| Objectives: Directed by the theory of collateral disorders, with endothelial dysfunction as foundation, this research established traditional Chinese medical syndrome animal models and cell models with stagnancy of collateral-qi, investigated the physiopathologic foundation of endothelial dysfunction with stagnancy of collateral-qi in a deficiency condition (SCQDC) and the intervening mechanism of different kinds of Herbs to dredge collaterals.Vascular endothelial dysfunction (ED) was a pathological process of vascular endothelial cells losing its normal function under the stimulation of different kinds of pathological factors (such as hypertension, hyperlipoidemia, diabetes mellitus, smoking, stress, etc.), and was closely related with the mechanism of oxidative damage, immunologic damage, and inflamed damage. It was mainly emerged to be dependent diastolic dysfunction of endothelium and disorder of vasoactive substances, and was the initiating agent and important element of cardialvascular and cerebrovascular diseases.The Traditional Chinese Medicine considered that the blood couldn't run normally in the blood vessel without the push of qi, and the deficiency and stagnation of qi could result in the interruption of blood running and induce vascular diseases.The three dimensional stereoscopic net system grounded on the theory of collateral disorders considered that the wider sense of collateral fell into qi-collateral and ending-vessel, ending-vessel was in charge of running blood, and its anatomic form was identical with the modern medical vascular system, therefore, the definition of vessel-collateral and vascular system disorders was put forward; qi-collateral was in charge of running meridian qi, had the function of supporting, protective defending, information conducting, and regulating. Stagnancy of collateral-qi in a deficiency condition was the early stage of ending-vessel pathological changes developing from functional pathological changes to substantial pathological changes. The ending-vessel's malfunction of self-adapting, self-adjusting, and self-balancing caused by Stagnancy of collateral-qi in a deficiency condition was the initiating agent of vessel-collateral and vascular system disorders, and would penetrate and affect the overall process of pathological changes, which had internal consistency with endothelial dysfunction, and also included the theory of Western Medicine that the regulating dysfunction of neruro-endocrine-immunity net would induce vascular diseases.This aroused us to combine the Traditional Chinese Medicine related theory of collateral disorders about collaboration between qi and blood with modern microexamination, investigate the law of NEI Network's regulating dysfunctional changes and its inner link with endothelial dysfunction with Stagnancy of Collateral-qi, and discover the intervening mechanism of different kinds of Herbs to dredge collaterals.Methods: This research used homocysteine to establish the preliminary model of endothelial dysfunction, adopted the approach of combining basic diet and compelling weight-loading swimming to induce the syndrome of deficiency of qi, eventually established the animal model of combining the syndrome of endothelial dysfunction and stagnancy of collateral-qi in a deficiency condition in a deficiency condition. On the basis of syndrome diagnostic standard of ending vessel-blood vessel system diseases established by clinical epidemiological survey, we evaluated the established model from the different aspects of syndrome theory of the Traditional Chinese Medicine, the changes of vascular endothelial function, and the reactivity changes of angiokinetic, and used single Chinese Traditional Medicine of supplementing qi, combined prescription of supplementing qi and activating blood circulation, and dredge collaterals complex herbs to work as the intervention of different level. On the basis of this experiment, we investigate the law of NEI Net's regulating dysfunctional changes and its inner link with endothelial dysfunction with Stagnancy of Collateral-qi in a deficiency condition , and discover the intervening mechanism of different kinds of Herbs to dredge collaterals. At the same time, we cultured the endothelial cell ECV -304 of human's venae umbilicalis, established in vitro Stagnancy of collateral-qi in a deficiency condition typed endothelial cell injured model by adding the sera endothelial dysfunction model rats with Stagnancy of Collateral-qi, used the above Herbs to dredge collaterals to intervene, and further investigate the Stagnancy of collateral-qi in a