| BackgroundChronic obstructive pulmonary disease（COPD）,is a common chronic airway inflammatory disease,characterized by persistent airflow limitation.According to statistics,the number of patients is close to 100 million in China.Cigarette smoke exposure is the main risk factor for COPD.Studies have shown that the inflammatory response is an important pathogenesis of COPD by cigarette smoke stimulating the respiratory tract.Chronic inflammatory reactions lead to airway remodeling and emphysema lesions,and are prone to acute exacerbations.Effective anti-inflammatory treatment can slow the progression of the disease and reduce the occurrence of acute exacerbation of COPD,thereby improving its quality of life.Currently,clinical treatment of COPD is mainly bronchodilator drugs,includingβ2 receptor agonists,anticholinergic drugs and theophylline drugs.High-risk,symptomatic COPD patients are recommended to use combination therapy with inhaled glucocorticoid（ICS）and bronchodilator drugs,but ICS treatment increases the risk of infection and exacerbates inflammation.Another anti-inflammatory drug is the phosphodiesterase-4（PDE-4）inhibitor Roflumilast（ROFL）.ROFL has adverse reactions such as diarrhea,nausea,and headache,which are rarely used clinically.In addition,unlike other airway inflammatory diseases（such as asthma）,inflammation in the development of COPD has not been effectively suppressed by existing anti-inflammatory drugs.The development of innovative,accurate and low-toxic anti-inflammatory drugs is the focus of COPD treat research.Chinese medicine has a long history of treating COPD.In the past ten years,there have been more than 100 clinical trials on Chinese medicine-assisted interventional COPD treatment.It has been confirmed by down-regulating the serum and sputum inflammatory mediators of patients（such as IL-8 and TNF-α）,Improve the quality of life of patients with COPD,improve lung function and reduce the frequency of acute exacerbations of COPD.Cordyceps sinensis is a valuable traditional Chinese medicine in my country.It contains many substances such as polysaccharides,adenosine,cordycepin,and selenium.Modern pharmacological studies have shown that Cordyceps sinensis has antibacterial,anti-inflammatory,anti-cancer,anti-fatigue,anti-aging and immune regulation effects.The record of Cordyceps sinensis in treating lung diseases began in"Compendium of Materia Medica"in 1694."Guifei Jing"suggests that Cordyceps sinensis has a selective treatment effect on the lungs.Meta-analysis of clinical studies shows that Cordyceps sinensis may have a certain improvement in lung function,symptoms and quality of life in patients with stage I-III COPD,but whether it has a definite preventive effect on Cordyceps sinensis in the development and acute exacerbation of COPD still lacking experimental basis.ObjectivesThrough the establishment of the COPD mice model,this study explored the effects of early intervention of cold extracts from fresh Cordyceps sinensis on lung function and airway inflammation in mice,and provided experimental basis for the prevention and treatment of COPD.Methods1.Sulfuric acid-phenol method was used to detect the total sugar content of the sample.Superoxide dismutase（SOD）kit was used to detect the SOD enzyme activity of the sample.2.In vitro experiments:Cigarette smoke exposure was an important inducement for the occurrence and development of COPD.Therefore,cigarette smoke extract（CSE）was used in vitro to stimulate human bronchial epithelial cells（16HBE）,IL-6,CXCL8/IL-8,and mucin 5ac（MUC5AC）gene expressions were detected.By immunofluorescence,MUC5AC protein expression was observed.3.In vivo experiments:（1）Male C57 mice aged 6 to 8 weeks were selected and divided into 6 groups.LPS combined with cigarette smoke mouth and nose exposure for 13 weeks was used to construct the COPD mice model.Drug intervention was started on the first day of smoke exposure in mice,and total extracts of Cordyceps sinensis（75 mg/kg/day,150 mg/kg/day,300 mg/kg/day）intervened in mice exposed to long-term smoke,and the Yupingfeng San（780 mg/kg/day）intervention group was regarded as the positive control.By analyzing the pathology of the lung tissue,the lung function indicators included the percentage of forced expiratory volume and forced vital capacity of 0.05 seconds in mice（FEV0.05/FVC）,the expiratory flow rate at 50%of the forced vital capacity（FEF-50%FVC）,the main airway resistance（Rn）,respiratory resistance（Rrs）,lung cytokine interferon-γ（IFN-γ）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,chemokine-1（CXCL1/KC）,the effect of total extracts of Cordyceps sinensis on inflammatory response in mice exposed to long-term smoke was evaluated.