| Objective:More and more scholars were paying attention to the pathogenesis of social psychological stress factors under the bio-psycho-social medical model.Based on the background,guided by collateral desease theory of Traditional Chinese Medicine(TCM),and through the clinical epidemiological survey on patients of Angina Pectoris of Coronary Heart Disease(APCHD),our study was to extract essential elements of syndromes using complicated system analysis method;to establish the standard about TCM differentiation of syndroms which combined qualitation with quantitation;to reveal the characters of pathogenesis according to the combining and distributing rules about syndromes;to establish the rat model of atherosclerosis(As)with collateral-qi deficiency according to standard of differentiation of syndroms,and evaluate objectively the concordance of syndromes of model animals with standard of differentiation of syndroms;to research the metabolic characters in model rats;to study the change rules of NEI network correlation factors and the relationship with As in model rats;to observe signaling pathway mechanisms of angiotensinⅡ(AngⅡ),which was the main factor of NEI network change caused by syndrome factor of collateral-qi deficiency,and which promoted vascular smooth muscle cells (VSMCs)proliferation;to evaluate Tongxinluo's effect on rat model of As with collateral-qi deficiency and on VSMCs proliferation promoted by AngⅡ. We also explored the influence of syndrome factor of collateral-qi deficiency to As according to the results from clinic patients and animals combined with cells,and illuminated initially the pathophysiologic basis on the rat model of As with collateral-qi deficiency,provided experiment evidences to support the research of the mechanisms about effect of social psychological factors on As. Meanwhile,we testified the scientific value of collateral desease theory.Methods:4 parts included:1 The study of quantitive standard for differentiation of syndrome to APCHD1.1 The clinical epidemiological survey in patients of APCHDBased on the study of literature and the consultants to experts,we bolted and classified the four physical examinations' information of TCM,instituted the clinical questionnaire,selected eight of GradeⅢ-A general hospitals in Beijing,Shanghai,Guangzhou,Ji'nan,Shenyang and Changchun as our investigating centers,carried out large sample clinical epidemiological survey in patients of APCHD from multicentres.During the investigation,we carried out strict quality control following principle of clinical epidemiology to ensure the reality and reliability of the data.1.2 The establishment of quantitive standard for differentiation of syndromes to APCHDThe study enrolled 403 subjects who visited during our investigation.They were suitable to the standard of APCHD.The 403 subjects were divided randomly into two groups by 3:1.One group,302 subjects,as the calculating group was to establish quantitive standard of differentiation of syndrome. Another group,101 subjects,as the examining group was to test prospectively diagnostic code of syndromes' quantization.The data of 302 subjects were filled in Matlab software,syndromes information was exercted,the contribution of symptoms to syndromes was calculated by entropy-based complex systems partition method(ECSPM).The thresholds on the differentiation of syndromes were set up by the analysis of receiver operator characteristic curve(ROC).The quantitive standards of differentiation of syndrome to APCHD was established successfully when those results of above research were obtained.Then the differentiation of tandards of syndromes prospectively was tested in another group.1.3 The research of combining and distributing rules of syndromes in APCHD.We analysed correlative grade of syndromes by ECSPM,and calculated the proportion of syndromes in 403 cases patients based on the established differentiation standard of syndrome,and then analysed the characters of pathogenesis according to the combing and distributing rules of the syndromes in patients.2 The establishment and evaluation of the rat model of As with collateral-qi deficiencyThe differentiation standard of syndromes in collateral-qi deficiency was established based on clinical epidemiological survey.According to the TCM theory of "excess fatigue consuming qi",basic diet plus loading swimming was applied to induce the syndromes of collateral-qi deficiency.Rats were given VitaminD3,high-methionine and high-grease diet to induce As model. And then the rat model of As with collateral-qi deficiency was induced by the two above factors' superposition.One hundred and five Wistar rats were divided into 7 groups randomly:①Control group.