deficiency condition typed vascular endothelial cell ' s damage mechanism and the effects of Herbs to dredge collaterals in cellular level.1 The establishment of model of endothelial dysfunction with stagnancy of collateral-qi in a deficiency conditionand the intervening effects of herbs to dredge collaterals.In this part, the model of endothelial dysfunction was established by inducing hyperhomocysteinemia with homomethionin diet, the approach of basic diet and compelling weight-loading swimming was adopted to induce the syndome of qi deficiency , three different kinds of Herbs to dredge collaterals (Renshen, shuangshen, Tongxinluo supermicro powder) were used to intervene, established animal model with syndrome of stagnancy of collateral-qi, observed and noted down TCM syndromes performance, detected the changes of vascular endothelial function and dependent diastolic reaction of vascular endothelium. 160 healthy male Wistar rats of cleanness level were randomly devided into 6 groups (30 rats in each group):①the Control Group, normal diet, same volume of 0.5 CMC was intragastrically administrated each day.②the HCY Group: fed with 3% homomethioni, food and water were given with no limitation, same volume of 0.5 CMC was intragastrically administrated each day.③the SCQDC group: 4 weeks later, HCY rats with basic diet were compelled to swim with the load of 5% its own weight.④the Renshen Group: the HCY rats were intraperitoneally injected 1ml /100g at the very beginning of modeling. The density of crude drugs was 0.008g/ml.⑤the Shuangshen Group: the HCY rats were intraperitoneally injected 1ml /100g drugs at the very beginning of modeling. The density of crude drugs was 0.075g/ml.⑥the Tongxinluo Group: the HCY rats were intragastrically administrated 1ml /100g at the very beginning of modeling. The density of crude drugs was 0.06g/ml.The general condition of rats was observed each day and the rats of all groups (excluding rats of the HCY Group) were given endurance tests after being given medicine 30 min; 3 rats from each group were anesthetized . The aorta thoracica of each rat was separated immediately, and taken the dependent diastolization function tests of blood vessel endothelium; another 3 rats were taken from each group. Cut abdominal cavity to separate mesentery, acetylcholine water solution was quickly injected from vena caudalis (40μg/kg). Detected the blood capillary's outspread condition; after the establishment of model, all the rats left were taken plasma and sera after a 7h fasting. Tested the level of angiotensinⅡ(AngⅡ), endothelin(ET ), thromboxane A2 (TXA 2), prostacyclin I2 (PGI 2), detected the content of NO and MDA in sera and the activities of SOD and GSH-PX, observed the morphological changes of aorta thoracica (including the general structure under light microscope and the ultramicrostructure under transmission electron microscope). Histochemical stain method was used to analyze the angiotensin converting enzyme in aorta thoracica tissue (ACE )and the expression of endothelium-typed nitrogen oxide synthase (eNOS ), No. 1 receptor of angiotensinⅡ(AT-1), endothelin receptor (ET-1)and peroxynitrosonegion(ONOO-).Results:①Compared with Control Group and HCY Group, and all the rats in SCQDC Group appeared the following symptoms: obvious hypodynamia, insensitive response, skin slacked, body weight decreased, fur on the back dimmed, nose and tail being erythroic and rarely lustrous, stool being soft and shaped, .In addition; the time of climbing pole was significantly less than that of the Control Group(128.88±49.27 s)(P< 0.05 ). The condition of the rats in all treatment groups was slighted improved, the weight of those rats appeared increasing, and the time of swimming and climbing pole prolonged a little.②The content of AngⅡ(140.31±20.43ng /ml )in plasma of rats in the HCY Group was significantly increased when compared with the content (70.27±10.09ng /ml )of the Control Group (P<0.01), the content of ET and TXA in the the HCY Group( 75.93±4.29 pg/ml, 509.96±323.79 pg/ml ) also had the tendency of increasing when compared with the content of Control Group (58.49±8.48pg /ml , 224.58±112.58pg /ml ), while the content of PGI 2 in the HCY Group (144.44±157.46 pg/ml) showed the tendency of decreasing when compared with that of the Control Group(216.29±64.94 pg /ml), but had no significant difference; the content of NO in the HCY Group(14.93±5.03μmol /l) was significantly decreased when compared with that of the Control Group(27.90±12.93μmol /l)(P<0.05). The AngⅡ, ET -1, and TXA 2 level in the SCQDC Group (306.13±127.87 ng/ml, 80.31±12.74 