Pharmacological And Biomarker Evaluations Of Active Ingredients In Artificial Cordyceps CPD0301 For The Treatment Of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease(COPD)is a slowly progressive disease characterized by an airflow limitation,which is largely irreversible.COPD mainly consists of two major distinctive criteria,called chronic bronchitis and pulmonary emphysema,associated with the decrease of lung function.COPD is a major cause of chronic morbidity and mortality throughout the world.The number of COPD patients has exceeded to 100 million in 2008,which is threating the people health in the world.The pathological mechanism of COPD is complex,including abnormal inflammation in the airway and lung parenchyma of COPD patients,along with oxidative stress,protease-anti-protease imbalance,immune imbalance and cell apoptosis leading to the reconstruction of airway and lung tissue.At present,the current main pharmacotherapies in COPD include inhaled corticosteroid,long-acting β2-adrenoceptor agonists(LAB As),long-acting muscarinic antagonists,and Phosphodiesterase 4(PDE4)inhibitors and antioxidant.Drugs such as antitussives,mucolytics,and bronchodilators that are used only to relieve the symptoms of bronchitis do not affect or reverse the pathological progress of COPD.PDE4 inhibitors such as roflumilast can only treat COPD inflammation.All of these first-line COPD medicines have been shown to have severe side effects in addition to their limited efficacy.Therefore,novel drugs with multi-targeted properties are needed to effectively combat the complex pathological course of COPD and eventually lead to a complete cure.Studies have shown that Traditional Chinese Medicine(TCM)is effective in preventing and treating COPD as well as its complications,delaying the damage of lung function and enhancing immunity.Cordyceps sinensis(C.sinensis),a kind of precious nourishing TCM,has a lot functions like invigorating the lung and kidney and relieving cough and phlegm.The artificial Cordyceps prepared by fermentation not only remains the pharmacological activities of the wild Cordyceps,but also has solved the problem of the serious shortage of the wild Cordyceps,such as the artificial Cordyceps powder capsules used in the clinical-Jinshuibao capsule and Bailing capsule,etc.However,there are few reports on the effective componentsand its mechanism and biomarkers of Cordyceps against COPD.In order to further improve the scientificity and effectiveness of Cordyceps in the treatment of COPD,in this project,we used the artificial Cordyceps as the drug substance to carry out the screening of the effective ingredients of Cordyceps CPD0301 against COPD and its mechanism,and its biomarkers in vivo.The projects of the subject are divided into three parts:(1)Screening of the effective ingredients in Cordyceps for COPD treatment;(2)Study on the pharmacological activity and mechanism of the effective ingredients for COPD treatment;(3)Biomarker selection for CPD0301 to explore the relationship between PK-PD in COPD model rats.1、Screening of the effective ingredients in CPD0301 for COPD treatmentThe study on the screening of the effective ingredients against COPD in Cordyceps CPD0301 includes the extraction and purification of the active parts,the qualitative and quantitative analysis of the active components,and the evaluation of the activity against COPD.The artificial Cordyceps powder was dissolved in ultra pure water and 95%ethanol according to the ratio of m/v=1:10 to obtain the corresponding water and alcohol extracts by ultrasonic extraction.(1)After the water extract was concentrated and freeze-dried under reduced pressure,10 kinds of nucleoside compounds were screened out and quantified by HPLC.Results are shown as Cytidine 0.753 mg/g,Adenine 0.248 mg/g,Guanine 6.520 mg/g,Uracil 0.215 mg/g,Hypoxanthine 0.252 mg/g,Uridine 0.395 mg/g,Adenosine 5.907 mg/g,2’-Deoxyadenosine 0.576 mg/g,Guanosine 4.234 mg/g,Thymidine 0.376 mg/g;(2)After the alcohol extract was extracted by ethyl acetate,column chromatography,concentrated and freeze-dried,the sterols of C sinensis were qualitatively analyzed by GC/Ms,and results show that ergosterol was the main sterols of C sinensis.Then,the ergosterol in C.sinensis was quantified by