Based On Molecular Docking Technology, The Mechanism Of Chicory Inhibiting Sputum Absorption Was Discussed

BackgroundThis research was part of content of Beijing Municipal Natural Science Foundation(No.7162117),which was supported by Beijing University of Traditional Chinese Medicine(2013-JYB22-XS-086).High purine diet has long been recognized as a risk factor for hyperuricemia.The intake of high purine food diet is closely associated with the prevalence and development of hyperuricemia.CNT2 plays a critical role in purine nucleoside absorption in intestinal,which is a central carrier responsible for the absorption of purine nucleosides.CNT2 is regarded as a potential drug target to treat the hyperuricemia,for inhibition of CNT2 activity would suppress the increase in serum uric levels derived from dietary purines.Traditional Chinese medicine can be safe and effective to prevent and treat the hyperuricemia in many channels and multiple targets.It still needs to investigate that Whether Traditional Chinese medicine reduce the level of uric acid by intervening the CNT2 activities in intestinal or not.Therefore,it has definite academic value and clinical significance to explore the mechanism of Traditional Chinese medicine to reduce the level of uric acid by intervening the CNT2 activities in intestinal and provide the basis for the prevention and treatment to the hyperuricemia.Based on the previous study,chicory has outstanding effect on lowering uric acid by reducing the generation and increasing the excretion of uric acid.The former research brilliantly expounds the mechanisms of chicory on controlling hyperuricemia from different points.The research will focus on the CNT2 in in intestinal to clarify the mechanism of chicory decrease uric acid level through reducing the absorption of purine nucleoside.The homology modeling and molecular docking technology was employed to virtual screen the chemical composition in chicory which combined with CNT2;and the experiment was used to inspect the effect of the targeted chemical composition in chicory inhibit the CNT2 activity.Objective1.Virtual screening the chemical composition in chicory combined with CNT2 intestinal,in order to provide theory basis for exploring the mechanism of chicory drop uric acid by intervention CNT2 activity in intestinal2.Validate the inhibit effective of the targeted chemical composition in chicory to the intestinal CNT2,in order to clarify the mechanism of chicory reduce uric acid level by decreasing the purine absorption and provide reference for the CNT2 as a novel target to prevent and treat the hyperuricemia in traditional Chinese medicine.Method and ContentThis study was focus on that based on the molecular docking to explore the mechanism of chicory reduce the level of uric acid by inhibit the purine nucleoside absorption from literature and experiential research.(?)Literature reviewIn the literature review part,we mainly discusses the relationship between the CNT 2 in intestine and hyperuricemia,the application of the homology modeling in the traditional Chinese medicine research and the employment of the cell model in drug transporter research.(?)Experiential researchChapter One Virtual screening of the chemical composition in chicory combining CNT21.Based on the chemical composition database,the homology model of hCNT2 was produced using a Vibrio cholera model as template,and then,to validate the model,docking studies were performed with its known inhibitor so that provide the three dimensional structure for virtual screening the composition in chicory.2.Molecular docking was employed to screen the small molecular compounds in chicory combined with intestinal CNT2,and analyzes the patterns between of them so that provide theory basis for exploring the mechanism of chicory drop uric acid by intervention intestinalCNT2 activity.Chapter Two Experimental research in the inhibit effect of the targeted chichory compounds on the intestinal CNT2 activity.1.IEC-6 cell model was established and evaluated by observation of the cell morphology,epithelial resistance,fluorescein sodium through rate in the laboratory conditions2.The inhibitory effect of the representative targeted chicory components on CNT2 activity,which is high in content and better in specificity,was explored.The content of adenosine,CNT2 substrate,in the basal layer of IEC-6 cell model was detected in HPLC.3.Quail everted intestinal sacs model was set up and the quail intestinal sac activities,incubating time,the appropriate intestine segment and the best concentration of substrate were investigated to provide a reliable model.Result1.The homologous modeling hCNT2 structure is reasonable and reliable,which could be able to recognize the inhibitor in a certain range of different activities.2.Using the hCNT2 homologous model as the receptor,the small molecule compound in chicory as the ligand,21 points more than the positive drug(scoring threshold-8.0)of the chicory compounds were screening out.The hydrogen bonding and hydrophobic interaction were predominant between the top ten compounds with hCNT2 model.3.A specific HPLC method was developed for analysis of the adenosine in the culture medium.The theophylline was internal reference and the mobile phase methanol and water were eluted in gradient.The specificity of the method was good at 0.7875-2.1?g/ml in linear range(R2 = 0.9936);the recovery rate was 80%above,the RSD of precision,repeatability and stability were less than 10%.4.The IEC-6 cell model was established.The effects of cichoric acid and Lactucopicin on the activity of CNT2 were observed by using the IEC-6 cell model.Cichoric acid could significantly inhibit the activity of CNT2 transferring adenosine(P<0.05).Conclusion1.The chicory contains the chemical compositions combined with intestinal CNT2.The intestinal CNT2 may be one of the targets for the chicory to reduce the level of uric acid.2.The chicory contains the chemical compositions which could inhibit the activity of CNT2.Reducing the absorption of purine in intestine by inhibiting the activity of CNT2 may be one of the mechanisms of chicory to reduce the level of uric acid.