ng/ml, 509.96±323.79 pg/ml) was significantly increased than the level in the Control Group (70.27±10.09ng/ml, 58.49±8.48pg/ml, 224.58±112.58pg /ml )(p<0.001, p<0.05,p<0.001); the content of PGI in the SCQDC Group(104.57±31.56pg /ml )was significantly decreased than the content in the Control Group (p<0.01),also the content of NO ( 8.91±3.02μmol/l) like this (p<0.001); the content of AngⅡin the SCQDC Group was much more increased than that in the HCY Group (p<0.05).After giving the three kinds of Herbs to dredge collaterals, the AngⅡ, ET-1 level in plasma of the SCQDC Group was significantly decreased when compared with the Control Group (p<0.01, p<0.05), the content of NO was significantly increased(p<0.05 ), the PGI 2 was increased with no statistical difference. The AngⅡ, ET -1 level of the Shuangshen Group was significantly decreased (p<0.01,p<0.001), the content of TXA 2 was decreased with no significant difference , and the content of NO was increased significantly (p<0.05).The AngⅡ, ET-1 level of the Tongxinluo Group was significantly decreased (p<0.001,p<0.001), the content of NO was increased significantly(p<0.01), whereas the TXA 2 level and PGI 2 level in plasma appeared different tendency, the formal was decreased, the latter was increased, and both had no statistical difference. the MDA level in the HCY Group (5.14±1.16nmol /ml ) was increased but no statistical significance when compared with that of the Control Group(4.38±1.25nmol/ml); however the content of MDA in the sera of rats of the SCQDC Group(11.44±2.80 nmol /ml ) was significantly increased, the level of GSH-Px and the activity of SOD in the HCY Group(1755.00±687.23U/ml, 293.54±12.74U/ml) and in the SCQDC Group ( 1475.78±242.89U/ml, 195.84±50.29U/ml) were also significantly lower than those of the Control Group(2409.58±341.84U/ml , 275.52±20.08U/ ml )(p<0.05,p<0.001).The content of MDA in the Renshen Group and the Tongxinluo Group was significantly decreased(p<0.001),whereas the MDA content of the Shuangshen Group had the tendency of decreasing , but had no significant statistical difference.and both SOD and GSH-Px activity in Renshen Group increased significantly(p<0.05,p<0.01).The SOD activity of the Shuangshen Group was apparently higher than that of the SCQDC Group(p<0.05),though the GSH -Px activity was higher than that of the SCQDC Group, yet had no statistical difference.The SOD and GSH -Px activity appeared varying degrees of increasing, and had significant statistical difference(p<0.05,p<0.01).③The light microscope analysis showed that minor edema could also be observed in the smooth muscle layer of aorta thoracica of rats in the HCY Group, and minor hyperplasia could be found under the tunica intima; minor edema could also be observed in the smooth muscle layer of aorta thoracica of rats in the SCQDC Group, minor inflammatory cell infiltration could be seen, and the arrangement of smooth muscle cells was in a mess, with the intervention of the three kinds of Herbs to dredge collaterals, the general structure was almost recovered to its normal condition.The Electron microscope analysis showed that some crests of mitochondria of vascular endothelial cells fused with membrane, and even disapeared; varying degrees of degranulation could be found in the rough surfaced endoplasmic reticulum; the counts of pinocytotic cells could be found decreased; the outer layer of karyotheca partly fused with the tunica interna. In SCQDC Group, the counts of pinocytotic cells and microvili on the free surface of vascular endothelial cells were significantly decreased, most crests of mitochondria fused with membrane, and even disapeared, and serious degranulation could be observed in the rough surfaced endoplasmic reticulum. Three kinds of herbs to dredge collaterals could relieve the above ultrastructural changes in different degree.The immunohistochemical stain analysis showed that the expression of ACE, AT-1 , ET-1, and ONOO- was enhanced, and xanthochromia signals could be observed in the kytoplasm,and disturbuted outer layer and internal layer of arterial tissue(including endothelial cells) mainly. Compared with the Control Group and the HCY Group, the eNOS expression of the SCQDC Group was significantly weakened, but the expression of AT -1, ET -1,and ONOO- was obviously enhanced. Three different kinds of herbs to dredge collaterals could weaken the expression of AT -1, ET -1,and ONOO, and enhance the eNOS expression in different degree.