| Part one The vasotonic effect of apigenin on isolated rat coronary arteriesObjective:1.To observe inhibitory effect of Apigenin?API?on isolated rat coronary artery?RCA?contraction induced by potassium chloride?KCl?,thrombin analogue?U46619?and vasopressin?VP?,and To observe the vasorelaxation effect of API on KCl,U46619and VP induced contraction of RCA.2.To study the effect of API on RCA by applying inhibitors.Inhibitors were used as follow:L-NAME,Indo,PD98059,Go6983,SB239063,CaCCinh-A01.3.By decalcification-calcium method,to study the relationship between the vasorela-xation of API on RCA and intracellular and extracellular calcium.4.By replacing all chloride ions,to esplore without chloride ions on apigen induced vasorelaxation on RCA.Methods:1.We anesthetize SD rats?adult males,260-290g?by injection of sodium pentobarbital?40mg/kg?.After the severed head,the heart was immediately removed and rapidly placed in a precooled physiological saline solution?PSS?of 4?,O2 saturation,and pH7.40.The coronary arteries were obtuse separated under stereoscopic microscopy,and the isolated coronary arteries were cut into vascular rings about 2mm long.The vascular rings were mounted on wire myograph using two tungsten wires?40?M?.Before the formal experiment,RCA were equilibrated in the bath for 1h and changed every 15min.After repeated contraction with 60mM KCl,if the difference between two consecutive RCA contractions is less than 10%and the contractions are greater than 2mN each time,the vascular ring activity and reaction are considered to be well repetitive.2.The concentration contraction curve was established by using KCl?20-108mM?,U46619?0.01-1?M?,VP?0.1-10?M?,every time when it reaches plateau difference is less than 10%,after 30min incubation of API?10,30,100?M?,re-established the above concentration-response curves,respectively by before preliminary hatch API of the RCA contraction caused by shrinkage agent which the maximum concentration of 100%,Taking maximum contraction caused by vasoconstrictors as 100%.The maximum inhibitory rate and the drug concentration required to inhibit the contraction of 50%was calculated(IC50).3.Adding KCl?60mM?,U46619?1?M?or the VP?1?M?to the chamber.Pre-contraction RCA,to make the final concentrations of API?1,3,10,30,100?M?,establishing the concentration contraction curve.The maximum contraction caused by vasoconstrictors was taken as 100%,calculating the maximal relaxation rate.4.Pre-contraction RCA with 60mM KCl or 1?M U46619.When it come to a platform,the 30?M API was added to relax the RCA.The RCA was rinsed and stabilized for 30 minuties,then established vasoconstriction again.L-NAME,Indo,Go6983,PD98059,SB239063 or CaCCinh-A01 was pre-incubated for 10 minutes,30?M API was added to observe the effects of inhibitors on RCA.The maximun contraction of the KCl or U46619 was calculated as 100%,and calculating the relaxation rate of API in the presence or absence of inhibitors.5.The normal PSS solution for calcium without PSS liquid containing EGTA hatch15 min,the extracellular fluid reach calcium state,replaced by liquid calcium containing no EGTA without PSS hatch 15min,again without replacement for excluding EGTA calcium 60mM KCl or 1?M U46619,reach the vasoconstriction plateau add 2.5mM CaCl2,after being vasoconstriction plateau again,elution blood vessels,as observed before.Add30?M API to preincubate for 15 to 20min,replicate the above operation,and observe the effect of preincubating different concentrations of API on the RCA.6.60mM KCl or 1?M U46619 RCA was added into chamber,when it come to plateau,then adding 30?M API.Pre-incubation with the normal PSS without Cl-for 30mimnuties.In this kind of liquid,NaCl,KCl,MgCl2,CaCl2 were respectively relaced with sodium chloride,potassium chloride,magnesium sulfate and calcium chloride.The 60mM K+or 1?M U46619 was added to stimulate the RCA,then API was added again to observe the effects of without Cl-on RCA.Results:1.The pre-incubation with API?10,30,100?M?can inhibit the contraction effect of KCl?20-108mM?,U46619?0.01-1?M?,VP?0.1-10?M?on RCA.The maximum contractions of RCA on KCl,U46619 and VP were?10.67±3.37?mN,?4.77±1.37?mN and?5.97±3.07?mN,respectively.IC500 was 22.36,20.71 and 14.21?M,respectively.Compared with the control,the differences were all significant?P<0.05?.2.API?1-100?M?concentration-dependently relaxed RCA precontracted with60mM KCl,1?M U46619,1?M VP,the maximum diastolic amplitude was?95.43±3.50?%,?92.49±4.48?%,?95.60±3.61?