| Objective: Under the guidance of Collateral Disease Theory and according to the TCM doctrine of"excessive rest on bed impairing Qi", a complex model named with vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type was established in this research. To approach the vascular endothelial function change in the state of"collateral-Qi deficiency and stagnancy"and regulative rule of general NEI network, at the same time, internal connection between of both. To illuminate the local immunology injury mechanism of endothelial dysfuntion in complex model. To reveal the molecular mechanism in the level of gene and protein that vascular endothelial dysfunction is relative closely with important chemical signal molecule belong to NEI network. Through the above research, it would illuminate the pathophysiologic mechanism of vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type caused by excessive comfortable life and the effective mechanism of different categories dredge-collateral herbs, and then, supply the scientific evidences for using the Collateral Disease Theory to guide vascular disease'prevention and cure.Methods: There are four sections in this research:1. The establishment of rat complex model named with vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type and the effect of dredge-collateral herbsPathological model was been established by using HHcy to injury vascular endothelium firstly, according to the TCM doctrine of"excessive rest on bed impairing Qi", the factor of hyperalimentation diet combinated with activity restraint was exerted into the pathological model and a complex model named with vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type was completed successfully. 120 numbers male Wistar rats were divided randomly into this following 8 groups: (1) Normal control group; (2)Vascular endothelial dysfunction model groups (HCY group); (3)Collateral-Qi deficiency and stagnancy model group (Syndrome model group); (4) Complex model group; (5) Ginseng group; (6) Siweitongluo group; (7) Tongxinluo group; (8) Simvastatin group. 15 numbers are in every groups. The rats of normal control group were feed with ordinary forage, however, the rats of HCY group were feed with forage contained 3% homomethionin, all of them could drink water freely. Every rat of syndrome model group, complex group and medication group was respectivly put into remoulded metabolic cage which space was regulated to keep rats quiet and turn round freely, all of rats were feed on hyperalimentation contained 3% homomethionin except syndrome model group. Every medication group rats was intragastric administration with corresponding dredge-collateral herbs and Simvastatin according to 1ml/100g weight from the beginning of experiment, on the contrast, the rats of normal control group and different model groups were intragastric administration with the same volume 0.5% CMC-Na. The whole experimental period was 10 weeks. In this sections, the complex model was objective evaluated by the following methods: semi-quantitived biology superficial syndrome scores, exhausted swimming time, respiratory frequency and heart rate, aortic tunica intima (endothelial cells) morphologic observation using light microscope and electron microscope respectivly, biochemical indicators related to vascular endothelial function were detected. In addition, the effect of three different dredge-collateral herbs on vascular endothelial function was observaed in detail.2. The general NEI network change of complex model rats and effect of dredge-collateral herbsAs an important incisivus point, these common chemical signaling molecules impacted on NEI network regulation were detected on the basis of complex model group rats, these indicators were following as: hypothalamic-pituitary-adrenal axle including CRH in the hypothalamus (ELISA) , ACTH in the plasma (RIA) and CORT in the serum (RIA) were detected; hypothalamic-pituitary-thyroid axis including TRH in the hypothalamus (ELISA), TSH and T3 and T4 in the serum (RIA) were detected; immunity cytokines collected from peripheral blood, including IL-1β(RIA), interferon-γ(RIA)and tumor necrosis factor-α(RIA)were also detected. To observe the change rule of NEI network regulation under the condition of"collateral-Qi deficiency and stagnancy"and the effect of dredge-collateral herbs, in addtion, reveal the internal relationship between NEI network disequilibrium and vascular endothelial dysfunction using the method of canonical correlation analysis.3. The immunity mechanism of complex model rats vascular endothelial cells injury and the effect of dredge-collateral herbsIn this section, the anti-vascular endothelial cells antibody content of rat's peripheral blood (ELISA), the expression of major histocompatibility complex-Ⅱmoleculeand ICAM-1/ VCAM-1 (immunohistochemical method) in the aortic endothelial cells were detected. These research on local vascular endothelial cells immunologic injury would prove proofs for interpreting complex