④The diastolic reactivity(diastolic percentage) analysis of the artery and blood capillary of rats in each group showed that the endothelial dependent diastolic percentage of blood vascular ring of the HCY Group ( 0.707±0.299 ) and the SCQDC Group( 0.501±0.176 )in vitro was significantly decreased compared with that of the Control Group( 1.024±0.184 ()p<0.05,p<0.001).Compared with the SCQDC Group, the endothelial dependent diastolic percentages of blood vascular ring in vitro of the Renshen Group, the Shuangshen Group, and the Tongxinluo Group were all increased in different degree(0.641±0.114, 0.825±0.151, 0.977±0.342()p<0.01,p<0.05,p<0.01). In addition , the outspreading amplitude of blood capillary of rats in the HCY Group and the SCQDC Group(1.67±0.23μm, 0.95±0.33μm)was significantly decreased compared with the Control Group( 4.50±0.94μm) (p<0.05,p<0.001); the outspreading percentage of blood capillary of rats in the HCY Group and the SCQDC Group(14.34±3.19%,10.74±3.26%)was significantly decreased compared with the Control Group(55.61±28.62%)(p<0.05,p<0.01); the outspreading amplitude of blood capillary of rats in the Renshen Group, the Shuangshen Group, and the Tongxinluo Group (2.35±1.24μm, 2.23±0.38μm, 3.76±0.92μm)were increased differently compared with the SCQDC Group(p<0.05, p<0.01), and the outspreading percentages of blood capillary were also increased, and the effect of Tongxinluo was most prominent(p<0.05).The result indicated that the symptims observed from the model rats established by combining methionine diet, basic diet, and weight loading swimming (such as listlessness, hair withered and yellow, loss of weight, etc.) conformed with the clinical diagnostic standard of Stagnancy of Collateral-qi in a deficiency condition , and the endothelial dysfunction was further exacerbated compared with the HCY Group, which proved that the endothelial dysfunction model with Stagnancy of collateral-qi in a deficiency condition was established successfully. After the intervention of different kinds of Herbs to dredge collaterals, the above symptoms could be improved in different degree.2 The NEI Net Changes of the endothelial dysfunction model rats with Stagnancy of collateral-qi in a deficiency condition and the intervening effects of Herbs to dredge collaterals.On the basis of endothelial dysfunction animal models with Stagnancy of collateral-qi in a deficiency condition established in the experiment One, Radio-immunity Approach was carried out to detect CRH, ACTH, GC , PRA, AngII, ALD in plasma and IL -1β, IL -2 , IL -6 in sera;ELISA Approach was used to detect NE , IL -10; Biochemistry Approach was adopted to detect the level of serum immune globulin IgG, IgA , IgM and alexin C3 , C4 , to calculate the index number of thymus and spleen , and to investigate the change rule of NEI Net related index; Bioinformatics Approach was used to analyze this model.The experimental animal, grouping, drug delivery and the methods of sampling was as above.Results: (1)PRA -ANG -ALD System: compared with the Control Group (0.865±0.251pg /ml ), the renin level in plasma of animals in the HCY Group and the SCQDC Group(0.590±0.175pg /ml , 0.403±0.125pg /) was significantly decreased(P<0.01); the ANG II level of the two groups(140.301 ±20.431pg/ml, 306.12±127.867pg /ml)was significantly increased compared with the Control Group(70.269±10.090pg /ml)(P<0.01,P<0.001), and the AngII level of the SCQDC Group was significantly increased compared with the HCY Group(P<0.01); After the intervention of three kinds of Herbs to dredge collaterals, the renin level in plasma was increased in different degree, but had no statistical difference; the AngII level in plasma (the Renshen Group:202.563±49.169 pg /ml,the Shuangshen Group: 212.490±46.364/ml, the Tongxinluo Group: 172.748±53.356 pg /ml)was decreased in different degree(P<0.01,P<0.01,P<0.001); the ALD level in plasma of the three treatment groups was increased in different degree, and the increasing level of the Tongxinluo Groups(0.834±0.255 ng/l)was apparently higher than the Control Group and the SCQDC Group(P<0.05), and the effect was also better than that of the Renshen Group(0.500±0.376ng /l)(P<0.05).