| BackgroundCOPD is a progressive disabling illness associated with an abnormal inflammatory response of the airways and the lung to noxious stimuli. It is characterized by persistent airflow limitation that is not fully reversible and airway inflammation.There were no clearly full understandings on the cytology, molecular and genetic etiology. The current treatments are insufficient, cannot delay the progression or reduce mortality.General practice databases indicate that almost 25% of the population has multiple morbidities and that this proportion increases substantially with age, so that most people over the age of 65 years have multimorbidity. Ischaemic heart disease, osteoarthritis, osteoporosis, type 2 diabetes, metabolic syndrome, chronic renal disease and Alzheimer's disease, as well as chronic obstructive pulmonary disease, they are all included. There is evidence strongly suggests that there may be common pathogenic pathways linking these diseases, closely related to the cellular senescence.Cellular senescence is a process that results from a variety of stresses, leading to a state of irreversible growth arrest. Senescent cells accumulate during aging and have been implicated in promoting a variety of age-related diseases.According to the research, cell senescence is the common cause of multimorbidity, Telomere shortening, activation of PI3K-AKT-mTor signaling, impaired autophagy, mitochondrial dysfunction, stem cell exhaustion, epigenetic changes, abnormal microRNA profiles, immunosenescence and low grade chronic inflammation ("inflammaging") are main mechanisms of cell senescence. Many of these pathways are driven by chronic oxidative stress. Increased oxidative stress is well documented in the lungs of COPD patients. This increase in oxidative stress is due to oxidants in cigarette smoke and activated inflammatory cells. Reactive oxygen species (ROS) are generated in oxidative stress, and activate PI3K, then, via activation of the kinase AKT, activates mTor, the phosphoinositide-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTor) pathway is activated. Then, the activated pathway inhibits autophagy via unc-51-like autophagy activating kinase-1 (ULK1) and reduces sirtuin-1 (SIRT1) activity, both of which accelerate the ageing process. mTor pathway is a key modulator of ageing and age-related disease. Activation of the mTor pathway may play an important role in multimorbidity and inhibition of this pathway offers a good future therapeutic opportunity.In summary, the inhibition of cell senescence has therapeutic effect on COPD and other multimorbidities.In China, many traditional Chinese medicines (TCM) modalities are regularly used in COPD patients. Several clinical trials have shown that TCM might have therapeutic effect for COPD patients including improvement of symptoms, quantity of life and lung function.Cordyceps sinensis (CS) or Dongchongxiacao in Chinese is a special type of mushroom, which is formed on an insect larva infected by the Cs fungus. It is a rare and precious medicinal mushroom having great medicinal value. Cordyceps sinensis has the functions of anti-aging, microorganisms, tumor, atherosclerosis and can regulate of endocrine, respiratory, immune, nervous system and kidney and liver.Now research on Cordyceps sinensis mainly focused on chemical constituents and pharmacological actions. There are nucleoside, polysaccharide, sterol, protein, amino acid, and polypeptide in Cordyceps sinensis, working in anti-inflammatory, antioxidant, antitumour, antiapoptosis, and immunomodulatory. But the specific mechanism of Cordyceps sinensis is not clear. Singh M found Cordyceps sinensis could decrease oxidative stress in human lung epithelial cells. Anti-oxidative plays an important role in Cordyceps sinensis's functions. The oxidative stress in the respiratory system is mainly derived from the smoke, has close association with cell senescence.Here, we investigate the inhibitory effect and mechanism of Cordyceps sinensis on the senescence of human airway epithelial cells induced by CSE (Cigarette Smoke Extract), to provide help to the prevention and treatment of COPD patients in future.Objectives1. To detect the influence of CSE on the cell survival rate of human airway epithelial cells.2. To investigate the cellular senescence induced by CSE.3. To detect the inhibition of CS on the cellular senescence induced by CSE.4. To explore the function of ROS/PI3K/AKT/mTor signalling pathway in the process of cellular senescence induced by CSE.5. To explore the mechanism of CSE and CS on cellular senescence.Methods1. To detect the appropriate dose and action time of CSE, we use MTT assays. Human airway epithelial cells (16HBE cells) cultured in vitro were treated with CSE in different doses and time points. The relative cell number was detected to evaluate cell growth.2. SA-?