| â… . Background and Purpose for the ResearchAcute myocardial ischemia (referred to as AMI) is a common manifestation ofcardiovascular disease and has a high clinical incidence rate. In terms of the frequent origin,pathogenesis and clinical manifestation of AMI, it can be classified into the category of"cardialgia" in Chinese medicine. Using the method of syndrome differentiation andtreatment of disease in Traditional Chinese Medicine (TCM), cardialgia can be clinicallyclassified into six types, cardiac blood stasis, internal obstruction of phlegrn and turbidity,accumulation and stagnation of yin-cold, deficiency of both vital energy and yin,deficiencyof heart-yang and kidney-yang and are treated accordingly. Of the six types, three types,cardiac blood stasis, internal obstruction of phlegm and turbidity, accumulation andstagnation of yin-cold are the most frequently seen during periods of cardialgia onset.It can be learned from literature retrieval that there is a lot of reportage on clinicalpractice and experimental research through western clinical treatment in combating AMIand clinical practice and basic research through Traditional Chinese medicine or through acombination of TCM with Western medicine in an attempt to cure AMI. Regarding curingAMI through Traditional Chinese medicine or through a combination of TCM with Westernmedicine, the majority of the study focuses on the following aspects:â‘ The clinicalpractice and experimental research by resorting to a certain TCM compound recipe(including classical recipes or contemporary recipes) in hope of fighting against AMI.â‘¡The study on the mechanism of action by a certain categorical prescription in TCM (such asthe categorical prescription of promoting blood flow and removing stasis, the categorical prescription of Modified Trichosanthes Fruit and Chinese chive Decoction) to treat AMI.â‘¢A study on the treatment and mechanism by other approaches (such as acupuncture) tocure AMI. The research mentioned above mainly studies TCM's curative effect andmechanism in treating AMI from a longitudinal angle. However, discussion of differenttreating principles on TCM cardialgia from a lateral angle, a comparison of curativemechanisms of action and the approaches, and a comparison of the emphasis in differentapproaches and mechanisms of action by the same treating principle and comparativestrong or weak effects of action have not been seen in reportage.Based on the present state of research on treating AMI by a combination of TCM andwestern medicine, this study tries to explore further, through a lateral angle, the mechanismof action and the ways by three different employed treating principles (the method ofpromoting blood flow and removing stasis, the method of aromatic herbs activating yangand the method of activating yang and getting rid of phlegm) to treat AMI. Afterexperimental research, this study observes the effect and mechanism of the correspondingrepresentative prescriptions (Drive out Stasis in the Mansion of Blood Decoction,Trichosanthes Fruit, Chinese chive, and Poinllia Decoction and Styrax Capsule forCoronary Disease) of three treating principles (the method of promoting blood flow andremoving stasis, the method of aromatic herbs activating yang and the method of activatingyang and getting rid of phlegm) to treat periods of cardialgia onset. With a combination oftechnique and method from molecular biology, immunohistochemistry, serumpharmacology, morphology, studies are carried out on the levels of cells and molecules. Theeffect and approach of three frequently employed treating principles to treat AMI areinferred. Similarities and differences in terms of acting approach by using different treatingprinciples and their differences in acting effect are compared. This study hopes to know,from a deeper level, the acting mechanism of TCM syndrome differentiation and treatmentto cure AMI and wants to embody the scientific implication of "heterotherapy forhomopathy ". At the same time, it will lay an operative foundation for the furoredevelopment of compound recipe for AMI in the principle of multiple treating target point.