| Objectives: Directed by the theory of collateral disease,according to diagnostic standard of vessel collateral-vascular system disease established through the clinical epidemiological investigation, to establish the rat model of endothelial dysfunction with collateral-qi stasis applied by chronic constraint stress and homomethionin diet. The rat model was evaluated systematically from different aspects of general condition of health, score of biological superficial syndrome,ethology, and the construction and function of blood vessel endothelium. At the same time, the changes of main indicatrix related to nerve–endocrine-immunization (NEI) network were observed, and the effect of different level of Tongluo drugs including compounds of Chinese herbal, compounds of components and single effective component on the rat model was explored. The purpose of our research was to elucidate the pathomechanism of the model with endothelial dysfunction and collateral-qi stasis, explore the effect of syndrome factor of TCM on the endothelial dysfunction, and evaluate systematically the effect of Tongluo drugs.Methods : 1 The establishment and evaluatement of the rat model of endothelial dysfunction with collateral-qi stasisIn this experiment, the rat model of collateral-qi stasis was established by chronic constraint. The rat model of pathology was established by homomethionin diet. On the basis of the result of experiment (data not shown in the paper) that acute constraint stress could raise blood serum HCY level and decrease NO level, the combinated rat model with endothelial dysfunction and collateral-qi stasis was established by chronic constraint stress and homomethionin diet. Forty healthy male Wistar rats of sanitary degree were randomly divided into 4 groups according to their body weight,①the normal control group, fed with normal diet.②the collateral-qi stasis group,fed with normal diet and given to repeated restraint stress for 6 hours every day.③the pathological model group,fed with 3% methionine diet.④the combinated model group (rat animal models of endothelial dysfunction and collateral-qi stasis), given to repeated restraint stress for 6 hours every day and fed with 3% methionine diet simultaneously. The whole experimental time is 6 weeks. The general condition of health including spirit, coat, appetite, weight was recorded. The test of intake of 1% cane sugar water at the beginning and endpoint of the experiment was measured. At the endpoint of experiment, the score of biology superficial syndrome, open-field and tail swing were carried out. The concentration of plasma endothelian (ET)was detected with radioimmunoassay, and serum nitro oxygen (NO) was detected with nitrate reductase. The morphological changes of aorta thoracica observed by light microscope and the ultramicrostructure changes of aorta observed by transmission electron microscope. Immunohistochemistry method was used to analyze the expression of endothelin-1(ET-1) in aorta tissue.2 The changes of NEI network of the rat model of endothelial dysfunction with collateral-qi stasisExperimental animal, the method of dividing groups and establishing model were the same as aboves. The concentrations of CRH, ACTH, CORT, PRA, AngII, ALD and serum IL-1β, IL-2, IL-6, TNF-αwere detected with radioimmunoassay. The concentration of NE, E,γ-INF, 5-HT, DA were detected with ELISA. The index number of thymus and spleen were calculated.3 Effect of Tongluo drugs on the combinated model ratsFifty healthy male Wistar rats of sanitary degree were randomly divided into 5 groups according to their body weight,①the combinated model group ,②Benazepril group ,administered at 10mg/Kg/d.③Xiebai group, administered at 1.2g crude drug /Kg/d.④Xiebaisiwei group, administered at 3.47g crude drug /Kg/d.⑤Tongxinluo group, administered at 1.2g crude drug/Kg/d. The time and method of established model were the same as aboves. The time of intragastric administration is 6 weeks. The combinated model group was given with 0.5%CMC-Na. The changes of biology superficial syndrome, the morphological changes and the ultramicrostructure changes of aorta, blood vessel endothelial function and the changes of NEI nethwork were detected.4 The research of expression of glucose regulating protein78(GRP78) mRNA and protein in aortic tissue of the combinated model rats and effect of Tongluo drugs on itEighty healthy male Wistar rats of sanitary degree were randomly divided into 8 groups according to their body weight,①the normal control group,②the collateral-qi stasis group,③the pathological model group,④the combinated model group,⑤Benazepril group,⑥Xiebai group,⑦Xiebaisiwei group,⑧Tongxinluo group. The method of establishing model and intragastric administration were the same as aboves. The expression of GRP78 mRNA and protein in aortic tissue of every group rats was detected by RT-PCR and WesternblotResults:1 The establishment and evaluatement of the rat model of endothelial dysfunction with collateral-qi stasis1.1 The general condition of health and score of superficial syndromeCompared with normal control group, the rats of collateral-qi stasis group showed depressed, reduction of diet, torpidity, lost of color pattern, increasing slowly weight, while the rats of pathological model group showed reduction of diet, lost of color pattern and increasing slowly weight except with obvious depressed. The rats of combinated model showed lazy, drowsiness, depressed and reduction of aggressive behavior.Compared with normal control group, the score of biology superficial syndrome of the collateral-qi