model rats'pathophysiologic mechanisms from another angle.4. TNF-αinduce HUVEC to secrete ET-1 and eNOS: p38 MAPK singal pathway play an import role and the effect of dredge-collateral herbsTo observe the role of p38 MAPK signal pathway which is in the course of TNF-αinducing HUVEC to secrete ET-1 and eNOS in vitro.The contents of ET-1 and eNOS derived from HUVEC culture supernatant after exerting into different concentration TNF-α(0,2.5,5,10,15,20ng/ml) in different time (0,1,2,4,8,12,24h) was detected by the method of radioimmunity and ELISA, respectively. The protein and mRNA of ET-1/eNOS in HUVEC lysate was extracted and their expression was detected by the method of Western blot and Real time quantitive RT-PCR after 12 hours added with TNF-α. Every groups was following as: (1) Normal control group; (2) TNF-α group (the final concentration of TNF-αis 10ng/ml); (3) SB203580 group (the final concentration of SB203580 is 25μmol/L, preincubate for 1h and then incorporate 10ng/ml TNF-α); (4)-(6) Tongxinluo group (the final concentration of Tongxinluo is 100μg/ml, 500μg/ml and 1000μg/ml in order, preincubate for 1h and then incorporate 10ng/ml TNF-α); (7)-(9) Simvastatin group (the final concentration of Tongxinluo is 0.1μmol/L, 1μmol/L and 10μmol/L in order, preincubate for 1h and then incorporate 10ng/ml TNF-α).Three same wells were designed in every groups. The expression of HUVEC phosphorylation p38 MAPK protein was detected by the method of Western blot after 10min, 30min and 60min incorporated into TNF-α, respectively. Every group was following as: (1) Normal control group; (2) TNF-αgroup (the final concentration of TNF-αis 10ng/ml); (3) SB203580 group (the final concentration of SB203580 is 25μmol/L, preincubate for 1h and then incorporate 10ng/ml TNF-α); (4) Tongxinluo group (the final concentration of Tongxinluo is 500μg/ml, preincubate for 1h and then incorporate 10ng/ml TNF-α); (5) Simvastatin group (the final concentration of Tongxinluo is 10μmol/L, preincubate for 1h and then incorporate 10ng/ml TNF-α). Three same wells were designed in every groups.Results:1. The establishment of rat complex model named with vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type and the effect of dredge-collateral herbsThe complex model was evaluated from the following many aspects after established: (1) biology superficial syndrome observation and scores: with the time prolonging, rats of complex model group had taken on tired and lazy step by step, limbs were scrunch, activities were sluggish, the skin was flaccid slightly and pelage was withered and dim; the colour of ears and tails was pale and lusterless; dejecta was soft but shaped. Furthermore, the scores of biology superficial syndrome were all increased significantly than normal control group (P<0.01); (2)compared with normal control group, rats'exhausted swimming time of syndrome model group and complex model group was all shorten obviously (P<0.05); (3)compared with nomal control group, rat's respiratory frequency and heart rate of syndrome model group and complex model group were speed up obviously (P<0.05 or P<0.01); (4) under the light microscope, the aortic endothelial cells of complex model group rats were swelling and maldistribution, with the density increased or decreased, endomembrane became into thickening and abound with inflammatory cell infiltration under of that. In addtion, the density of smooth muscle cell was enhanced and arrangement disordered. Under the electron microscope, most of cristae and endomembrane of mitochondria in endothelial cells were fusion or disappeared, with rough endoplasmic reticulum expanding into spherical or oval-shap and obvious degranulation phenomenon, at the same time, the amount of pinocytosis vesic was reduced significantly. (5)the index detection correlated to vascular endothelial function: compared with normal control group, the contents of ET in rats'blood plasma and vWF/sICAM-1 in rats'serum were all increased significantly for HCY group and complex model group (P<0.01 or P<0.05), however, the contents of NO in serum were decreased obviously (P<0.01). In total, this complex model has both the appearance of collateral-Qi deficiency and stagnancy and character of endothelial cells dysfunction, reflecting the conjunction of"diseases"and"syndrome of TMC", so, the establisment of model was successful and it supplied the experimental platform for approaching its pathophysiologic mechanism more deeply.The morphous of aortic tunica intima (endothelial cells) in syndrome model group rats was injury in different degree, furthermore, the level of NO in serum was decreased and the level of vWF/sICAM-1 were increased significantly, all of these had indicated that vascular endothelial dysfunction could be induced by only collateral-Qi deficiency and stagnancy. After combinating syndrome of TCM with HHcy, the sICAM-1contents in serum of complex model group rats were increased (P<0.05), although there were no chang obviously in the level of