â…¡.Research Methods1,The protective effect of different treating principles during periods of cardialgiaonset on rats' cardiac muscle tissue in time of AMI caused by Pituitrin (Pit).In order to probe the protective effect of three different treating principles duringperiods of cardialgia onset on the cardiac muscle tissue in time of AMI, this study hasselected corresponding representative prescriptions (Drive out Stasis in the Mansion of Blood Decoction, Trichosanthes Fruit, Chinese chive, and Poinllia Decoction and StyraxCapsule for Coronary Disease) of three treating principles (the method of promoting bloodflow and removing stasis, the method of aromatic herbs activating yang and the method ofactivating yang and getting rid of phlegm) and has had them applied to AMI rat modelresulting from Pit, and by observing the effect of these three prescriptions on PitAMI-induced rats' plasma ET, mRNA expression of cardiac muscular tissue eNOs andelectrocardiogram of the rats, and can therefore deduce the mechanism of the three differenton fighting AMI by improving the blood circulation of cardiac muscle tissue during periodsof cadialgia onset.(1) The AMI model of rat building by injection of PitThe-determination of Pit dosage used in model building. Various doses of Pit are injectedinto the caudal veins of the rats and this is recorded. The ECG change of the rats iscarefully observed. If the ST portion of the ECG is elevated farther than 0.1mv, it can beconcluded that this is an ECG of myocardial ischemia.(2) Having an intra-gastric administration to SD rats weighing 180～220g by means ofDrive out Stasis in the Mansion of Blood Decoction, Trichosanthes Fruit, Chinese chive,and Poinllia Decoction and Styrax Capsule for Coronary Disease for 7 consecutive days.Under drugged state, the rats are given Pit injection into the caudal veins, leading to AMIin rats. After that, ECG change of the rats in the next 15s～10min after Pit injection isnoted down. Meanwhile, blood is drawn out from the heart, serum separated, the amount ofblood plasma ET in rats is detected by means of radio-immunity method, the mRNAexpression of eNOs in the cardiac muscular tissue determined through the method ofRT-PCR.2,The influence of different treating principles during periods of cardialgia onset onapoptosis-related gene, bcl-2, and mRNA expression of bax in AMI rats' cardiac musculartissue.After the intra-gastric administration and Pit injection in the above-mentioned manner,the chest is cut open and the heart is taken out. Tissues in the myocardium of apex of heartare selected. Apoptosis-related gene, Bcl-2, mRNA expression of Bax are detected bymeans of RT-PCR method. At the same time, the ratio of bcl-2/bax is calculated.3,The protective effect of different treating principles during periods of cardialgia onseton the hypoxia and oxygen-regaining trauma experienced by isolated cadiocyte in neonateratsBy studying the interfering effect of medicine-containing serum of the correspondingrepresentative prescriptions (Drive out Stasis in the Mansion of Blood Decoction, Trichosanthes Fruit, Chinese chive, and Poinllia Decoction and Styrax Capsule forCoronary Disease) of three treating principles (the method of promoting blood flow andremoving stasis, the method of aromatic herbs activating yang and the method of activatingyang and getting rid of phlegm) during periods of cardialgia onset on post hypoxia andoxygen-regaining trauma in isolated cardiocyte of neonate rats, the cell survival rate, thecell apoptosis rate, cell MDA content, LDH activity, the protective effect of the threedifferent treating principles during periods of cardialgia onset on hypoxia andoxygen-regaining trauma experienced by cardiocytes can be deduced.(1) Cultivation of primary generation rat cardiocyte: Suckling rats of albino rats as old as3 to 5 days belonging to the strain of Wistar are selected.Their chests are cut open, andmyocardiac tissues of apex of heart are taken. After digestion of trypsin liquid (0.06%),the digestive liquid is collected. After centrifuging, the clear supernatant liquid is discarded.After sedimentation, 1640 culture liquid containing calf serum (20%) is added to make cellsuspension. The cariocytes are purified by means of differential speed adherence method.After filtration through 200-hole sterile stainless mesh, they are subcultured in the cultureflask and cultivated in CO2gas incubator (37℃, 5%CO2). Rats' cardicytes cultivated for 5to 7 days are ready for experiments.