stasis group and the combinated model group increased significantly (P<0.01). Compared with the pathological model group, the score of the combinated model group increased (P<0.05).Compared with normal control group, the appetite of the collateral-qi stasis group, the pathological model group and the combinated model group were significantly more lowered (P<0.01). And the body weight increased slowly (P<0.01). Compared with the pathological model group and collateral-qi stasis group, the changes of the combinated model group was obviously lowered (P<0.01).1.2 The result of ethologyCompared with the normal control group, the score of the open-field of the collateral-qi stasis group, the pathological model group and the combinated model group had no difference.Compared with normal control group, the immobility time of the tail swing of the collateral-qi stasis group and the combinated model group were prolonged significantly and the struggling frequency were reduced significantly (P<0.01), the pathological model group had no difference.Compared with normal control group, the intake of cane sugar water of the collateral-qi stasis group, the pathological model group and the combinated model group were significantly lowered (P<0.01 or P<0.05).1.3 The changes of function and morphous of blood vessel endothelium in every groupCompared with the content of ET(142.91±8.72pg/ml)in plasma of rats in normal control group, the content (163.19±24.74 pg/ml) of the collateral-qi stasis group, the content (161.70±13.96 pg/ml)of the pathological model group and the content(178.25±21.85 pg/ml) of combinated model group were significantly increased (P<0.05). Compared with the content of NO(45.22±12.42umol/L) in serum of rats in normal control group, the content (25.68±10.92 umol/L) of the collateral-qi stasis group, the content(26.82±13.03 umol/L) of the pathological model group and the content(24.91±9.95 umol/L) of combinated model group were significantly lowered (P<0.05 or P<0.01). The difference between the combinated model group and the collateral-qi stasis group was not obvious. Compared with the normal control group, the content of HCY in serum of rats in the collateral-qi stasis group, the pathological model group and the combinated model group increased significantly (P<0.05).HE staining showed that:The collateral-qi stasis group: endothelial cells were lightly engorge. Smooth muscle were lightly edema.The pathological model group: endothelial cells were engorge, maldistribution. The density increased. There were many lymph-cell and inflammatory cells in the tunica intima. Smooth muscle were edema.The combinated model group: endothelial cells were engorge, maldistribution. The density increased. There were many lymph-cell and inflammatory cells in the tunica intima. Smooth muscle were obvious edema.Immunohistochemical staining showed that:The expression of ET-1 was not founded in the arterial tissue of the control group.The expression of ET-1 in the arterial tissue of the collateral-qi stasis group, the pathological model group and the combinated model group were enhanced.Transmission electron microscope showed that:The collateral-qi stasis group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum was broaden. The phenomenon of degranulation was obvious. The number of pinocytosis vesic was significantly decreased.The pathological model group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum was lightly broaden. The phenomenon of degranulation was obvious. The number of pinocytosis vesic was decreased.The combinated model group: some crests of mitochondria of vascular endothelial cells fused with membrance, and even disappeared. Rough endoplasmic reticulum and the pinocytosis vesic could not be founded almost. 2 The changes of NEI network of the rat animal models of endothelial dysfunction with stasis of collateral-qi2.1 Hypothalamic-Pituitary-Adrenal Axis:Compared with normal control group, the CRH level of the collateral-qi stasis group, the pathological model group and the combinaed model group had no statistical difference (P>0.05). The ACTH level reduced and the CORT level increased significantly(P<0.01). Compared with the collateral-qi stasis group and the combinated model group, the ACTH level of the combinated model group had no difference(P>0.05). And the CORT level increased significantly (P<0.01).2.2 Renin-Angiotensin-Aldosterone System:Compared with normal control group, the PRA and AngII level of the collateral-qi stasis group and the combinated model group increased significantly(P<0.01or P<0.05). The difference between the collateral-qi stasis group and the pathological model group was not obvious(P>0.05). Compared with the collateral-qi stasis group and the combinated model group, the ALD level of the combinated model group increased significantly(P<0.01).2.3 Trisplanchnic Nerve -Adrenal Medulla System:Compared with normal control group, the NE, E level of the collateral-qi stasis group and the combinated model group increased significantly(P<0.01). the difference between the pathological model group and the control group was not obvious(P>0.05). Compared with the pathological model group, the NE, E level of the collateral-qi stasis group and the combinated model group increased(P<0.01 or P<0.05).2.4 Monoamine neurotransmitter:Compared with normal control group, the 5-HT, DA level of the collateral-qi stasis group, the pathological model group and the combinated model group decreased significantly (P<0.01). The difference between the pathological model group and the collateral-qi stasis