②As group.③Collateral-qi deficiency group(loading swimming group).④As with collateral-qi deficiency(complex model group) group.⑤Large dose Tongxinluo group.⑥Small dose Tongxinluo group.⑦Simvastatin group.After the establishment of rat models,general condition,score of biology superficial syndrome,climbing-time,open-field experiment,and electrophysiology were detected to evaluate objectively the major syndromes' information(debilitation,short breath,fatigue,palpitation)in model rats with collateral-qi deficiency.At the same time,the HE staining,Masson staining,electron microscope, immunohistochemical staining of arota were carried out.Total cholesterol(TC), low density lipoprotein(LDL)and homocysteine(HCY)were detected.The changes of metabolin were analyzed by metabonomics.All those indexes were to evaluate the degree of As.3 The NEI network changes of the As model rats with collateral-qi deficiency and the intervening effect of HerbsThe collateral-qi deficiency model,As rats model and complex model had been instituted as the study objects to investigate the change rules of indexes related to NEI network and the influence of the indexes to As.The dividing of rat groups and the drugs observed were as above.At the finished time point of the model,the rats were anesthetized and blood preparation was collected through arteria carotis cannula,plasma or blood serum was separated.The indexes were detected as following:Hypothalamo-pituitary-adrenal axis(HPAA):corticotropin releasing hormone(CRH)of hypothalamus,adrenocorticotrop(h)ic hormone(ACTH)in plasma,corticosterone(CORT)in serum were detected by Radio-immunity Approach.Norepinephrine and epinephrine in serum was detected by ELISA Approach.Renin-angiotensin-aldosterone system(RAAS):plasma renin activity(PRA),aldosterone(ALD)in plasma,AngⅡin aorta and plasma were detected by Radio-immunity Approach.Cytokine:interleukin(IL)-6 in serum was detected by Radio-immunity Approach,and interferon(IFN)-γin serum was detected by ELISA Approach.Paraffin sections of aorta pectoralis was used to detect the expression of proliferating cell nuclear antigen(PCNA)by Envision immunohistochemistry kit.4 The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on itAngⅡwas selected,which was the main effect factor of NEI network derangement induced by the intervention of collateral-qi deficiency.It was studied by us that the signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it.The research could also provide the experimental evidence of the mechanism of the syndrome factor influncing vascular lesion.The primary culture VSMCs of rat by the tissue sticking mass method was used for the study.The proliferation capability of VSMCs was detected using MTT method.The cell cycle of VSMCs was detected using flow cytometry.The nuclear translocation of signal transducer and activator of transcription(STAT)3 was observed with laser confocal microscopy.The expression of proteins of Janus Kinase(JAK)2, STAT3,phospho -JAK2 and phospho-STAT3 was detected with Westernblot technology.Such drugs as AG490,an special inhibitor of JAK2,Tongxinluo and Simvastatin were selected.Result:1 The establishment of quantitive standards of differentiation of syndrome to APCHD1.1 The extraction of syndromes and the establishment of the quantitive standard of APCHDBased on the analyses of syndrome without supervision,eight syndromes were obtained in the patients with APCHD.The contribution of symptoms to syndromes(the number at the low right of symptoms)and thresholds(the nmuber at the low right of syndroms)as followings:①collateral-qi stasis5: oppressed feel in heart and chest4,preference for sighing3,depressed emotion3,restlessness2,viry pulse1;②collateral-qi deficiency6:vague pain in heart and chest1,cardiopalmus2,short breath2,mind lassitude3, debilitation3,lazy to speak2,pale tongue1,weakened pulset1;③phlegm turbid4:cerebaria2,muco-greasy in mouth2,excessive phlegm3,muffled in chest and gastric cavity2,body fat1,distress in limbs2,whiteness greasy of musci2,viry and slide pulse1;④blood stasis3:localized pain in chest as acanthus and angor3,eclipse of facial expression2,cyanochroia in labia oris3,purplish tongue with ecchymosis or petech3,unsmooth pulse2;⑤yin asthenia7:feverish of five centres3,insomnia3,multi-dream3,hectic fever and night sweat3,sour soft in waist and knee2,few musci or no musci(1), pulse fine and cipher1;⑥heat accumulation6:flushing2,dry mouth2,bitter taste of mouth3,suitable red2,constipation2,red tongue with yellowish fur1,yellow tongue fur1pulse cipher2;⑦yang asthenia5:facial expression white2,urine schedule length1,mild facial edema and swollen limbs1, chilly3,limbs cold3,pale tongue and fat1,pulse heavy slow and powerless1;⑧phlegm fever3:mouth sticking and greasy2,pulse slide and cipher1, yellow tongue fur with greasy dirt2,cough yellow phlegm2. 