(2) Establishing modeling for hypoxia and oxygen-regaining of rats' cardiocytes.After pouring out the nutrient medium in which the rats' caridocytes are growing well, it isrinsed with anaerobic cultivation saturated with pure nitrogen gas (99.99%). While puttinganaerobic cultivation into the culture flask, nitrogen gas is filled into the flask for 10seconds in an attempt to displace the air in the flask. The flask is kept corked and put in anenvironment at a temperature of 37℃for 4 hours. This is anaerobic. After that, with asimulation of mixed gas saturation with 95%O2 and 5%CO2 is filled into nutrient medium.In this way, the anaerobic cultivation is displaced. It is kept in the incubator (5%CO2) at atemperature of 37℃for incubation for 2 hours. This is called oxygen regaining. By meansof MTT, the survival rate of cadiocytes before and after hypoxia and oxygen-regaining ismeasured; the MDA content of cadiocytes and LDH content of cellular supernate fluidbefore and after hypoxia and oxygen-regaining are also measured to determine whether themodeling is successful.(3) Preparing the making the medicine-containing serum: A intra-gastric administrationis given to rats with Drive out Stasis in the Mansion of Blood Decoction, TrichosanthesFruit, Chinese chive, and Poinllia Decoction and Styrax Capsule for Coronary Disease for 3consecutive days. 50 minutes after the last intra-gastric administration, blood is drawn fromthe rats' heart, centrifuged and the serum is separated. After deactivation, the serum is put away in the refrigerator at a temperature of—80℃for future use.(4) By means of MTT method, medicinal serum concentration used to interfere withhypoxia and oxygen-regaining of rats' cadiocytes is measured.(5) The rats' cardiocytes in various experimental groups are treated beforehand for 3hours with medicine-containing serum whose concentration has been determined byExperiment (4). After that, it is kept in an anaerobic condition for 4 hours before it issupplied with oxygen again for 2 hours.(6) The MDA content and activity of LDH of cardiocytes in various groups are measuredwith automatic biochemical instruments.(7) Cardiocytes after hypoxia and oxygen-regaining are taken and by means of MTTmethod, the survival rate of cardiocytes in various groups is measured.(8) Through the method of TUNEL, the apoptosis rate of cardioctyes is determined.Cardiocytes after hypoxia and oxygen-regaining in various groups are taken and fixed byparaformaldehyde solution (4%). Next, the blockage of endogenous peroxydase andcellular permeation are performed. After that, TUNEL reactive mixed solution is droppedfor markers. This is put under the fluorescence microscope for an analysis of results andthe cellular apoptosis index is figured out.â…¢. Research Findings1. A model is established of rats' AMI resulting from Pit needed for this research.The measuring of ECG and statistical analysis show that this is a successful modeling. Itis determined that the actual Pit dosage for modeling is 1.5u/kg (injection into the rats'caudal vein) and 30s～10min after injection, typical AMI ECG occurs, manifesting itselfin the elevation of ST portion, arrhythmia, even accompanied with the occurrence of Qwave.2. The corresponding representative prescriptions of the three different treatingprinciples during periods of cardialgia onset have a marked curative effect on rats' ECGsuffering from AMI resulting from Pit. In high doses, the method of promoting bloodflow and removing stasis is the most effective; in low doses, the method of aromaticherbs activating yang gets the best result. The second best is the method of promotingblood flow and removing stasis, and what comes next is the method of activating yangand getting rid of phlegm.3. All the three different treating principles during periods of cardialgia onset canfight against AMI by means of reducing the level of ET in the plasma. Comparativelyspeaking, among the three, there is a difference in the ability to reduce the level of ET in the plasma during AMI. The method of promoting blood flow and removing stasis worksthe best in reducing ET.4. All the three different treating principles during periods of cardialgia onset canexert a protective influence on tissues of myocardium suffering from AMI by promotingthe expression of eNOs gene. But there is a marked difference in the effect among thethree treating principles. It is as follows: In high doses, the method of activating yang andgetting rid of phlegm is the most effective, far better than the others: the method ofpromoting blood flow and removing stasis and the method of aromatic herbs activatingyang. In low doses, the method of aromatic herbs activating yang is the most effective,far better than the others: the method of activating yang and getting rid of phlegm and themethod of promoting blood flow and removing stasis. The method of activating yang andgetting rid of phlegm is far better than the method of