ET in plasma and NO/vWF in serum compared to HCY group rats. One hint form that was non-linear superposition effect between both of syndrome of TCM and HHcy which could injury endothelial cells more seriously.Three category dredge-collateral herbs all could improve complex model rats'biology superficial syndrome, prolong rats'exhaused swimming time and cut down rats'respiratory frequency/heart rate, displayed its ascendency in relieving the appearance of collateral-Qi deficiency and stagnancy, group comparison was no difference (P>0.05), but Simvastatin had no effect. In the aspect of improving vascular endothelial function and repairing patho damage, these dredge-collateral herbs all could lessen aortic tunica intima (endothelial cells) pathomorphology injuries in complex model rats as well as Simvastatin, cut down the contents of ET in plasma and vWF/sICAM-1 in serum (p<0.01 or P<0.05), accordingly, to protect vascular endothelial function. By the way, the contents of NO in Tongxinluo group rats'serum was increased compared with HCY group, but no change in the Ginseng group and Siweitongluo group, manifested that compound recipe had more extensive regulative effect in improving endothelial function.2. The general NEI network change of complex model rats and effect of dredge-collateral herbsHypothalamic-pituitary-adrenal axis: compared with normal control group, the contents of CORT in HCY group rats'serum were increased significantly; in syndrome model group, the contents of CRH in rat's hypothalamus were decreased (P<0.01) and ACTH in plasma was increased obviously (P<0.01), and the same results were standed out in complex model group (P<0.01); compared with the HCY group, the contents of ACTH in plasma and CORT in serum were all increased obviously for complex group rats.Hypothalamic-pituitary-thyroid axis: compared with normal control group, the contents of TRH in hypothalamus and TSH in serum were all no change (P>0.05) for HCY group and syndrome model group rats, however, the contents of T3/T4 in serum all were decreased (P<0.05 or P<0.01); in complex model group, the contents of TRH in hypothalamus and TSH/ T3/ T4/ in serum were all decreased notably (P<0.05 or P<0.01). Compared with HCY group, the contents of TRH in hypothalamus and TSH/T4 in serum were decreased for complex group rats (P<0.05 or P<0.01).Immunity cytokines in peripheral blood: compared with normal group, the contents of IFN-γin HCY group rats'serum were increased significantly (P<0.05), but the contents of IL-1βand TNF-αwere no changed (P>0.05); in syndrome model group, the contents of IL-1βand TNF-αwere increased obviouly (P<0.05 or P<0.01), but there was no changed in IFN-γ(P>0.05); in complex model group, the contents of above cytokines were all increased (P<0.05 or P<0.01) and had signicicant deviation compared with HCY group (P<0.05 or P<0.01).According to the above results, both of HHcy and"collateral-Qi deficiency and stagnancy"could have an influence in HPAA, HPTA and immunity correlation factors, at the same time, they could have non-linear superposition effect. In this study, the HPAA function of complex model group rats was accentuation, on the contrast, the function of HPTA was under the inhibitory state, furthermore, these immunity correlation factors were extrordinarily increased most obviously.The effect of dredge-collateral herbs: compared with complex model group, the contents of ACTH in Ginseng rats'plasma were decreased (P<0.05); the contents of CRH in hypothalamus were increased in Siweitongluo group and Tongxinluo group rats (P<0.05 or P<0.01), respectivly, with the level of CORT in serum decreased (P<0.01); the contents of ATCH in Tongxinluo group rats'plasma were decreased obviously (P<0.01); however, there were no any change about all of the index in the Simvastatin group rats (P>0.05). Compared with complex model group, the contents of T3/T4 in Gingeng group rats'serum were increased significantly (P<0.05); in the Siweitongluo group, all the index were increased (P<0.05 or P<0.01) except that the conternts of T3 in serum was no changed; All the above index were increased in Tongxinluo group rats (P<0.05 or P<0.01). Compared with complex model group, the contents of IFN-γ/TNF-αin Ginseng serum were decreased mignificantly (P<0.05 or P<0.01); the contents of TNF-αalso were decreased in Siweitongluo group rats'serum; all the immunity cytokines detected were decreased in Tongxinluo group rats'serum (P<0.05 or P<0.01); Finally, the contents of IL-1β/TNF-αin Simvastatin group rats'serum were cut down (P<0.05). These results have proved that different category dredge-collateral herbs could regulate NEI network functional disorder of complex model rats in different degrees, among of them, Tongxinluo's effect was the most extensive and significant. Regretly, the effect of Simvastatin was no good.The canonical correlation analysis between these biochemical indicators of endothelial dysfunction and common chemical signal molecules in NEI network