1.2 The combining and distributing rules of syndroms in APCHDThe results of correlative grade of syndromes:the syndrome of collateral-qi stasis had the strong correlation with heat accumulation,blood stasis,phlegm fever,phlegm turbid,yin asthenia,and the syndrome of collateral-qi deficiency had the strong correlation with phlegm turbid,yang asthenia,blood stasis,yin asthenia.The distributing proportion of syndromes:all the patients have either collateral-qi stasis syndrome or collateral-qi deficiency syndrome.The collateral-qi deficiency syndrome accounts for 57.6%(232 cases)of all investigated patients(403 cases),collateral-qi stasis accounts for 31.5%(127 cases),10.9%(44 cases)has both collateral-qi deficiency and the collateral-qi stasis.However,blood stasis accounts for 60.3%(243 cases),which was thought the major syndrome in previous study,phlegm turbid(heat)accounts for 43.4%(175 cases),gasthenia accounts for 14.9%(60 cases).1.3 To raise the viewpoint about the pathogenesis of APCHD based on the combining and distributing rules of syndromesBased on the above results,either collateral-qi stasis or collateral-qi deficiency was the initiating agent of APCHD and breakthrough the whole process.Phlegm and blood stasis were not only the patho-products induced by collateral-qi stasis or collateral-qi deficiency,but also influenced the developing of the disease conversely.Meanwhile,collateral-qi stasis could induce pyretic generation,damage yin,depress vital energy and yin asthenia. Deficiency of vital energy for a long timemay developped to yang asthenia. Both collateral-qi stasis and collateral-qi deficiency were the basic syndromes, and heat accumulation,blood stasis,phlegm turbid(fever),yin asthenia,yang asthenia and yin asthenia,which were derived from collateral-qi stasis or collateral-qi deficiency,were the accompanied syndromes.2 Establishment and evaluation of the rat model of As with collateral-qi deficiency2.1 The evaluation of model rats of collateral-qi deficiencyCompared with the control group,the rats of collateral-qi deficiency group appeared low-spirited,eye-closed,superficial few gloss,and extrados appearance.The score of biology superficial signs was significantly higher than control group(P＜0.01).Fatigued swimming time was significantly decreased than that on the first day(P＜0.01).The rats of collateral-qi deficiency group also displayed the shorter climbing time,the longer stay-time in central frame and lower crossing and rearing score in open-field test(P＜0.05,P＜0.01).All the results reflected the cardiopalmus,short breath, debilitation,mind lassitude of collateral-qi deficiency syndrome had the concordance with the model rats with collateral-qi deficiency syndrome.2.2 The evaluation of model rats of AsRats of As model group only appeared weight breakdown.The biology superfocial signs score of As group had no significant difference.Open-field test and physiologic function also had no significant change(P＞0.05).HE staining and Masson staining showed endothelial cell partly loss, endomembrane thickened,VSMCs hyperplasy and arrange disorder,collagen fiber proliferation,local tubal walls lumina outstanding and form fiber hyperplasy As.SEM and TEM showed endothelium injured,and there was deposition on surface.Bilblast of VSMCs dissolved,and endoplasmic reticulum distended.Aortic immunityhiscochemstry showed theα-actin masc cells decreased obviously.While the TC,LDL and HCY increased obviously. All those results showed the rat model had the pathological features of plaque fiber proliferation in earlier period of As.2.3 The evaluation of complex model ratsRats of the complex model group appeared energetic dispirited,insensitive response,skin slacked,hair on the back falled off,et al.The score of complex model group was highest,which was significantly higher than both As group and collateral-qi deficiency group(P＜0.01).The climbing-time,respiratory frequency and heart rate,the rearing score