promoting blood flow and removingstasis.5. All the three different treating principles during periods of cardialgia onset canexert a protective influence on the tissues of myocardium by regulating up the ratio of bcl—2/bax, therefore regulating up mRNA expression of the apoptosis-suppressing bcl-2gene, or/and regulating down mRNA expression of apoptosis-promoting bax, thereforereducing the apoptosis of cardiocytes. There is a wide difference among the three treatingprinciples concerning the effect of bcl-2/bax ratio up-regulation. The manifestation is: themethod of aromatic herbs activating yang is the most effective, the second best is themethod of activating yang and getting rid of phlegm and what comes next is the method ofpromoting blood flow and removing stasis. At the same time, for those rats in the groupsgiven the representative medicine according to the method of activating yang and gettingrid of phlegm and the method of aromatic herbs activating yang, the bcl—2/bax ratio ofthe tissues of myocardium is markedly higher than the control groups.6. Cardiocytes in neonate rats, if suffering from hypoxia for 4 hours and then givenoxygen for 2 hours, will experience obvious trauma in cardiocytes. It can observed that thebeat frequency and amplitude become markedly lower, and the survival rate of cardiocytesis dramatically reduced. The MDA content and LDH activity show a significant rise,displaying a successful modeling.7. The medicine-containing serum of the corresponding representative prescriptionsaccording to the three treating principles during periods of cardialgia onset can interferewith the hypoxia and oxygen-regaining of rats' cardiocytes. All of them can markedlyreduce the MDA content in the rats' cardiocytes resulting from hypoxia andoxwen-regaining trauma, showing all the three treating principles have the function of anti-oxidation and removing oxygen free radicals. There is a wide difference among thethree treating principles concerning the effect. The method of aromatic herbs activatingyang is the most effective, better than the others: the method of activating yang and gettingrid of phlegm and the method of promoting blood flow and removing stasis.8. The medicine-containing serum of the representative prescriptions according to themethod of aromatic herbs activating yang and the method of activating yang and getting ridof phlegm can markedly reduce LDH activity in rats' cardiocytes suffering from hypoxiaand oxygen-regaining trauma, but the medicine-containing serum of the representativeprescriptions according to the method of promoting blood flow and removing stasis exerts aless influence on the LDH activity than the medicine-containing serum of the representativeprescriptions according to the method of aromatic herbs activating yang and the method ofactivating yang and getting rid of phlegm. The effect is also less effective. This means thatconcerning the protection effect on cardiocyte hypoxia and oxygen-regaining trauma, themethod of aromatic herbs activating yang is the most effective. The method of activatingyang and getting rid of phlegm is the second best and the method of promoting blood flowand removing stasis is the third best choice.9. The medicine-containing serum of the representative prescriptions according tothe three treating principles can dramatically reduce the cardiocyte apoptosis index of dueto hypoxia and oxygen-regaining. Among the three, the medicine-containing serum of therepresentative prescriptions according to the method of aromatic herbs activating yang isthe most effective. The second best is the medicine-containing serum of the representativeprescriptions according to the method of activating yang and getting rid of phlegm. Thethird best choice is the medicine-containing serum of the representative prescriptionsaccording to the method of promoting blood flow and removing stasis. It follows that all thethree frequently employed treating principles during periods of cardialgia onset can exert aprotective influence on cardiocytes suffering from hypoxia and oxygen-regaining traumathrough fighting against cardiocyte apoptosis. Of the three treating principles, the methodof aromatic herbs activating yang is the most effective. The second best is the method ofactivating yang and getting rid ofphlegrn and the third best one is the method of promotingblood flow and removing stasis.10. The medicine-containing serum of the representative prescriptions according tothe three treating principles can dramatically improve the survival rate of cardiocytesuffering from hypoxia and oxygen-regaining trauma. The effect of themedicine-containing serum of the representative prescriptions according to the method ofaromatic herbs activating yang and the method of activating yang and getting rid of phlegm is far better than the medicine-containing serum of the representativeprescriptions according to the method of promoting blood flow and removing stasis.