%?P<0.05?.RC500 value was?19.89±2.15??M,?27.19±1.04??M,?7.043±1.35??M.The control had no significant effect on RCA under the same condition.3.The diastolic percentage of 60mM KCl presystolic RCA was?66.79±3.42?%,and the preincubated inhibitors Indo?0.01mM?and SB239063?3?M?had no significant effect?P>0.05?.Preincubation with inhibitors Go6983?0.1?M?,PD98059?3?M?,CaCCinh-A01?10?M?,L-NAME?0.01mM?,API to 60mM KCl presystolic relaxation ratio were?27.89±5.10?%,?29.41±4.11?%,?19.34±6.10?%,?30.34±2.13?%,respectively,the difference was significant?P<0.05?.The diastolic percentage of API to 1?M U46619presystolic RCA was?80.83±4.11?%,and the preincubation with inhibitors Indo?0.01mM?and SB239063?3?M?had no significant effect?P>0.05?.Preincubation with inhibitors Go6983?0.1?M?,PD98059?3?M?,CaCCinh-A01?10?M?,L-NAME?0.01mM?,API presystolic ratio of 1?M U46619 were?41.13±5.21?%,?39.07±5.15?%,?38.18±3.97?%,?38.58±6.20?%,respectively,with significant differences?P<0.05?.4.In calcium-free PSS,60mM KCl stimulation can cause a small range of RCA contraction,the maximum range of RCA contraction after calcium complex is?4.41±0.06?mN,and the maximum range of RCA contraction after calcium complex is?1.00±0.09?mN after pre-incubating 30?M API,which can inhibit the range of RCA contraction after calcium complex,and the maximum range of RCA contraction after calcium complex is?1.00±0.09?mN,the difference is significant?P<0.05?.In the calcium-free PSS,1?M U46619 prestimulation could hardly cause RCA contraction.After calcium complex,the maximum contraction range of RCA was?3.19±0.11?mN,and after incubating 30?M API,the maximum contraction range of RCA after calcium complex was?0.73±0.11?mN.The difference was significant?P<0.05?.5.In the normal PSS,the maximum relaxation precentage of 60mM KCl or 1?M U46619 pre-contracted RCA was?66.79±3.95?%or?65.20±4.12?%,respectively.The maximum relaxation precentage of 60mM K+or 1?M U46619 pre-contracted RCA was?41.83±5.08?%or?44.10±2.13?%,after pre-incubation with witnout Cl-PSS 30min.Compared with the control,the difference was significant?P<0.05?.Conclusion:1.API?10,30,100?M?can inhibits contractions induced by KCl,U46619 and VP;API relaxed RCA pre-contracted by KCl,U46619 and VP.2.CaCCinh-A01 and Cl-free PSS can weaken the relaxation effect of API on RCA,indicating that the CaCCs may be involved in the mechanism of API on RCA.Both protein kinase C inhibitor Go6983 and extracellular signal-regulated kinase inhibitor PD98059 could weaken the relaxation effect of API on RCA,indicating that protein kinase C and ERK1/2 signaling pathways may participate in the mechanism of API on RCA.L-NAME can weaken the diastolic effect of API on RCA,indicating that the mechanism of API relaxing RCA may be related to NO.3.The relaxant effects of API may be related to calcium antagonism,mainly to inhibit extracellular calcium influx.Part two Effect of apigenin on CaCCs of isolated rat coronary smooth musclesObjective:To study the effect of API on CaCCs current of RCASMCs.Methods:1.RCASMCs isolation:Coronary artery of rats after the separation,quickly go to preheat to 37?,1 mg/ml DTT,1 mg/ml albumin,0.5 mg/ml papain,continuous oxygen,oxygen bubbles shoulds not be too big,thermostatic water bath heating,incubation 25 to28min,to contain 0.3 mg/ml collagenase H,1 mg/ml albumin,0.6 mg/ml of collagenase F,0.7ml liquid cell separation,continue to advance hatch 5-8 min,large for oxygen bubbles.Then add the preheated cell separation solution to 1ml,continue to preincubate for2-4min,add the separation solution at 4?to the top of the test tube,centrifuge the separated cells twice,discard the supernatant,and get fresh RCASMCs.It was stored in the refrigerator at 4?for standby use.2.Using the Whole-cell patch clamp to record the RCASMCs CaCCs current.After we record the stable CaCCs current,10?M CaCCinh-A01 was added.After recording current is CaCCs current,record the normal CaCCs current again,add 30?M API and observe the effect of API on CaCCs current.Results:We record the stable peak current of RCASMCs CaCCs current was?561.55±102.58?pA and the current density was?26.30±1.38?pA/pF,when test potential is+100mV.After adding 10?M CaCCinh-A01,the peak current density could be suppressed,and it was proved that the recorded current was CaCCs current,and the inhibition rate was?56.72±3.75?%?P<0.05?.After adding 30?M API,CaCCs current was inhibited,and the inhibition rate was?66.75±11.20?%,which was significantly different from that of the normal group?P<0.05?.30?M API can suppress RCASMCs CaCCs current.Conclusion:30?M API can suppress RCASMCs CaCCs current. |
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