of open-field of complex model rats had the same results as the above.At the same time,the swimming-time of complex model group was significant lower than that on the first day of the same group.The stay-time in central frame of open-field was longer and crossing score was lower than control group(P＜0.05,P＜0.01).As degree of complex model was more serious than As model according the observation of HE staining,Masson staining and ultramicrostructure.The masc cells expressing of aorticα-actin in complex model group also decreased,and the TC,LDL,HCY increased highly.All those results showed that there were both collateral-qi deficiency syndrome and As in complex model,whose degree was more seriously.2.4 Metabonomics resultsCompared with control group,As model group,collateral-qi deficiency group,complex model group metabolin spectra had obvious difference.The potential biomarkers were identified initially.According the identification results,bile acid and phospholipid increased in As model,while decreased in collateral-qi deficiency model.Bile acid increased in complex model,while phospholipid decreased in it.Amino acids and carnitine all increased in three models.2.5 Drugs interventionTongxinluo could improve the indexes related to the syndrome,such as improve the biology superficial signs score(P＜0.01),prolong the fatigued swimming time and climbing time(P＜0.01),shorten the stay-time in central frame,improve the crossing score and rearing score(P＜0.05),and degrade the respiratory frequency and heart rate(P＜0.01).At the same time, Tongxinluo might also improve the change of shape and structure of aorta, promote the phenotypic modulation of VSMCs from contractile phenotype to synthetic phenotype,reduce the level of TC,LDL and HCY(P＜0.05,P＜0.01).Simvastatin mainly improved the locally pathological change of aorta. Although Simvastatin had the effect on the fatigued swimming time and climbing-time,whose effect was not as good as Tongxinluo.Simvastatin had no effect on biology superficial signs score,respiratory frequency and heart rate,open-field test in combinted model.3 The study about the changes of NEI network in collateral-qi deficiency As rats and the intervention of Tongxinluo 3.1 The changes of HPAAChanges of model rats:compared with control group,tissue CRH,blood plasma ACTH,serum CORT in As group had statistic difference(P＞0.05), ACTH and CORT in collateral-qi deficiency group increased obviously(P＜0.01),CRH only appeared decrease tendency(P＞0.05),and CRH decreased obviously(P＜0.01),ACTH and CORT increased obviously in complex model group(P＜0.01).The intervention of drugs:compared with complex model group,large dose Tongxinluo could increase obviously CRH level(P＜0.05),decrease ACTH level(P＜0.05).Large and small dose Tongxinluo could obviously decrease CORT level,and Simvastatin had no obvious effect(P＞0.05)。3.2 The changes of Sympathesis-Adrenal Medulla Axis.Changes of model rats:compared with the control group,the indexes of E,NE in As group had upgrade tendency but had no significant difference (p＞0.05).In collateral-qi deficiency group,the index of E,NE upgraded significantly(P＜0.01),which was significantly different compared with arteriosclerosis group(P＜0.01).To compare with normal group,the index of E,NE in complex model group is obviously heighten(P＜0.05).The intervention of drugs:compared with the complex model group, E,NE of large dose and low dose tongxinluo group obviously decreased (P＜0.05).But E,NE of Simvastatin group have no significant difference3.3 The changes of RAASChanges of model rats:compared with control group,the PRA,angⅡand aldosterone in blood of As group have no significant change(P＞0.05). The PRA,angⅡin blood of collateral-qi deficiency group obviously increased(P＜0.05),and ALD had no significant difference(p＞0.05).The PRA,angⅡand ALD in blood of the complex model group obviously increased(P＜0.05,P＜0.01)Compared with the control group,AugⅡin aorta of As group obviously increased(P＜0.05).AngⅡin aorta of collateral-qi deficiency group had upgrade tendency,but no significant difference(P＞0.05).AngⅡin aorta of the complex model group obviously increased(P＜0.01).The intervention of drugs:compared with the complex model group, Simvastatin could lower the level of PRA and angⅡ(P＜0.05).large and small dose Tongxinluo could lower the level of PRA,angⅡand ALD(P＜0.05,P＜0.01)。