| Objective:Through affinity "fishing",protein identification and affinity analysis,we found that the creatine kinase(CK-MM)is the target of Ginsenoside in skeletal muscle tissue,and the target has no interaction with the prototype and Panax ginseng saponins,saponins and glycosides have interaction,which with the original Panaxdiol(PPD)was the strongest.On this basis,the further experiments using a variety of affinity detection method to confirm the interaction between PPD and CK-MM,and based on the target of anti fatigue physical interpretation of ginsenoside pharmacodynamicsMethods:Experiment 1:Determination of protopanaxadiol and creatine kinase affinity constantThe use of biological film interferometry(BLI),micro thermal swimming technique(MST)and isothermal titration calorimetry(ITC)between PPD and CK-MM of all kinds of precise determination of affinity constants,including affinity(KD)with the dose ratio(n),enthalpy change((?)H),entropy((?)S).Determine the relationship between the two.Experiment 2:molecular docking protopanoxadiol and creatine kinaseMolecular docking software based on PPD and CK,allowing free conformation change,calculate the docking results of PPD and CK binding sites and binding mode for the interaction between the two data provide further evidence.Experiment 3:the influence of protopanaxadiol on creatine kinase activity in vitroThree different strains of CK-MM from:CK-MM,rabbit CK-MM and mouse skeletal muscle homogenate,which mixed with different concentrations of PPD,analyze the effect of PPD on the activity of CK-MM.Experiment 4:the influence of protopanaxadiol in vivo on creatine kinase activityIntragastric administration of PPD mice after taking double hindlimb skeletal muscle,determination of skeletal muscle homogenate in CK-MM activity by Creatine Kinase Kit,by Western blotting(Wester-Blot,WB)on the expression of CK-MM and Q-PCR method,and using high performance liquid chromatography(HPLC)determination of content of creatine in the organization,to explore the produced biological biological effects when PPD combined with CK-MM.Experiment 5:the influence of protopanaxadiol on energy metabolism in miceThe normal mice treated with PPD,compared with mice swimming before and after skeletal muscle tissue adenosine three phosphate(ATP),two AMP(ADP),adenine ribonucleotide(AMP),creatine phosphate and lactic acid content,analysis of the generation of protopanaxadiol will help delay the lactic.Experiment 6:the influence of protopanaxadiol on mice swimming and hypotonic hypoxiaThrough the experiment of mice and asphyxia swimming test,verify whether PPD has effect of preventing the pathophysiological process involving energy metabolism.Results:Experiment 1:Show that the BLI detection between PPD and CK-MM affinity(KD)value of 2.53 X 10-5M;ITC detection of the binding constant(K)was 5.38±0.924×105 M-1 reaction(N)combined ratio of 1.05±0.0212 Sites,changes of enthalpy(△H)-3494±95.70cal/mol,entropy the change of(△S)14.5cal/mol/deg;MST technology measure combination Kd was 1.34±0.179 ×10-7M.Experiment 2:The FTMap analysis showed that PPD and creatine kinase has three potential binding sites,except the ADP locus(S1,S1 ’and S1"),there are neighboring sites(S2,S2’ and S2")and two dimer interactions;using Glide module Schrodinger software platform for the simulation of protopanaxadiol this and several potential binding sites for molecular docking,found protopanaxidiol tend to combined with the S2/S2 ’sites.Protopanaxidiol by hydroxyl,carboxyl and His191,Glu231,Glu232(S2)or the formation of hydrogen bonds with Arg320,Gln318,Ser285(S2")hydrogen bond formation.Experiments 3:Whether the mouse,human or rabbit derived CK-MM in vitro,PPD can increase the enzyme activity,so the maximum increase of about 10%.However,between PPD concentration and CK-MM enzyme activity is not a linear relationship,but the relationship between the inverted "U";as the concentration of PPD in vitro enzyme gradually increased live gradually increased,but the concentration of PPD increased,the enzyme activity began to declineExperiments 4:PPD gavage has no obvious effect to the expression of CK-MM in skeletal muscle tissues and mRNA,but the high dose of PPD can improve the CK-MM muscle activity,increased by about 5%;at the same time,PPD dose dependently increased the content of creatine in skeletal muscle tissue,about the maximum increase about 10%.Experiment 5:For the non treatment group mice,compared with before swimming,the content of lactic acid in skeletal muscle after swimming in significantly increased,about 48%;for the treatment group mice,compared with before swimming,the content of lactic acid in skeletal muscle after swimming in about 18%.Compared to the mice that did not give the drug,the group of mice had higher levels of creatine and energy storage in the skeletal muscle tissue before swimming.After swimming,the ratio of energy storage and ATP/ADP in mice that did not swim in the group was lower than those for the drug.Experiment 6:Compared with the control group,PPD could significantly prolong the swimming time in weight and the time of death caused by asphyxia.In the experiment,the survival time of mice in low,middle and high dose group increased by 7.6%,12.9%and 15.3%;the swimming test in weight,the swimming time of mice in the middle and high dose group respectively increased 37.3%and 42.5%.Conclusion:1.CK-MM is the target of Ginsenoside in skeletal muscle tissue,PPD is one of the good metabolic products of the Ginsenoside in vivo when combined with the target.2.After PPD combined with CK-MM,it can improve the enzyme’s activity of CK-MM.When the body is relatively static,CK-MM activity increased,it can increase the content of phosphocreatine in tissue,enhance the body’s energy reserve;when the body is in a state of energy consumption,the improved energy reserves can provide extra energy support to body,and delay the formation of lactic acid in body,the pathophysiology of hypoxia and stimulated by high energy consumption will be extended as well. |
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