showed that there had statistical significance in the first canonical correlation coefficient (P<0.01), indicated their intimate correlation both of NEI network dysfunction and vascular endothelial dysfunction.3. The immunity mechanism of complex model rats vascular endothelial cells injury and the effect of dredge-collateral herbs Compared with the normal control group, the contents of AECA in syndrome model group rats'serum were increased, but no significant difference in statistics (P>0.05); however, it was increased significantly in HCY group and complex model group (P<0.01) and group comparison of both group was no difference (P>0.05). Compared with normal control group, the positive staining gray scales of endothelial MHC-Ⅱ/ ICAM-1/ VCAM-1 in HCY group and syndrome group and complex model group rats were all decreased obviously (P<0.05 or P<0.01).It could be showed that the increased contents of AECA in serum and enhanced expression of MHC-Ⅱ/ ICAM-1/ VCAM-1 in artery had promoted and participated in the proceed of complex model rats'vascular endothelial dysfunction. Perhaps, these abnormal molecular expression was one of immunity foundation which induced in vascular endothelial dysfunction.All of the dredge-collateral herbs could make the contents of AECA in serum degraded significantly and restrain the overexpression of MHC-Ⅱ/ ICAM-1/ VCAM-1 in aortic endothelial cells of complex model group rats, interestingly, there were no difference compared with Simvastatin.4. TNF-αinduce HUVEC to secrete ET-1 and eNOS: p38 MAPK singal pathway play an import role and the effect of dredge-collateral herbsFrom the beginning of 2 hours, different concentration TNF-αcould induce ET-1 expression of HUVEC supernatant to increase in different degrees with the time prolong (P<0.05 or P<0.01) and achieve the peak amplitude at 12h. Compared with the normal control group (0ng/ml), different concentration TNF-αcould make the ET-1 of cell supernatant increased in 4h, 8h and 12h (P<0.05 or P<0.01), the concentration of 10 ng/ml was the most significant, yet no different among of them (P>0.05). From the beginning of 4 hours, different concentration TNF-αcould induce eNOS expression of HUVEC supernatant to decrease in different degrees with the time prolong (P<0.05 or P<0.01); Compared with the normal control group (0ng/ml), the different concentration of TNF-α(5～50ng/ml) could make the eNOS of cell supernatant decreased in 4h, 8h and 12h (P<0.05 or P<0.01), but no different among of them (P>0.05).Compared with the normal control group, the expression of ET-1 protein and mRNA in TNF-αintervention cells was increased obviously (P<0.05 or P<0.01), on the other hand, the expression of eNOS protein and mRNA was decreased obviously (P<0.01); compared with TNF-αgroup, B203580, one special inhibitor for p38 MAPK, and all of medication groups could cut down the intracelluar expression of ET-1 protein and mRNA (P<0.05), meanwhile, heighten the expression fo eNOS protein and mRNA (P<0.01).Compared with normal control group, TNF-αcould induce the expression of intracelluar p-p38 MAPK protein to increase in different time (P<0.01). Compared with TNF-αgroup, the SB203580 could depress p-p38 MAPK expression significantly at 10min and 30min, respectivly (P<0.05 or P<0.01), but the inhibitory action was quiet at 60min. Both of Tongxinluo and Simvastatin could depress the p-p38 MAPK expression induced by TNF-αat all different time (P<0.01), it was the most obvious at 60min (P<0.05). These results indicated sufficiently that the signal pathway of p38 MAPK palyed an important role in the process of TNF-αinducing vascular endothelial cells injury. Meanwhile, Tongxinluo could depress intracelluar p38 MAPK phosphorylation and activation induced by TNF-αin different time, demonstrated that its action mechanism of improving endothelial function was concerned with p38 MAPK signal transduction pathway.Conclusion:1. The establishment of collateral-Qi deficiency and stagnancy rat model due to excessive easy and comfortable is uniqueAccording to the"excessive rest on bed impairing Qi"pathogenesis of TCM and status quo that some persons whose living habit was excessive easy and comfortable and easily to induce cardiovascular and cerebrovascular diseases, the establishment of collateral-Qi deficiency and stagnancy rat model due to excessive easy and comfortable is unique in this study, distinguished from using the fatigue method to induce syndrome of deficiency of Qi formerly. It will be useful for approaching the above crowd vascular diseases'pathophysiologic mechanism and clinical therapy more deeply.2. The establishment of complex model named with vascular endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type provide an experimental platform for studying its pathophysiologic mechanismPathological model was been established by using HHcy to injury vascular endothelium firstly, according to the TCM doctrine of"excessive rest on bed impairing Qi", the factor of hyperalimentation