Compared with the complex model group,AngⅡin aorta of simvastatin, large and low dose Tongxinluo group obviously decreased(P＜0.05,P＜0.01).3.4 The changes of cytokinesChanges of model rats:compared with normal control group,IFN-γin As and collateral-qi deficiency group obviously decreased(P＜0.05),while IL-6 had no significant difference(P＞0.05).IFN-γin the complex model group obviously decreased(P＜0.01),and IL-6 obviously increased(P＜0.05).The intervention of drugs:compared with the complex model group, Simvastatin could obviously raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05).The low dose Tongxinluo could obviously raise the level of IFN-γ(P＜0.01),while the large dose Tongxinluo could obviously both raise the level of IFN-γ(P＜0.05)and decrease the level of IL-6(P＜0.05)。3.5 The expressing of PCNA in aortaPCNA masc cells In the control group and collateral-qi deficiency group had been found.While plenty of PCNA masc cells in As group and complex model group had been found.The count of masc cells in the complex model group was obviously higher than that in As group(P＜0.01).All drugs could reduced the masc cells(P＜0.01)。4.The signal transduction mechanism of the VSMCs proliferation induced by AngⅡand the effect of Tongxinluo on it4.1 The influence of AngⅡto the proliferation of rat VSMCsCompared with DMEM group,10-5,10-6,10-7,10-8mol/L of AngⅡcould obviously promote VSMCs proliferation(p＜0.05,P＜0.01).Compared with 10-8mol/L of AngⅡgroup,the OD value of 10-5,10-6,10-7mol/L group obviously rised(P＜0.01).The OD value of 10-5,10-6,10-7mol/L of AngⅡhad no significant difference.4.2 The interventional effect of drugs on proliferation of rat VSMCs induced by AngⅡAG490(10,20,50,100,200μmol/L),Simvastatin(0.01,0.1,1,10μmol/L),Tongxinluo(100,200,500μg/ml)could inhibitted proliferation of rat VSMCs induced AngⅡat the concentration of 10-7mol/L(P＜0.05,P＜0.01),which was dose dependent.4.3 The effect of AngⅡon the cell cycle of VSMCs and the interventional effect of drugs on itCompared with DMEM group,the percentage of cells in phase S significantly increased after the incubation of AngⅡat the concentration of 10 -7mol/L,while the percentage of cells in phase G0/G1 bviously decrease(P＜0.01).All drugs could decrease the percentage of cells in phase S,and increased the percentage of cells in phase G0/G1(P＜0.01)4.4 The influence of AngⅡto nuclear translocation of STAT3 induced by AngⅡand effect of drugs on itIn the DMEM group,STAT3 was detected well-distributedly in the kytoplasm and nucleus.After 120min incubated by AngⅡ,STAT3 was detected mainly in the nucleus,which showed us the nuclear translocation of STAT3.However,After 10min incubated by AngⅡ,the nuclear translocation of STAT3 was not observed.All drugs could inhibited the nuclear translocation of STAT3 after 120min incubated by AngⅡ.4.5 The influence of AngⅡto the protein expression of JAK2/STAT3 signal passway and effect of drugs on itCompared with DMEM group,the protein expression of p-JAK2 increased obviously after 10 min incubated by AngⅡ(P＜0.05),while the protein expression of p-STAT3 had no significant difference.The protein expression of both p-JAK2 and p-STAT3 increased obviously after 120 min incubated by AngⅡ(P＜0.05,P＜0.01) Compared with AngⅡgroup,AG490 could reduce the protein expression of p- JAK2 and p- STAT3 after 10 min and 120 min incubated by AngⅡ(P＜0.05).Both large dose and median dose Tongxinluo could reduced the protein expression of p- JAK2 and p- STAT+3 after 120 min incubated by AngⅡ(P＜0.01,P＜0.05).Simvastatin and samll dose Tongxinluo only reduced the protein expression of p- STAT3 after 120 min incubated by AngⅡ(P＜0.05).The whole protein of JAK2 and STAT3 had no significant difference(P＞0.05)Conclusion:1 Guided by the theory of collateral disease,based on literature study, experts consultant and large sample clinical epidemiological survey in patients of APCHD from multicentres,the study was to establish quantitive differentiation of syndromestandard,on base of the data analysis without supervision using ECSPM,which avioded the subjective bias of simple literature study and experts'experience.Because the nonlinear and complex relationship existed in symptoms of syndrome,ECSPM could reflect the complex interactive relationship between symptoms more precise,compared with the method of dealing with liner relationship in previous study,which makes a found to establish the syndrom model and study the pathophysiologic basis.2 Studying the pathogenesy of TCM about APCAD on basis of the combining and distributing rules of syndromes,Our results showed that collateral-qi stasis or collateral-qi deficiency existed