Anti-asthma Compounds In Traditional Chinese Medicines-Screening And Mechanism Study

Asthma is an airway disease that affects about 300 million people worldwide,a total that is expected to rise to about 400 million over the next 15-20 years.Asthma is a chronic airway inflammatory disease,which requires long-term medications or lifelong treatments for therapy.It is difficult to prognosticate the curative effects of the treatment.In addition,the limited knowledge of drug screening models makes it difficult to develop new medicines for asthma as well.In Traditional Chinese Medicine(TCM) there are hundreds of different kinds of herbs used for the treatment of asthma.The empirical observations accumulated for over 2000 years by the clinical practices of generations of TCM physicians are valuable.Many herbal medicines are still used to treat patients'illnesses and improve their quality of life.However, empirical records and explanation are not enough.TCM needs to be evaluated by the general methods of basic biomedical sciences and by drug development models in conventional medicine.Modern medicine requires that the mechanisms of the herbs be explained at the molecular or cellular level,which can direct the clinical use and development of natural compounds.β₂-adrenergic receptor(β₂-AR) agonists have been widely used as bronchodilator treatment in asthma for decades.The agonists produce effective bronchodilatation through airway smooth muscle relaxation and reduce exacerbations.In this study,by transfectingβ₂-AR gene into a CHO cell line, which has cyclic adenosine monophosphate response element-directed reporter gene,a cell-basedβ₂-AR agonist functional screening system was established. This system is consist of CHOβ₂-AR-CRE-GFP test cell line and CHOCRE-GFP control cell line and can used for high throughput screening ofβ₂-AR agonists from compound libraries.Here the screening system was applied to the primary screening of 123 Traditional Chinese herbs.As a result,there were 25 positive herb extracts withβ₂-AR agonist activity. In this study,we took fuzi(Radix Aconiti Lateralis Preparata) as an example.With the cell-basedβ₂-AR agonists functional screening system combined with HPLC-ESI-MS,theβ₂-AR agonist in fuzi was purified and identified to be higenamine.It could dose-dependently relax the isolated guinea pig trachea strip precontraction with acetylcholine(p＜0.01).Further in vivo studies also displayed that hignamine could protect experimental asthma model induced by histamine in guinea pigs to prolong the latent periods of asthma (p＜0.01).The combination ofβ₂-AR agonists and glueoeortieoids is more efficacious in asthma and COPD than either alone or other combination therapies.It is evidenced in this study that glycyrrhizin(GL),which is the most abundant constituent in dried Glycyrrhiza glabra roots and has a glueocorticoid-like action,was capable of increasing theβ₂-AR mRNA level and the efficiency ofβ₂-AR agonists.The interaction amongβ₂-AR agonist and GL resulted in complementary anti-inflammatory effects in airway cells.Data reported here included the combined additive effect ofβ₂-AR agonist and GL on TNF-αinduced NF-κB activation(p＜0.01).Inhibition of I-κBαdegradation and IL-8 production was significantly greater when the two drugs were combined. GL could enhance the relaxing effect ofβ₂-AR agonists on isolated guinea pig tracheal strips In vitro.In vivo studies of the combination also showed a synergistic anti-asthma effect.Combined treatment was significantly more effective thanβ₂-AR agonist alone in the inhibition of atomized histamine induced asphyxial convulsions.Considering the vast applications of licorice and its therapeutic potential,results reported here demonstrated the important pharmacology of GL in combination with aβ₂-AR agonist,offering the possibility of therapeutic application in the treatment of asthma.A cell line named HEK293-β₂-AR,which overexpressedβ₂-AR,was established by transfecting pcDNA3.1/β₂-AR into HEK293 cells.The AnnexinV/PI staining result showed that salbutamol could induce the apoptosis of HEK293-β₂-AR cells(p＜0.01).GL could inhibit the cell apoptosis induced by salbutamol in early satge(p＜0.01).Nevertheless,GL had no effect on the late apoptotic cells.In the MTT assay,GL dose-depently inhibited the cell death induced by salbutamol.GL also protected HEK293-β₂-AR cells from the nucleu aberration,which was induced by salbutamol and proved by DAPI staining of the nucleu.Further investigation of the apoptosis genes by realtime-PCR showed that GL could regulate the ratio of Bcl-2/Bax mRNA and maintain it at a normal level so as to inhibite the apoptosis induced by salbutamol.These results may give a clue to further the study about molecular mechanizm of the GL andβ₂-AR agonist combined therapy.The cell-basedβ₂-AR agonist functional screening system in this study was used to screening leadβ₂-AR agonists in TCM natral compound library.The bioactive compound evaluation system,which consists of extraction, purification,identification and pharmcological analysis processes,can provide an approach to the intensive study of natralβ₂-AR agonists at molecular,cellular, organic and in vivo levels.To a certain extent,this system may help the explanation of the mechanism in TCM.And it may provide more opportunities to develop new ideas for the modernization of TCM.