group was not obvious(P>0.05). Compared with the collateral-qi stasis group, the 5-HT, DA level of the combinated model group decreased(P<0.01or P<0.05).2.5 The index number of spleen and thymus:Compared with normal control group, the index number of spleen and thymus of the collateral-qi stasis group lowered (P<0.05), the index number of spleen of the pathological model group and the combinated model group increased (P<0.01), the index number of thymus lowered(P<0.05). The index number of thymus of the combinated model group was the lowest in four groups.2.6γ-INF, TNF-α, IL-1β, IL-2, IL-6:Compared with normal control group, theγ-INF level of the pathological model group and the combinated model group lowered(P<0.01). Compared with the collateral-qi stasis group, theγ-INF level of the combinated model group lowered (P<0.05). There were no obvious difference on the level of TNF-α, IL-1β, IL-2 and IL-6 among the four groups (P>0.05).3 Effect of Tongluo drugs on the combinated model rats 3.1 Score of biological superficial syndrome ethology:After 6 weeks of administration, compared with the combinated model group, the score of biological superficial syndrome of Benazepril group had no obvious change(P>0.05). The score of Xiebai group, Xiebaisiwei group and Tongxinluo group decreased significantly(P<0.01 or P<0.05). Compared with Benazepril group, the three groups decreased(P<0.01). There were no obvious difference in three groups(P>0.05).Compared with the combinated model group, the body weight of Benazepril group had no obvious difference(P>0.05). Compared with the combinated model group and Benazepril group, the weight of Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly(P<0.05 or P<0.01 ).Every drug group could increase the appetite of the combinated model rats.Compared with the combinated model group, the intake of cane sugar water of Benazepril group had no obvious difference(P>0.05). Compared with the combinated model group and Benazepril group, the intake of Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly(P<0.01).Compared with the combinated model group, the struggling frequence of Benazepril group increased(P<0.01), the immobility time decreased, but it had no difference(P>0.05). The immobility time of three Chinese medicine shortened, the struggling frequence increased(P<0.01). They had obvious difference with Benazepril group(P<0.05).3.2 Endothelial function and morphous:Compared with the combinated model group, ET level of every medicine groups decreased significantly(P<0.01 or P<0.05). There were no obvious difference in every medicine groups. NO level of Benazepril group, Xiebaisiwei group and Tongxinluo group increased, but it had no obvious difference(P>0.05). NO level of Xiebai group increased(P<0.01). Compared with Benazepril group, NO level of Xiebai group increased(P<0.05).Compared with the combinated model group, every medicine group could improve the general structure and endothelial cell ultramicrostructure of aorta throacica, especially Tongxinluo group did.3.3 The indexs related to NEI networkMonoamine neurotransmitter: Compared with the combinated model group, 5-HT level of every drug group increased, especially Xiebai group, Xiebaisiwei group and Tongxinluo group increased significantly (P<0.01). There was no obvious difference between Benazepril group and the combinated model group(P>0.05). DA level of every drug group increased obviously(P<0.01).Hypothalamic-Pituitary-Adrenal Axis: Compared with the combinated model group, CORT level of Benazepril group, Xiebaisiwei group and Tongxinluo group decreased significantly(P<0.01 or P<0.05), Xiebai group had no obvious difference(P>0.05). Compared with Benazepril group, the CORT level of Xiebaisiwei group and Tongxinluo group decreased (P<0.01). The difference between Xiebaisiwei group and Xiebai group was obvious(P<0.01).Trisplanchnic Nerve -Adrenal Medulla System: Compared with the combinated model group, NE and E level of every drug group decreased(P<0.01 or P<0.05). Compared with Benazepril group, NE level of Xiebaisiwei group decreased(P<0.01). E level of Xiebaisiwei group and Tongxinluo group decreased (P<0.01 or P<0.05). The effect of Xiebaisiwei group in decreasing NE level was better than Xiebai group (P<0.01).Renin-Angiotensin-Aldosterone System: Compared with the combinated model group, PRA, AngII and ALD level of every drug group decreased. Compared with the Benazepril group, AngII level of Xiebai group decreased(P<0.05), ALD level of Xiebaisiwei group decreased( P<0.05). Compared with Xiebai group, ALD level of Xiebaisiwei group and Tongxinluo group decreased(P<0.05 or P<0.01).Cell factor, thymus and spleen index number: Compared with the combinated model group,γ-INF level of every drug group increased(P<0.05 or P<0.01). The effect of increasingγ-INF of Tongxinluo group was better than Xiebai group( P<0.05). Compared with the combinated model group, TNF-αlevel of every drug group decreased and the index number of thymus increased , but they had no obvious difference (P>0.05). Compared with the combinated model group, the index number of spleen, IL-1β, IL-2 and IL-6 of every drug group had no obvious difference(P>0.05).4 The expression of GRP78 in aortic tissue of rats with endothelial dysfunction and collateral-qi stasis and effect of Tongluo drugs on itCompared with the control group, the expression of GRP78 mRNA of the collateral-qi stasis group, the pathological model group and the combinated model group strengthened significantly (P<0.05). There was no obvious difference among three groups.Compared with