that PARP-1inhibition by inhibitor3-AB can inhibit NF-κB activation and IL-8expression induced by H2O2.ObjectiveIn the present study, we aimed to investigate the role and mechanism of PARP-1in mechanical stretch-induced inflammation in human bronchial epithelial cells (HBEpiCs).MethodsHBEpiCs were simulated by mechanical stretch in our experiment. Cells under static were used as the control. PARP-1expression was interfered by small interfering RNA(siRNA).The expression of inflammatory cytokine related gene and protein of stretch-preconditioned and static cells were detected separately by quantitative reverse transcription polymerase chain reaction (RT-PCR), western bloting, enzyme linked immunosorbent assay (ELISA), small interference RNA (siRNA) and immunofluorescence. Oxidative stress was evaluated by DHE staining. DNA damage was assessed by comet assay.Results1. IL-8and vascular cell adhesion molecule-1(VCAM-1) expression were regulated by hyperstretch in a time-dependent manner.HBEpiCs were stimulated by hyperstretch for various times. Hyperstretch induced a time-dependent regulation of IL-8and VCAM-1expression. Compared with cells under static condition,24-h hyperstretch could significantly increase the mRNA and protein expression of VCAM-1, and the ELISA also showed that IL-8secretion was upregulated by hyperstretch stimulation (P<0.05).2. Hyperstretch could increase PARP-1expression and activity by inducing superoxide production and DNA damage.Compared with the static cells, PARP-1expression was significantly increased by hyperstretch, and PARP-1activity was also upregulated (P<0.05).There was almost no superoxide production in cells under static, while hyperstretch stimulation could significantly induce oxidative stress (P<0.05).In the cells under static, there was almost no DNA in the tail, while hyperstretch could increase the DNA content in the cellular tail, which suggested that hyperstretch could induce DNA damage in HBEpiCs.3. Silencing of PARP-1attenuated hyperstretch-induced IL-8and VCAM-1upregulation as well as monocytes adhesion, which were related to the inhibition of nuclear factor-kappa B (NF-κB) translocation.Hyperstretch stimulation markedly increased IL-8secretion and VCAM-1expression as compared with static cells. However, after PARP-1silencing, IL-8secretion and VCAM-1expression were significantly reduced (P<0.05). The negative control of PARP-1siRNA had no significant effect on the expression of inflammatory cytokines.Compared with cells under static condition, stimulation of HBEpiCs with hyperstretch resulted in a significant increase in THP-1cell adherence; while pretreatment of HBEpiCs with PARP-1siRNA before hyperstretch stimulation resulted in a significant decrease in monocyte adhesion.NF-κB p65was mostly cytoplasmic before hyperstretch stimulation, but its localization quickly changed to the nucleus with stimulation. After PARP-1silencing, p65remained primarily cytoplasmic after hyperstretch treatment.Conclusion1. Hyperstretch could induce inflammatory response and superoxide production as well as DNA damage in HBEpiCs.2. PARP-1silencing decreased IL-8and VCAM-1expression, partly through inhibition of NF-κB translocation.3. PARP-1played a fundamental role in hyperstretch-induced inflammation.4. PARP-1silencing could be used as a potential therapeutic approach to reverse bronchial epithelial inflammation in asthma. |
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