diet combinated with activity restraint was exerted into the pathological model and set up a complex model. The results show off that rats'biology superficial syndrome were changed obviously, exhausted swimming time were shorten and respiratory frequency and heart rate were decreased significently. So, these appearances could basically reflect the characteristic of collateral-Qi deficiency and stagnancy writed in the diagnostic criteria of"Collateral-Vascular system diseases"; the pathematology observation of aortic tunica intima (endothelial cells) and blood biochemical indicators detection confirmed that vascular endothelial dysfunction was existed in the complex model rats. Aggregate analysis indicated that the establishment of complex model was successful and it supplied a good experimental platform for pathophysiologic mechanism research.3. Both of general NEI network regulative disequilibrium and regional vascular endothelial cells immunity injury are common important pathophysiologic mechanism of vascular endothelial dysfunction genesis and developmentIn this study, these critical chemical signal molecules related to NEI network in complex model vivo were changed abnormally, including the HPAA function accentuation, the HPTA function restraint and immunity cytokines abnormal increased. The canonical correlation analysis confirmed that it was related to vascular endothelial dysfunction, indicating general NEI network regulative disequilibrium come into being an important pathophysiologic mechanism of vascular endothelial dysfunction genesis and development under the state of collateral-Qi deficiency and stagnancy. At the same time, the contents of AECA in serum and expression of MHC-Ⅱ/ ICAM-1/VCAM-1 in artery were increased or enhanced, that was not only immunity foundation of vascular endothelial injury but also one of partial pathophysiologic mechanism in vascular endothelial dysfunction genesis and development.4. The TCM syndrome factor of collateral-Qi deficiency and stagnancy play an important role in the development of vascular endothelial dysfunctionThe TCM syndrome factor of collateral-Qi deficiency and stagnancy could induce not only vascular endothelial dysfunction but also NEI network regulative disequilibrium and reinforce the expression of MHC-Ⅱ/ ICAM-1/ VCAM-1 in vascular endothelial cells. The non-linear superposition effect comed from syndrome factor and HHcy could injury endothelial cells more seriouly. Therefore, it is very important to prevent and cure vascular diseases that correcting infaust living habit and style, adjusting food and drink construction and enhancing physical exercise appropriately.5. The dredge-collateral herbs display sufficiently an integration regulative dominance for vascular endothelial dysfunctionThree different category dredge-collateral herbs all could improve the TCM syndrome of collateral-Qi deficiency and stagnancy in complex model rats obviously, lessen endothelial cells injury degrees and protect endothelial function efficiently. Meanwhile, dredge-collateral herbs could depress HPAA hyperfunction, reinforce HPTA function and cut down abnormal increased immunity cytokines of complex model rats in different degrees, accordingly, to improve NEI network disequilibrium state. At the same time, under the regulation of dredge-collateral herbs, the contents of AECA in serum and adhesion molecules in aortic endothelial cells, such as MHC-Ⅱ/ICAM-1/VCAM-1 all were decreased. So, it could be extracted that dredge-collateral herbs'critical mechanism of action for improving endothelial dysfunction belong to collateral-Qi deficiency and stagnancy type was related to regulate general NEI network equilibrium and regional immunologic injury mechanism. Therefore, duing to the effect of not only improving efficiently"region/vascular endothelial dysfunction"but also regulating"whole/NEI network", dredge-collateral herbs have more therapy dominance than positive control drugs which improve regional pathological injury merely.6. The research of cellular level offer experimental proofs for illuminating molecular mechanism of correlation between critical chemical signal molecules abnomal change in NEI network and vascular endothelial dysfunctionIn this study, TNF-αcould induce intracellular expression of ET-1 protein and mRNA and restrain expression of eNOS protein and mRNA in HUVEC by the signal transduction pathway mediated by p38 MAPK and thereby lead to vascular endothelial dysfunction, it offer experimental proofs for illuminating molecular mechanism of correlation between critical chemical signal molecules abnomal change in NEI network and vascular endothelial dysfunction. In addtion, Tongxinluo could cut down abnormal expression of ET-1 in HUVEC cultural supernatant and intracellular, but increase the expression of eNOS. Its action mechanism of improving endothelial function was concerned with impacting on p38 MAPK signal transduction pathway.
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