â…£. Conclusions1. All the three different treating principles during periods of cardialgia onset, themethod of promoting blood flow and removing stasis, the method of aromatic herbsactivating yang and the method of activating yang and getting rid of phlegm can, throughvarious ways,â‘ Regulate the secretion of angiokinesis factor and reduce the synthesis andsecretion of ET, a blood vessel-constricting factor, fight against coronarospasm and maintaincirculation volume in the coronary artery.â‘¡Regulate the expression of cardiocyteapoptosis-related genes.â‘¢Suppress the generation of oxygen free radicals or/ andstrengthen the elimination of oxygen free radicals.â‘£Improve the survival ability (survivalrate) of cardiocytes in hypoxia and oxygen-regaining trauma.⑤Exert the influence offighting against AMI by suppressing the cardiocyte apoptosis during hypoxia andoxygen-regaining trauma.2. The various functions and ways used to fight against AMI in the three differenttreating principles during periods of cardialgia onset are mutually interrelated andcoordinated. The manifestations areâ‘ . In the aspect of protecting myocardium, on onehand, it can fight against arteriospasm, maintain circulation volume in the coronary arteryand ensure the blood supply in the myocardium by reducing the synthesis and secretion ofET, a blood vessel-constricting factorl on the other hand, it can improve the activity ofeNOS, promote the synthesis of endogenous eNO and maintain the myocardial function ofrelaxation and contraction by regulating the mRNA expression of eNOS in the myocardium,The synergistic effect of the two can protect the function of the myocardium.â‘¡In theaspect of fighting against cardiocyte apoptosis, the first part is to regulate the ratio ofcardiocyte apoptosis-related genes. On one hand, the expression of apoptosis-suppressinggene, bcl-2, is regulated up. On the other hand, the expression of apoptosis-promoting gene,bax, is regulated down. By way of suppressing the formation of free radicals or/ andstrengthening the elimination of oxygen free radicals, the synergistic action of the two cansuppress cardiocyte apoptosis, improve the survival rate of cells, thus exerting a protectiveinfluence on cardiocyte hypoxia and oxygen-regaining trauma.3. Of the three different treating principles against AMI during periods of cardialgiaonset, different approaches will produce radically different effects. The manifestations are:â‘ The focus of the method of promoting blood flow and removing stasis is to fight againstAMI by reducing ET. Its effect is markedly better than the method of activating yang and getting rid of phlegm and the method of aromatic herbs activating yang.â‘¡The emphasisof the method of activating yang and getting rid of phlegm and the method of aromaticherbs activating yang is to suppress the formation of oxygen free radicals or/and strengthenthe elimination of oxygen free radicals; promote the mRNA expression of eNOS, increasethe synthesis of NO, a blood vessel stretching substance; fight against the cardiocyteapoptosis due to hypoxia and oxygen-regaining trauma. Through these approaches, thecardiocytes are protected, and hypoxia and oxygen-regaining trauma is avoided. For theseapproaches, these two methods are far better than the method of promoting blood flow andremoving stasis.4. Of the these different employed treating principles against AMI during periods ofcardioalgia onset, the method of aromatic herbs activating yang and the method ofactivating yang and getting rid of phlegm are, in various ways, far better than the method ofpromoting blood flow and removing stasis. This is especially true of the method ofaromatic herbs activating yang. Its effect is the most noticeable concerning the function andeffect in fighting against AMI by various approaches. This is in accordance with the clinicalpractice. When there is the attack of coronary heart disease and cardiogmus, the first choicein emergency medical treatment is usually the method of aromatic herbs activating yang(medication). |
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