HPLC,results show that the content of ergosterol is 5.547 mg/g;(3)C.sinensis powder been ethanol extracted was extracted again in ultra pure water by ultrasonic extraction according to the ratio of m/v=1:10.After alcohol precipitation,deproteinization and decolorization,the polysaccharide components were determined by sulfuric acid-phenol assay,results show that the content of polysaccharide is 4.4%;(4)The anti-COPD activity of nucleosides,sterols(ergosterol)and polysaccharides was evaluated by using NO as the evaluation index.The results showed that all of them inhibited the NO secretion in CSE-induced RAW264.7 cells.However,nucleosides and sterols were superior to polysaccharides.In view of a large number of literature reports on the anti-inflammatory and anti-oxidation effects of polysaccharides,the follow-up experiments mainly focused on nucleosides and sterols(ergosterol).二、Study on the pharmacological activity and mechanism of the effective ingredients for COPD treatmentThe study on the pharmacological activity and mechanism of the active components on COPD include the establishment of COPD model both in vitro and in vivo,the determination of pharmacological activity and the exploration of related signaling pathways.For the establishment of COPD model in vitro,we used cigarette extract(CSE)to stimulate RAW264.7 cells or 16HBE cells,then the cells were treated with active ingredients for 24 h,and the anti-inflammatory effect of the active ingredients in C.sinensis was investigated by ELISA and RT-PCR;the level of ROS,cell surface antigen and cell apoptosis was determined by flow cytometry;the related signal pathway proteins and apoptosis related proteins were determined by Western blot.Secondly,we established the COPD animal model by intraperitoneal injection of CSE once a week for three weeks.After gavaged with effective ingredients for 21 days,the mice/rats were killed and collected the BALF,plasma and lung tissue.The number of inflammatory cells in BALF were counted.The related enzymes and cytokines in BALF and plasma were measured by ELISA,the morphological changes of lung tissue was studied by HE and Masson staining,and the related proteins were measured by immunohistochemistry and Western blot1.Pharmacological activity and potential mechanism of nucleosides on COPDThe results show that nucleosides reduce the secretion of NO,TNF-α,IL-6,IL-1p in 10%CSE-induced RAW264.7 cells,and inhibit iNOS,TNF-α,IL-6,IL-1βmRNA level.Nucleosides also improve the lung feature,reduce the infiltration of inflammatory cells in BALF,and inhibit the secretion of NO,TNF-α and IL-1β in BALF and plasma in CSE-induced mice.Moreover,results show that nucleosides increase the expression of SIRT1 and reduce the expression of NF-KB/p65,indicating that SIRT1-NF-κB/p65 signaling pathway plays an important role in the anti-inflammatory effect of nucleosides.2、Pharmacological activity and potential mechanism of ergosterol on COPDThe pharmacological activities and potential mechanism of ergosterol on COPD include the effect on anti-inflammatory,anti-oxidation,anti-apoptosis,cell polarization and protease.(1)The experimental results show that ergosterol not only has anti-inflammatory and anti-oxidation effect through inhibiting the secretion of inflammatory factors(like NO,IL-6 and TNF-α),reducing the infiltration of inflammatory cells in the lung,reducing the ROS level,and inhibiting the activity of oxidative stress associated enzymes(CAT,MDA and SOD)in BALF,but also has anti-apoptosis effect.Annexin V-FITC/PI double staining and DAPI staining shows that ergosterol inhibits the apoptosis of 16HBE cells induced by CSE;TUNEL staining showed that ergosterol inhibits the apoptosis of lung tissue in CSE-stimulated mice;Western blot and immunohistochemistry results show that ergosterol upregulates Bcl-2,while down-regulate Bax and cleaved caspase 3/7/9 and cleaved PARP to inhibit apoptosis,which was regulated by inhibiting the activation of NF-κB/p65.