(2)CRH -ACTH -GC Functional Ax : the CRH and GC level of the HCY Group (CRH : 4.769±1.913 ng /ml , GC : 15.584±1.926μg/l)were all increased compared with the Control Group (CRH: 4.195±0.599ng/ml,GC: 13.921±1.142μg/l), but had no statistical difference(p>0.05), and the change of ACTH level was also indistinct.The CRH level in CRH of the SCQDC Group(6.548±1.620ng /ml ) was significantly increased(p<0.01), and apparently higher than that of the HCY Group(p<0.05). At the same time, the ACTH (3.918±0.333pg/ml) and GC (10.320±2.992μg/l) level of the SCQDC Group were significantly decreased compared with the Control Group (ACTH:4.431±0.762 pg/ml)(p <0.05), the GC level was apparently lower than that of the HCY Group(p<0.01).After the intervention of three different kinds of Herbs to dredge collaterals, the CRH level was decreased in different degree, and the effects of the Shuangshen Group and the Tongxinluo Group (5.370±1.599 ng /ml ,4.494±0.802ng /ml , 4.383±1.100ng /ml)were superior to the Renshen Group(p <0.01).The ACTH level was observed to have the tendency of increasing, but had no statistical difference. The GC level of the three treatment groups was increased in different degree (the Renshen Group: 13.953±2.800μg/l, the Shuangshen Group: 14.357±2.528μg/l, the Tongxinluo Group:20.576±5.684μg/l)(p<0.001,p<0.01,p<0.001).In addition, the NE level of the HCY Group and the SCQDC Group was increased compared with the Control Group. After the intervention of three different kinds of Herbs to dredge collaterals, the NE level showed the tendency of decreasing, but had no statistical difference .(3)The Immune System :①the immunoglobulin fam: IgG and IgA of the HCY group were significantly increased compared with the Control Group(p<0.05, p<0.01), while IgM had the tendency of increasing, but had no statistical difference. The content of alexin C3 and C4 of the HCY Group and the SCQDC Group was decreased(p<0.05, p<0.001); IgG and IgA content of the SCQDC Group was decreased compared with the HCY Group(p<0.05, p<0.001),and the alexin C4 level of the Tongxinluo Group was significantly increased compared with the SCQDC Group(p<0.05).②The interleukin Fam: the IL -1βand IL -6 level of the SCQDC Group were significantly increased compared with the Control Group (p<0.01, p<0.01), and were also higher than that of the HCY Group (p<0.001, p<0.01). The IL -10 level of the HCY Group showed the increased tendency, but had no statistical difference . After the intervention of three different kinds of Herbs to dredge collaterals, the IL -1βlevel of the Shuangshen Group and the Tongxinluo Group were decreased in different degree compared with the SCQDC Group(p<0.01).Compared with the SCQDC Group, the content of IL -6 of the Tongxinluo Group was apparently lower(p<0.05).③Thymus and spleen : the thymus index of the HCY Group and the SCQDC Group was decreased in different degree compared with the Control Group(p<0.05, p<0.001), and the spleen index was increased compared with the Control Group (p<0.05). After the intervention of three different kinds of Herbs to dredge collaterals, the thymus index was increased in differernt degree, and had statistical difference(p<0.001,p<0.01, p<0.05), but the spleen index was not significantly changed. ( 4 ) The bioinformatics analysis: the subsystem composed of the NEI Net indices and the endothelium functional indices in the SCQDC Group interrelated according to the principle of optimality of complex system(such as: ET and CRH, ACTH, GC, ALD; Ang II and ET, IL-1; PGI2 and ALD; NO and ALD,four subsystems), and gradual change regulatin could also be observed. In the Control Group , the HCY Group , and the Tongxinluo Group, the same interrelation between subsystems could not existed, which indicated that overstrain and damage of qi were the leading factor of causing the disorder of NEI Net, and the endothelial dysfunction with Stagnancy of collateral-qi in a deficiency condition might be closely related with the imbalance of NEI Net which might be one of the pathophysiological foundations of the occurance and development of endothelial dysfunction.Experimental result indicated that the vascular endothelium function was observed damaged in the HCY Group, and AngII was increased; compared with the HCY Group, the increasing of AngII level of the SCQDC Group was particularly significant, which indicated that the stagnancy of collateral-qi in a deficiency condition was the important inducing factor of the increasing of AngII .At the same time, we observed that the decreasing of renin and aldosterone, and no activation of RAAS System, whereas NE and Ang II showed close relationship of increasing instantaneously, which indicated that the excitation of sympathetic nerve was the leading cause of the increasing of Ang II. And thymic atrophy and increasing of the expression of inflammatory factor were also observed, which reflected that the dysfunction of vegetative nerve caused by overstraining played an important role in the occurrence of vascular diseases. However the regulatory function of NEI Network was observed seriously disturbed which resulted in the hypothalamus function overexcited, and the