the control group, the collateral-qi stasis group and the pathological model group, the expression of GRP78 protein of the combinated model group strengthened significantly (P<0.05).Compared with the combinated model group, the expression of GRP78 mRNA and protein of every medicine group were repressed obviously(P<0.05 or P<0.01). There was no difference among drug groups(P>0.05).Conclusion:1 Establishment and evaluation of rat model with endothelial dysfunction and collateral-qi stasisAccording to diagnostic standard of vessel collateral-vascular system disease established through the clinical epidemiological investigation, The combinated rat animal model of endothelial dysfunction and collateral-qi stasis was established successfully with the method of chronic constraint stress and homomethionin diet on the basis of the fact that acute constraint stress could raise blood serum HCY as foundation. The model rats were evaluated synthetically by many semi-quantitative and quantitative methods including the general condition of health, the score of biology superficial syndrome, indicatrixes of ethology, the construction and function of blood vessel endothelium. The study indicated that the combinated model could show macroscopic syndrome of collateral-qi stasis and the changes of construction and function of blood vessel endothelium. This showed that the establishment of combinated model was successful. The study showed that syndrome of collateral-qi stasis of TCM played an important role influencing endothelial function.2 The changing characteristics of NEI network of rat model with endothelial dysfunction and collateral-qi stasisOn the basis of analysis and investigation of NEI network in rats of normal control group, collateral-qi stasis model, pathology model and combinated model of collateral-qi stasis with endothelial dysfunction, to explore the changing characteristics of NEI network, and analyze the effect of the syndrome facor of collateral-qi stasis on the vasculopathy. The study showed that: hypothalamic-pituitary-adrenal axis and renin-angiotensin-aldosterone system were activated in different degree, and Monoamine neurotransmitter and the ability of immune system inhibited in collateral-qi stasis group, pathological model group and the combinated model group. Trisplanchnic nerve -adrenal medulla system was activated both the collateral-qi stasis group and the combinated model group, while the trisplanchnic nerve -adrenal medulla system in the pathological model group was not activated. The combinated model showed nonlinear superposition effect between the collateral-qi stasis model and the pathology model. The result indicated that syndrome of collateral-qi stasis of TCM paid no attention previously was the important influcence factor. The disorder of indexes related to NEI network was possible pathological basis of rat model with endothelial dysfunction and collateral-qi stasis..3 The effect of Tongluo drugs on the combinated model with endothelial dysfunction and collateral-qi stasisThe combinated model were cured with Xiebai, Xiebaisiwei and Tongxinluo. Three species of Tongluo drugs could improve general condition of health and score of superficial syndrome, raise the intake of cane sugar water, and improve depressed emotion and endothelial function in the combinated model rat. At the same time, they could repress the activations of hypothalamic-pituitary-adrenal axis, trisplanchnic nerve -adrenal medulla system, renin-angiotensin-aldosterone system, raise Monoamine neurotransmitter and enhance the ability of immune system. The study also showed that the effect of Chinese herbs was better than Benazepril in improving praxiology, endothelial function and regulating NEI network. The effect of Tongxinluo was better than any other medicines, which was related to compatibility of Tongxinluo due to the pathological link of vasculopathy. This results authenticated determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs was scientific, and disease-syndrome-method-drug of TCM was dependent. The effect of Tongluo drug on the combinated model with endothelial dysfunction and collateral-qi stasis showed regulation mechanism of re-establishing balance of multi-system in body through the improving the disorder state of multi-system belong to the NEI network in the morbid state, which provided the method to reveal the effective regularity of compound recipe of TCM.4 The change of GRP78 in aortic tissue of the combinated model with endothelial dysfunction and collateral-qi stasisThe recent research indicated that endoplasmic reticulum stress was the common pathway both mitochondrion stress and nuclear stress. Endoplasmic reticulum stress was one self-protective mechanism of cell, while the dysfunction, over- intensity and long-time stress could cause the celluar disorder, even celluar death. The study showed the expression of GRP78 mRNA and protein of the collateral-qi stasis group and the combinated model group was strengthened significantly. There species of Tongluo drugs could reduced the expression of GRP78 mRNA and protein of combinated model group, which was not different with Benazepril. The result indicated the endoplasmic reticulum stress was the one of common pathways of syndrome of collateral-qi stasis and the combinated model with endothelial dysfunction and collateral-qi stasis. There species of Tongluo drugs might improved the endothelial function through reducing the expression of GRP78 mRNA and protein of combinated model group.
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