in all patients of APCAD, which was higher than the percentage of blood stasis,phlegm muddy(heat), heat accumulation,yin asthenia,yang asthenia etc.The results showed that stagnancy or deficiency caused by the changes of collateral-qi might be initiating agent of APCAD.Phlegm muddy(heat)and blood stasis were the patho-products derived of collateral-qi stasis or collateral-qi deficiency, which had been paid attention and researched for several decades by domestic medical society.Studying the change of collateral-qi of APCAD was different from that that study such blood stasis and phlegm muddy,etc,which showed the feature of qi related to blood in collateral desease theory,and had the guidance significance to explore the pathomechanism of various kinds of social-psycho factors under the bio-psycho-social medical model.3 Based on the differentiation standard of syndromes established by clinical epidemiological survey,we eatablished syndrome model of collateral-qi deficiency,using basic diet plus forced loading swimming.The model rats were evaluated objectivly by the methods of the general condition observation,semi-quantity score of biology superficial signs,fatigued swimming-time,climbing-time,ethology,electrophysiology,according to main symptoms of collateral-qi deficiency.The complex model was established using As rat model overlain basic diet plus forced loading swimming.The model rats had both local pathological feature in the earlier period of As,and the representation of collateral-qi deficiency syndrome.The three models showed difference in the metabolism spectra using the metabonomics technology.This study also lain the foundation for exploring pathophysiologic basis of collateral-qi deficiency syndrome and it's effect on AS.4 This study had explore the changes rules of factors related to NEI network in the rat model of As with collateral-qi deficiency.The function of HPAA was in the chaos in the condition of collateral-qi deficiency.Adrenergic nerve -adrenal medulla system and RAAS were activated.However AS model had not such significant changes.The syndrome factor of collateral-qi deficiency not only caused the indexes related to NEI network to change more significantly in complex model rats,but also promoted more increase of AngⅡin aorta,which caused by pathological factors.Total effect was that PCNA which reflected the VSMCs proliferation in the complex model rats was obviously higher than simple As model,which revealed that syndome factors of collateral-qi deficiency could cause the factors related to the NEI network to be disorder and then influence As.5 The study selected AngⅡas the interventional factor,which was the main factor related to the NEI network influenced by the syndrome factor of collateral-qi deficiency,and explored the influence of AngⅡto the proliferation of rat VSMCs,which would provide the experimental evidence about the pathomechanism of syndrome of collateral-qi deficiency.The results showed that AngⅡcould obviously promote rats proliferation of VSMCs, which was related to the influence to the cell circle.Meanwhile,AngⅡalso promoted protein expression of p-JAK2 and p-STAT3 of VSMCs,while the effect above could be stocked by AG490,which was specific inhibitor of JAK2.Those results showed that the effect of AngⅡpromoting the proliferation of VSMCs was related to signaling pathway of JAK2/STAT3.6 The effect of Tongxinluo and Simvastatin on the complex model rats showed that they could both improve local pathological change,and inhibit proliferation of VSMCs caused by AngⅡthrough blocking JAK2/STAT3 signaling pathways.Tongxinluo also significantly improved the pathological changes caused by syndrome factor of collateral-qi deficiency,such as ethology,electrophysiology etc,while Simvastatin had no such effect. Especially Tongxinluo showed the integrated regulation effect in improving the disorder of NEI network caused by intervention of syndrome factor of collateral-qi deficiency,which was better tha Simvastatin.These results manifested that Tongxinluo showed double-effect on improving both local and global pathological change,which was different from Simvastatin.These results also possessed much more widespread meaning to prevent and care human body complex pathological and physiological phenomenon,which caused by various kinds of social psychological stress factors and biological pathogenic factors under modern living conditions and pathology factors.
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