pituitary function degraded, at the same time the adrenocortical function was also degraded, which indicated from another angle that the imbalance of NEI Net could be one of the pathophysiological foundations of the occurance and development of the endothelial dysfunction with Stagnancy of Collateral-qi in a deficiency condition .3 The changes of JNK/c-Jun /HO-1/CO pathway of the arterial tissue of model rats with Stagnancy of collateral-qi in a deficiency condition and the intervention effects of Herbs to dredge collaterals3.1 The changes of HO-1/CO of aorta thoracica of rats with Stagnancy of collateral-qi in a deficiency condition and the intervention effects of Herbs to dredge collateralsIn this part, both HO-1 expression and CO in the arterial tissue were detected, and the effects of Herbs to dredge collaterals was also observed. Rats were randomly devided into five groups:①the Control Group : 0.5%CMC -Na intragastrically administrated;②the SCQDC Group;③t he Renshen Group;④the Shuangshen Group;⑤the Tongxinluo Group,And the intervention methods of Group②,③,④, and⑤were the same to the Experiment One. 30 min after the ending of the experiment, all the animals were put to death with arterial tissue sampled. Sodium Hydrosulfite Approach, RT-PCR, Westernblot, and Immunohistochemical Approach were used separately to detect the content of CO and the expression of HO-1mRNA and albumen in the arterial tissue of the rats in each group, and immunofluorescence double-labelling confocal microscope was used to observe the intrastitial location of HO-1.Results: showed that the COHb content of the SCQDC Group was significantly decreased(p<0.01), the HO-1mRNA and albumen expression were down(p<0.01,p<0.001),its positive signals were mainly observed in the endochylema of endothelial cells and smooth muscle cells, and the intervention of three different kinds of Herbs to dredge collaterals, the HO-1mRNA and albumen expression were observed increasing in different degree(p<0.001), the intervening effects of the Tongxinluo Group on increasing the albumen were superior to the effects of the Shuangshen Group and the Renshen Group, and the Tongxinluo also could significantly increase the CO content of the tissue(p<0.01). The results indicated that the Herbs to dredge collaterals could induce the production of internal CO by ameliorating or recovering the HO System to exert their functions of antioxidation, improving the vasorelaxation and contractile function, etc. and further improve the endothelial dysfunction coming with Stagnancy of Collateral-qi in a deficiency condition .3.2 The changes of JNK /c-Jun pathway of the arterial tissue of model rats with Stagnancy of collateral-qi in a deficiency condition and the intervention effects of Herbs to dredge collateralsIn this part, the experimental animals and grouping were the same to 3.1. RT-PCR, Westernblot, and Immunohistochemical Approach were used separately to detect c-Jun mRNA and albumen expression in the arterial tissue and the phosphorylation condition of c-Jun, and immunofluorescence double-labelling confocal microscope was used to observe their intrastitial location.Results:①RT -PCR analysis showed that the JNK and c-Jun mRNA level of the SCQDC Group(19.67±4.163, 26.33±6.110) were significantly decreased compared with the Control Group (P<0.001); the JNK and c-Jun mRNA level of the Shuangshen Group and the Tongxinluo Group were increased in different degree (P<0.05, P<0.01, P<0.001); Western Blot Analysis showed that the JNK and phosphorylation c-Jun level of the SCQDC(33.67±2.082, 23.00±1.000) were decreased compared with the Control Group(P<0.001); after the treatment of the different kinds of Herbs to dredge collaterals, the JNK and phosphorylation c-Jun level were apparently higher than those of the SCQDC Group (P<0.01, P<0.001).②The results of immunohistochemistry and analysis with laser scanning confocal microscope showed that minor JNK and p-c -Jun positive signals were observed in the arterial tissue of rats in the Control Group. The expression of JNK and c-Jun of the SCQDC Group was significantly decreased, and the JNK and p-c-Jun positive signals in the arterial histiocyte were weakened; the JNK positive signals were located in the hyalomitome of arterial histiocyte of rats, while the p-c-Jun positive signals were located in the cell nucleus of the arterial tissue of rats. In the three treatment groups, the JNK and p-c-Jun expression were observed increased. The JNK and p-c-Jun positive signals were mainly located in the endothelial cells and arterial smooth muscle cells, most JNK and p-c-Jun positive