The Function And Mecfianism Of Ophiocordyceps Sinensis In Airway Epithelial Cell Senescence In COPD

BackgroundChronic Obstructive Pulmonary Disease(COPD)is a common inflammatory disease in clinic that is characterized by persistent respiratory symptoms and airflow limitation,and can further develop into chronic pulmonary heart disease and respiratory failure.Its morbidity and mortality rates are rising year by year.COPD has been currently the fourth leading cause of death in the world,but is projected to be the 3rd leading cause of death by 2020.More than 3 million people died of COPD in 2012,accounting for 6%of all deaths globally.COPD represents an important public health challenge that is both preventable and treatable.Being a country with a large population,China has been one of the countries with high incidence rate of COPD The etiology for COPD is airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases.Both chronic airway inflammation and airway remodeling are the main pathological features of COPD.Therefore,the two aspects are also the main directions of prevention and treatment in COPD at present,but the clinical effect is not still ideal.Several studies have found that cellular senescence plays an important role in the pathogenesis of chronic pulmonary diseases,including COPD,asthma,lung cancer and pulmonary fibrosis.Cellular senescence is a fundamental property for all cells,which is manifested as the stagnation of cell growth under the condition of cellular stress.In general,cellular senescence can be viewed as a protective mechanism of the body against the occurrence of cancer.Other studies have found that cellular senescence is the pathological basis in some chronic diseases,such as cerebral nervous system diseases(e.g.,Alzheimer’s disease),cardiovascular system diseases(e.g.,atherosclerosis),musculoskeletal system diseases(e.g.,osteoporosis)and COPD.The process of cellular senescence is associated with decreased physiological function and protein degradation,which is irreversible.In particular,cellular senescence affects regeneration ability by reducing the number of hematopoietic stem cells,and aging cells release many cytokines to further enhance the inflammatory response.Thereby cellular senescence promotes the occurrence and development of airway inflammation and airway remodeling in COPD patients.In the preliminary cell experiment we have demonstrated that the existence of cellular senescence was in CSE-induced COPD models in vitro.Therefore,we think that cellular senescence may exist in the airway epithelial cells of COPD patients,and may be involved in the pathogenesis and progression of COPD.However,the specific mechanism of cellular senescence in COPD is still unknown and needs further study.We will explore the prevention and treatment of COPD from the perspective of cell senescence.Effectively inhibiting the senescence of COPD airway epithelial cells is expected to become a new clinical therapeutic strategy for COPD.Ophiocordyceps sinensis(O.sinensis)has been widely used in traditional Chinese medicine.O.sinensis has many bioactive constituents,such as polysaccharides,cordyceps sinensis,glycol,aminophenol,ergosterol,etc.The chemical constituents extracted from O.sinensis have various pharmacological activities,such as protection of kidney and liver,anti-inflammatory,antiviral,antioxidant and anti-apoptosis.In COPD patients,cultured O.sinensis can inhibit chronic airway inflammation,improve lung function,correct the imbalance of Thl/Th2 ratio,and have two-way immune regulation effect.It also can reduce respiratory muscle fatigue,and improve exercise endurance.Although the various functions of O.sinensis have been widely used in the treatment of COPD,but Its inhibitory effect on cell senescence in COPD is rarely reported.In previous cell experiments,we found that cigarette smoke extract(CSE)can induce cellular senescence of human bronchial epithelial cells in vitro,while cultured O.sinensis can alleviate airway epithelial cell senescence caused by CSE to some extent.Therefore,this study will further investigate how O.sinensis regulates cellular senescence in COPD airway epithelium cells in vivo and in vitro.ObjectivesThe objective of the study is to evaluate the inhibitory effect of O.sinensis liquid on the cellular senescence of airway epithelial cells in CSE-induced COPD both in vitro and in vivo,and the possible mechanism was discussed.MethodsLung tissues of patients with COPD were collected,and morphological methods(HE staining and immunohistochemical staining)were used to confirm the existence of COPD airway epithelial cell senescence.The protein expression levels of P16 and P21 in the airway epithelial cells of 30 patients with COPD were detected by western blotting.The senescence of airway epithelial cells was observed by SA-β-gal staining and BrdU staining.Fluorescence quantitative PCR and western blotting were used to determine the appropriate time and concentration of CSE induced senescence in human bronchial epithelial cells(16HBE).The 16HBE fumigation model was successfully constructed to detect the influence of O.sinensis in the 16HBE pretreated with CSE.COPD model rats were treated with O.sinensis at various concentrations to detect the proteins/mRNAs expression changes of P16 and P21 in airway epithelial tissues.The COPD rat model was prepared by LPS endotracheal drip and passive fumigation,and confirmed the success of modeling by morphology(HE staining and immunohistochemical staining).In addition,the function of O.sinensis on airway epithelial cells in COPD rats was observed by detecting both the mRNA and protein expressions of P16 and P21 by means of immunohistochemical staining,fluorescence quantitative PCR and western blotting.The different effect of different O.sinensis doses on the proteins/mRNAs expression of P16 and P21 in airway epithelial cells of COPD rats was observed.To explore the possible mechanism of O.sinensis inhibiting airway epithelial cell senescence with the proteins expression level of BCL-2,P53,HSP90 and HIF-1α.ResultsIt was found that the proteins and mRNAs expression levels of P16 and P21 in airway epithelial cells of COPD patients were higher than those of the normal group by immunohistochemical staining,real-time quantitative PCR and western blotting.In this experiment,we found that the appropriate concentration of CSE acting on the 16HBE was 2.0%and the appropriate time was 6h.Cell experiments confirmed that O.sinensis could alleviate CSE-induced cellular senescence.It was found that the protein expressions of P16 and P21 in the O.sinensis intervention group were lower than those in the CSE-induced cell group,and O.sinensis could promote the proliferation of 16HBE cells,and the differences were statistically significant.Pathological changes in lung tissue of COPD model rats were observed by HE staining,which confirmed the successful construction of COPD model.Real-time quantitative fluorescence PCR and western blotting were used to detect the proteins/mRNAs expression of P16 and P21,and it was found that the expression levels of P16 and P21 were also increased in airway epithelial cells of COPD model rats.Compared with the control group,the results were statistically significant.The protein/mRNA expression levels of P16 and P21 in lung tissues of rats treated with different doses of O.sinensis were significantly reduced compared with those in the COPD model group,and the differences were statistically significant.We found that the expression of Bcl-2 protein existed in all the 16HBE cells of the three groups,and the expression of Bcl-2 protein was highest in the CSE group.After treatment with Ophiocordyceps sinensis(O.sinensis),the expression of Bcl-2 protein was reduced in CSE+O.sinensis group,and the difference of Bcl-2 protein expression between the two groups was statistically significant.The expression of P53 protein was the highest in 16HBE cells of the CSE group,followed by the CSE+O.sinensis group,and the lowest in the control group,with statistically significant differences among the three groups.The expression trend of HSP90 protein in the three groups was the same as that of Bcl-2 protein and P53 protein.The expression of HIF-la protein in the CSE group was the highest.The expression of HIF-la protein was no statistical difference between the control group and the CSE+O.sinensis group,but there was statistical difference between the CSE group and the CSE+O.sinensis group.ConclusionsThe CSE can induce cellular senescence of COPD airway epithelial cells.O.sinensis can inhibit CSE-induced cellular senescence both in vitro and in vivo.O.sinensis may inhibit the cellular senescence of COPD airway epithelial cells through the anti-apoptotic pathway P53/BCL-2/HSP90/HIF-1α.