(2)Macrophages are prone to cell polarization when is stimulated:M1 polarization(promoting inflammation)and M2 polarization(inhibiting inflammation).In this chapter,results showed that ergosterol reducegs the expression of typical cytokines(ROS,IL-6,TNF-α)and their corresponding mRNA(iNOS,IL-6,TNF-α)in M1 macrophage cells,while increases the expression of typical cytokines(IL-10,TGF-β)and their corresponding mRNA(IL-10,TGF-β)in M2 macrophage cells.The results of flow cytometry,immunohistochemistry and Western blot show that the ergosterol reduces the expression of M1-marker CD40 while increases the expression of M2-marker CD 163.All the above results showed that ergosterol could promote the transformation of RAW264.7 cells from Ml to M2.In addition,the experimental results showed that ergosterol activates the expression of HDAC3 mRNA and protein,inhibit the expression of P300,CBP,PCAF mRNA and protein;ergosterol can also inhibit the activity of IKKβ kinase and then inhibit the activation of NF-κB/p65.In conclusion,ergosterol regulates the polarization of macrophages through HDAC3-NF-κB/p65 signaling pathway.Macrophages,an important immune cell,play a role in the immune response,beyond all question,play a critical role in the treatment of COPD.三、Biomarker selection for CPD0301 to explore the relevance between PK-PD in COPD model ratsThe study of biomarkers in vivo and PK-PD correlation in COPD model rats includes the selection of biomarkers in vivo and the study of its kinetic parameters in COPD model rats.According to the study of the preliminary screening of nucleosides,polysaccharides,sterols in CPD0301 against COPD as well as the literature research,ergosterol was finally determined as the biomarker of CPD0301 against COPD,and the PK-PD correlation of artificial Cordyceps CPD0301 in the COPD model rats was further explored.The results show that the content of ergosterol in the plasma of rats fed with different concentrations of CPD0301 powder by gavage changed accordingly.The content of ergosterol in the plasma of rats was in consistent with that of C.sinensis fed.In brief,the content of ergosterol in the plasma with high concentration of C.sinensis was high,and that of ergosterol in the plasma with low concentration of C.sinensis was low.This result was consistent with the pharmacological activity which was in a concentration dependent manner.In addition,compared with water as the solvent and three times a day of small dose(450 mg/kg),20%hydroxypropyl-β-cyclodextrin as the solvent and one time a day of large dose(1.35 g/kg)has a higher AUC0-∞(1031.3 ng/mL·h),which is 5 times to the former,while the time to reach Cmax of the latter one was significantly delayed.This study provides a theoretical basis for the research and development of new dosage forms of C.sinensis or ergosterol for COPD patients to improve the efficacyTo sum up,we firstly screened the effective ingredients of C.sinensis in the treatment of COPD and,by means of HPLC,GC/MS and chemical reaction,10 kinds of nucleosides,ergosterol,and polysaccharides were isolated and screened.Subsequently,we carried out a systematic study on the pharmacological activity and molecular mechanisms of nucleosides and ergosterol on COPD both in vitro and in vivo.The results showed that nucleosides had a better anti-inflammatory effect,which could reduce inflammatory cells infiltration and the release of inflammatory mediators.Ergosterol showed significant regulatory effects on anti-inflammatory,anti-oxidation,anti-apoptosis,protease balance and immune regulation by inhibiting inflammatory cell infiltration and the release of inflammatory factors,reducing the level of oxide,improving the activity of antioxidant enzymes,inhibiting the expression of apoptosis-related proteins,inhibiting the activity of metalloproteinases and affecting the polarization of macrophages.The mechanism of nucleosides and ergosterol is related to the activation of histone deacetylase and the inhibition of NF-KB/p65.This study provides a basis for the pharmacological study of C.sinensis in the treatment of COPD.Finally,we determined ergosterol as the biomarker of C.sinensis,and explored its pharmacokinetic characteristics in COPD rat model.The results showed that C.sinensis demonstrates a concentration dependent pharmacological role,and when 20%hydroxypropyl-β-cyclodextrin was used as the solvent,AUC0-∞ was increased,and the time to reach Cmax was delayed,which provided a reference for the development of new formulations of C.sinensis and its clinical administration.