signals were observed in the same cell. All the above results indicated that the content of COHb of endothelial dysfunction with Stagnancy of collateral-qi in a deficiency condition was significantly decreased, the HO -1mRNA and albumen expression were down regulated, and JNK and c-Jun mRNA or JNK and phosphorylation c-Jun protein level were significantly decreased, which indicated that the HO-1/CO System of the endothelial dysfunction with Stagnancy of collateral-qi in a deficiency condition was restrained. And the protective effects to cells of Herbs to dredge collaterals were likely to be exerted by improving the activity of JNK/c-Jun pathway, and produce its effects mainly through phosphorylation c-Jun.4 The expressive changes of JNK /c-Jun /HO -1 in the ECV -304 cells induced by sera of the animal models with Stagnancy of collateral-qi in a deficiency condition and the intervention effects of Herbs to dredge collateralsIn this part, the sera from the animal models of the SCQDC Group was used to hatch endothelial cell ECV -304 of venae umbilicalis, and investigate the effects of sera from the animal models of the SCQDC Group to JNK /c-Jun pathway , the effects of Herbs to dredge collaterals of up regulating the HO-1expression of endothelial cells, and the intervention of three different kinds of Herbs to dredge collaterals. The experiment was divided into the following groups:①the Control Group: rats sera from Control Group was added;②the Model Sera group: in this group rats sera of same density from the SCQDC Group was added;③the Model Sera+Renshen Group: Renshen was added to hatch; 2h later, rats sera of same density from the SCQDC Group was added;④the Model Sera+Shuangshen Group: Shuangshen were added to hatch; 2h later, rats sera of same density from the SCQDC Group was added;⑤the Model Sera+Tongxinluo Group: Tongxinluo was added to hatch; 2h later, rats sera of same density from the SCQDC Group was added;⑥the Model Sera+SP600125(the antagonist of JNK specificity)Group : SP 600125 was added into the 1640 culture medium without sera; 2h later, rats sera of same density from the SCQDC Group was added;⑦the Renshen+SP 600125+Model Sera Group: Renshen was added into the 1640 culture medium without sera; 6h later, SP 600125 was added; 2h later, rats sera of same density from the SCQDC Group was added;⑧the Shuangshen+ SP600125+Model Sera Group: Shuanshen was added into the 1640 culture medium without sera; 6h later, SP600125 was added; 2h later, rats sera of same density from the SCQDC Group was added;⑨the Tongxinluo+SP600125+Model Sera Group: Tongxinluo was added into the 1640 culture medium without sera; 6h later, SP600125 was added; 2h later, rats sera of same density from the SCQDC Group was added. 12h, 24h, and 48h after the culture, kits were used to detect the release rate of corpuscular lactate dehydrogenase(LDH)of each group and the content of CO in the supernatant of cell culture; RT -PCR and western blot were used to detect the albumen phosphorylation condition of c-Jun and the expressive changes of HO-1, JNK , c-Jun mRNA , and albumen in the animal cells of each group. Results:①the LDH leakage rate of the Model Sera Group and the sp600125 Group were observed significantly increased 48h later(p<0.05), and the COHb content of the Model Sera Group was also observed significantly increased 48h later. Compared with the Model Sera Group, the leakage rate of the Tongxinluo Group was observed significantly decreased 48h later (p<0.01), and the COHb content was also observed significantly increased (p<0.05). Compared with the Model Sera+SP600125 Group, the COHb content of the Model Sera+SP600125+Shuangshen Group and the Model Sera+Tongxinluo Group were observed significantly increased 48h later(p<0.05).②RT -PCR results showed that the expression of JNK, c-Jun and HO-1mRNA of the Model Sera Group were significantly increased at different time compared with the Control Group (P<0.05, P<0.001), and compared with the Model Sera Group, the three kinds of Herbs to dredge collaterals could up regulate the expression of JNK, c-Jun and HO-1mRNA in different degree at different time(P<0.05,P<0.01,P<0.001), whereas Tongxinluo's effects of up regulating the expression of JNK , c-Jun and HO-1mRNA were superior to the effects of the Shuangshen Group and the Renshen Group(P<0.001,P<0.05,P<0.01. Compared with the Model Sera+sp600125 Group, the expression of JNK, c-Jun mRNA of the Shuangshen+SP600125+Model Sera Group and the Tongxinluo+SP600125+Model Sera Group was observed significantly increased 24h an...
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