（2）The acute exacerbation of COPD was mostly related to bacterial or viral infections.Among them,the acute exacerbation of COPD caused by influenza virus infection accounted for 7.83%.Therefore,mice infected with influenza virus were used to construct the acute pneumonia model and were given total extracts of Cordyceps sinensis（75 mg）/kg/day,150 mg/kg/day,300mg/kg/day)intervention,oseltamivir phosphate（60 mg/kg/day）intervention as a positive control.By analyzing the lung index,lung pathology and lung inflammation mediators IFN-γ,IL-1β,IL-6 and KC indicators,the effect of total extracts of Cordyceps sinensis on acute viral pneumonia was evaluated,and the effect of total extracts of Cordyceps sinensis on influenza virus that was potential factor causing the acute exacerbation of COPD was discussed.Results1.Sulfuric acid-phenol method was used to detect the total sugar content of the sample,the total sugar content was 18.69%.Superoxide dismutase（SOD）kit was used to detect the SOD enzyme activity,the SOD enzyme activity was 14736.5U/m L.2.CSE was used to stimulate 16HBE cells for 48h.Compared with IL-6（1.07±0.47）,IL-8（1.25±0.24）,MUC5AC（1.05±0.57）m RNA of the normal control group,IL-6（13.35±2.06）,IL-8（5.34±0.96）,MUC5AC（6.49±0.73）m RNA expressions were significantly increased（p<0.001）.In the total extracts of Cordyceps sinensis intervention group（0.25,0.50,1.00 mg/m L）,IL-6（6.54±0.99）（4.72±1.01）,MUC5AC（2.35±0.10）（1.44±0.65）m RNA expressions of the total extracts of Cordyceps sinensis intervention group at 0.50 mg/m L and 1.00 mg/m L were significantly decreased compared with the model group（p<0.05）.In the immunofluorescence experiment,the total extract of Cordyceps sinensis at 1.00 mg/m L can significantly inhibit the overexpression of MUC5AC protein of 16HBE.3.Long-term smoke exposure mice model:（1）Compared with the normal control group,the pathology of the lung tissue of mice of the model group showed a large number of inflammatory cell infiltration of the lung tissue,the rupture of the alveolar wall,and the alveolar cavity fusing into a larger cystic cavity and the formation of a typical pulmonary bullae,the thickening of the bronchial wall,the exfoliation of bronchial epithelial cells,and obstruction of the lumen and other typical pathological changes of COPD,which suggested that the model was successful.Compared with the model group,the lung pathology of the total extracts of Cordyceps sinensis intervention group at 300 and 150 mg/kg/day showed a decrease in inflammatory cell infiltration,a decrease in the number of pulmonary bullae,and a regular structure of the bronchial wall.（2）Compared with FEV0.05/FVC（65.05±2.1）and FEF-50%FVC（15.62±1.31 m L/s）of the normal control group,the model group’s lung function indicators FEV0.05/FVC（52.66±5.29）and FEF-50%FVC（9.69±2.65 m L/s）were significantly decreased（p<0.05）.Compared with the model group,the lung function indicators FEV0.05/FVC（63.23±3.13）and FEF-50%FVC（14.59±1.7 m L/s）of the total extracts of Cordyceps sinensis intervention group at 300 mg/kg/day were significantly improved（p<0.05）,FEV0.05/FVC in mice improved by 10.57%.The success of the COPD model mice was judged by the pathological changes and the significant decrease in lung function in mice of the model group.（3）Compared with IL-6（26.06±2.51 pg/m L）,KC（79.48±29.66 pg/m L）,IL-1β（31.14±8.3 pg/m L）of the normal control group,the lung IL-6（40.18±7.13 pg/m L）,KC（156.02±15.15 pg/m L）and IL-1β（60.81±8.55 pg/m L）in mice of the model group were significantly increased（p<0.05）.Compared with the model group,IL-6（27.33±6.22 pg/m L）,KC（108.19±20.98 pg/m L）and IL-1β（38.1±11.27 pg/m L）expressions were significantly decreased of the total extracts of Cordyceps sinensis intervention group at 300 mg/kg/day（p<0.05）;Compared with the model group,KC（122.15±15.51 pg/m L）expression of the total extracts of Cordyceps sinensis intervention group at 150 mg/kg/day was significantly decreased（p<0.05）.Compared with the normal control group（39.22±11.16 pg/m L）,lung IFN-γ（53.91±13.02 pg/m L）expression of the model group was increased,but there was no statistical difference（p>0.05）.Compared with the normal control group,lung IFN-γ（74.87±11.41 pg/m L）expression of the total extracts of Cordyceps sinensis intervention group at 300 mg/kg/day and IFN-γ（66.18±13.32 pg/m L）of the total extracts of Cordyceps sinensis intervention group at 300 mg/kg/day were significantly increased（p<0.05）.Compared with the model group,there was no significant difference（p>0.05）.4.Acute pneumonia model of influenza virus-infected mice:（1）Compared with the normal control group,the lung pathology in mice of the model group showed obvious congestion of lung tissue,the thickening of the alveolar wall,the infiltration of a large number of white blood cells and red blood cells around the alveolar space and bronchi,presenting the typical interstitial pneumonia,which indicated the model was successful;Compared with the model group,the pathology of the total extracts of Cordyceps sinensis（75 mg/kg/day,150 mg/kg/day,300 mg/kg/day）intervention group had no obvious changes.（2）Compared with the normal control group（30.22±4.81 pg/m L）,lung IFN-γ（130.91±25.98 pg/m L）in mice of the model group was significantly increased（p<0.001）.Compared with the model group,the total extracts of Cordyceps sinensis intervention group at 300 mg/kg/day（87.9±15.38）was significantly decreased（p<0.05）.ConclusionThe cold aqueous extract of fresh Cordyceps sinensis early intervened in smoke-exposed mice,by reducing the expression of IL-6,KC,and IL-1βinflammatory factors in the lungs,reducing the airway inflammation in mice,and delaying the decline of lung function in mice. |
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