Based On The Uniform Design Of The Four Components Of Ephedra And Other Effective Components Of The Chinese Traditional Medicine To Affect The Optimal Compatibility Of Vascular Activity

Objective:To study the effect of pseudoephedrine hydrochloride in ephedra,schisandrol in schisandrin,ferulic acid in angelica,tetramethylpyrazine in Chuanxiong and their combination on the tension of isolated rat thoracic aorta rings.The optimal combination of the four components was optimized,and the possible mechanism of its action was explored,and the correlation between composition and vasoactive activity was explored.Methods:(1)Study on single component effects:The isolated thoracic aortic rings were prepared using SPF grade male Wistar rats weighing(300±20)g.Vascular ring tension changes were recorded by a multi-channel physiological recording instrument.The vasoconstriction model was constructed by KCl-pretreat method.The effects of concentration gradients of pseudoephedrine hydrochloride,schizandrol A,ferulic acid,and tetramethylpyrazine on vascular tone were observed by cumulative dosing method,and the dose-effect curves were recorded.(2)Study on the effect of compatibility combination and optimization:Using the U12*(1210)table to design 4-level and 12-factor experiments.The vasodilation rate was used as an index to obtain the relationship between 12 combinations of 4 components and vasoactive activity.Using stepwise regression method to fit the equations of relaxation rate(Y)with tetramethylpyrazine(X1),ferulic acid(X2),schisandrin(X3),and pseudoephedrine hydrochloride(X4),and using mathematical software to optimize the combination of the 4 components and to predicte the optimal solution.(3)Pharmacodynamic verification of the best combination:The endothelium-intact vascular rings and the endothelium-denuded vascular rings were prepared at the same time.After the removal of the endothelium was detected,the vasoconstriction model was constructed using KCl pretreatment.The effect of the best combination on the tension of the two types of blood vessel rings was observed,and the efficacy was compared with the above predicted values.At the end of the validation experiment,the endothelium-intact vascular rings and KCl-model vascular rings were removed from the constant temperature bath and fixed with 4%paraformaldehyde together with blank vascular rings to make paraffin sections.After HE staining,the morphological structure of vascular ring tissue was observed under a biological microscope.(4)Study on endothelium-dependent relaxation:The endothelium-intact vascular rings were incubated separately by non-selective nitric oxide synthase(NOS)inhibitor L-Arg(NO2)-Ome·HCl(L-NAME,3×10-4 mol·L-1),endothelium-derived NOS inhibitor N?-Nitro-L-arginine(L-NNA,3×10-4 mol·L-1)and cyclooxygenase(COX)inhibitor indometacin(INDO,1×10-5 mol·L-1)for 15 minutes to observe the effect of each inhibitor on the optimal combination of diastolic phenylephrine(PE,1×10-6 mol·L-1)precontracted vascular rings.(5)Study on non-endothelium-dependent relaxation:The calcium-containing Krebs-Henseleit(KH)solution was replaced with the calcium-free KH solution,and the effect of the best combination on the contraction of blood vessels of PE(1 × 10-6 mol·L-1)and CaCl2(0.5,1,1.5,2,2.5,3 mmol·L-1)was observed.Results:(1)Study on single component effects:In vitro,pseudoephedrine hydrochloride,schizandrol A,ferulic acid and tetramethylpyrazine all had relaxation effect on vascular rings stimulated with KCl(6×10-2 mol·L-1)and it Was dose-dependent within a certain range.Among them,Schizandrin A had the strongest relaxation effect,tetramethylpyrazine was the second,ferulic acid was the third,and pseudoephedrine Hydrochloride was the weakest.(2)Pharmacodynamic verification of the best combination:The 12 combinations of the four components obtained through uniform design have relaxation effects on the pre-contracted vascular rings of KCl(6×10-2 mol·L-1).Mathematical modeling was performed using stepwise regression method.Tetramethylpyrazine(X1)was eliminated,while ferulic acid(X2),schizandrin A(X3),and pseudoephedrine hydrochloride(X4)entered the model.The multiple linear regression equation can be expressed as:Y=28.1188+0.1124 X2+0.1834 X3+0.0736 X4.Through optimization,the best combination was predicted to be X2=104 mol·L-1,X3=130 mol·L-1,X4=97.5 mol·L-1,and the corresponding relaxation rate(Y)was predicted to be 70.8%.(3)Verification of the best combination of combinations:The best combination has a significant relaxation effect(78.16%±8.89%)on the contraction of the intact vascular rings caused by KCl(6×10-2 mol·L-1).It was close to the predicted value of 70.8%.After the removal of endothelium,the diastolic effect was significantly decreased(46.75%± 14.07%).Compared with the intact endothelium group,there was a significant difference(P<0.01).After observing the pathological slices of HE staining in each group,it was found that in the blank group,the inner vascular rings were uniform and flat,the surface was smooth,the endothelial cells were intact and neatly arranged,and there was no local defect;the smooth muscle cells of the media were thick and long spindle-shaped;the outer membrane was intact.In the KCl model group,the intima was rough.The endothelial cells protuberant,and they partially fell off and arranged irregularly.The tunica media thickens and curls and the smooth muscle cell morphology is irregular.The connecting space increases,and the arrangement is disordered;the outer membrane partly falls off.In the administration group,the inner membrane surface was smooth,and the endothelial cells were arranged neatly and partially;the medial membrane was slightly curled and thickened compared with the KCl model group.The smooth muscle cells arranged more neatly,and the connection gaps increased.Compared with the KCl model group,the improvement was obvious;the outer membrane partly falls off.(4)Study on endothelium-dependent relaxation:After the addition of NOS inhibitor L-NAME or L-NNA in advance,the optimal combination of vasodilatory effects was significantly inhibited,and there was a significant difference compared with the control group without inhibitor pretreatment(P<0.01).After incubation with COX inhibitor INDO,the diastolic effect of the best combination did not change significantly compared with the control group(P>0.05).(5)Study on non-endothelium-dependent relaxation:In the calcium-free K-H solution,the vasoconstriction rate caused by PE(1 ×10-6 mol·L-1)was not significantly different from that in the control group after incubation with the optimal combination(P>0.05).After adding CaCl2(0.5,1,1.5,2,2.5,3 mmol·L-1),the vasoconstriction rate increased significantly in a dose-dependent manner.After incubation with the best combination,the contractile effect caused by CaCl2 was significantly inhibited,and there was a significant difference compared with the control group(P<0.01).Conclusion:This study shows that in vitro,pseudoephedrine hydrochloride,schizandrol A,ferulic acid,and tetramethylpyrazine have different degrees of vasodilating effects on rat thoracic aortic rings.Uniform design and stepwise regression were used to optimize the combination of pseudoephedrine hydrochloride,schizandrol A,ferulic acid and tetramethylpyrazine.The best combination was obtained.The best combination has partial endothelium-dependent effects on the vasodilation of isolated rat thoracic aorta.In addition,the degree of intimal smoothness of the rat vascular rings and the irregular pathological states of endothelial cells and smooth muscle cells were improved.The diastolic effect is the result of multiple interactions.This study shows that there are two possible mechanisms.One way is through the endothelium-dependent NO-cGMP pathway,affecting the release of NO on the endothelial cells.The other way is through the inhibition of receptor operated calcium channel(ROCC)and voltage dependent calcium channel(VDCC)on vascular smooth muscle,which leads to the decrease of Ca2+ influx and reduction of the concentration of intracellular Ca2+.The in-depth mechanism needs further study.