-gal (Senescence-Associated ?-galactosidase) staining was used to detect cellular senescence. P16 and P21 were too, by detections of immunofluorescence, PCR (Polymerase Chain Reaction), and western blotting. Meanwhile, the protein expressions of P16 and P21 were detected by the gradient CSE treatment.3. CS(Cordyceps sinensis) was added 2 hours before CSE treatment, and then cellular senescence was detected. SA-p-gal staining, immunofluorescence, PCR, and western blotting test were used, compared to the cells only treated by CSE.4.ROS (Reactive Oxygen Species), PI3K/AKT/mTor and their phosphorylated proteins were detected after CSE and CS treatment to testify the signaling pathway, by using ROS fluorescent staining kit and western blotting. Meanwhile, the protein expressions of p-AKT and p-mTor were detected by the gradient CSE treatment.5. Inhibitors of ROS and PI3K were used to explore the mechanism of CSE and CS on cellular senescence. ROS fluorescent staining, PI3K/AKT/mTor, and their phosphorylated proteins were detected to observe the influence on the signaling pathway by inhibitors. We detected SA-?-gal staining and protein expressions of P16 P21 to prove the cellular senescence.Results1. Cell survival rate was inhibited by CSE in a time and dose-dependent manner. According to the survival curve, stimulated by 2%CSE for 24 hours could be used in the follow-up experiments.2. Activation of cellular senescence was increased by CSE treatment.SA-?-gal positive cells ratio was obviously increased (p<0.05), immunofluorescence of P16 and P21 was enhanced, P16 and P21 mRNA levels were significantly increased (p<0.05, p<0.05, respectively),the protein expressions of P16 and P21 were increased(p<0.05, p<0.05,respectively), compared to the control group. CSE promotes the expression of P16 and P21 in 16HBE cells in a time and dose-dependent manner (p<0.05, p<0.05, respectively).3. Activation of cellular senescence was decreased by CS treatment before adding CSE. After adding CS, SA-?-gal positive cells ratio was obviously decreased (p<0.05), immunofluorescence of P16 and P21 was weakened, P16 and P21 mRNA levels were significantly decreased (p<0.05, p<0.05, respectively), the protein expressions of P16 and P21 were reduced (p<0.05, p<0.05, respectively).4. Activation of ROS and P13K/AKT/mTor signaling pathway was increased by CSE treatment, and decreased when Cordyceps sinensis was added, evidenced by ROS fluorescence and PI3K/AKT/mTor and their phosphorylation detection(p<0.05. p<0.05,respectively).The expressions of p-AKT and p-mTor were promoted by CSE in a time and dose-dependent manner(p<0.05, p<0.05,respectively).5. Blocking ROS by NAC could weaken the ROS fluorescence obviously, reduce the expressions of p-AKT and p-mTor (p<0.05, p<0.05, p<0.05, p<0.05, respectively), therefore, the activation of ROS and PI3K/AKT/mTor signaling pathway was decreased. Blocking ROS by NAC could reduce the cellular senescence in CSE group and CS+CSE group, evidenced by lessening of SA-?-gal positive cells ratio and protein expressions of P16 and P21(p<0.05, p<0.05, p<0.05, p<0.05,respectively). Blocking PI3K by Ly294002 could reduce the expressions of p-AKT and p-mTor obviously (p<0.05, p<0.05, p<0.05, p<0.05, respectively), so the activation of signaling pathway was decreased. But it had no obvious effect on ROS. Blocking PI3K by Ly294002 could reduce the cellular senescence in CSE group and CS+CSE group, evidenced by lessening of SA-P-gal positive cells ratio and protein expressions of P16 and P21(p<0.05, p<0.05, p<0.05, p<0.05,respectively).Conclusions1. Cell survival rate was inhibited by CSE in a time and dose-dependent manner.2. Cellular senescence could be induced by CSE.3. CS can inhibit the CSE-induced senescence in vitro.4. ROS and PI3K/AKT/mTor signaling pathway may play an important role in the cellular senescence induced by CSE.5. Blocking ROS/mTor signaling pathway can alleviate CSE-induced cellular senescence in human bronchial epithelial cell. |
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