| Objective: Under the guidance of Collateral Disease Theory, the TCM quantitative standards of differentiation of symptoms and signs for classification of syndrome on vascular endothelial dysfunction (VED) were established. On the base of the above, the animal model and the cell model of collateral-QI deficiency type with VED were made. The changes of VED and the imbalance of NEI network due to collateral-QI deficiency and the internal association between them were investigated. Meanwhile, the effect of serum from combined model rats on the endothelial cytoskeletal protein, F-actin, and the permeability of cultural monoptychial endothelium were observed. The effect ofcollateral-QI deficiency on endothelial cells and the mechanism of the different kinds of Herbs dredging collaterals were researched. All of the work will provide the foundation of treatment and prevention of vascular diseases by collateral disease theory of Traditional Chinese Medicine (TCM)Methods:1 The establishment of syndrome standards for classification of TCM differentiation of symptoms and signs on VED"The clinical questionnaire of TCM syndrome of vascular endothelial dysfunction"was established on the basis of literature research, expert consultation, and grades and scores of examination by TCM four methods . Then clinical epidemiological investigations were carried out. The inclusive criteria is as follows:①the age is above 40 years old;②at least with one of diagnosis as folloiwing: simple hypertension, hyperlipidemia, overweight or obesity, smoking, and metabolic syndrome;③NO<59umol /L and ET>53.38pg/ml. The case will be eliminated with one of diagnosis as folloiwing:①target organ damages such as heart, brain, kidney, nerve, ocular fundus;②serious liver or kidney dysfunctions;③pregnancy or breast-feeding women or psychotic patients. The special messenger was responsible for the investigation. The database was established using Epidata3.0. Four hundred patients selected were randomly divided into Group A and Group B, with 300 cases in Group A and 100 cases in Group B. The data of Group A were used for the establishment the quantitative standards of differentiation of symptoms and signs and retrospective test. The data of Group B were used for the prospective test of this standard. The clinical investigating data of 300 cases in A group were analyzed with entropy-based partition method for complex systems. According to the results of symptoms clustering, the TCM information of symptoms of VED was extracted, and the syndromes were summarized. The contributing scores of each symptom to its syndrome were calculated. ROC curve analysis method was applied to determine the threshold of syndromes. The retrospective and prospective tests of quantitative standards for differentiation of syndromes were conducted according to the research methods in clinical epidemiology diagnostic test. The combination rule and pathogenesis converting rule of syndromes were summed up through analyzing the correlative values between various syndromes.2 Establishment of rat models of VED due to collateral-QI deficiency and the intervention research of Herbs dredging collaterals.According to the TCM standards established, under the guidance of TCM theory of "over-strain consuming essence" and "hunger damaging essence". rats in our study were given homomethionin diet to establish VED models, then"Basic Diet combined loaded Swimming"was applied to superimpose the syndrome of collateral-QI deficiency. As a result, the model of combining disease and syndrome, complex model, was established. Male Wistar rats were randomly divided into the following seven groups, and there were 15 rats in each group : (1) control group; (2) pathological model group (HCY group); (3) complex model group (stagnancy of collateral-QI deficiency + HCY); (4) Ginseng group (complex model + Ginseng); (5) Siweitongluo group (complex model + Siweitongluo); (6) Tongxinluo group (complex model + Tongxinluo); (7) Benazepril group (complex model + benazepril). These models were evaluated by the methods of quantitation or half-quantitation score of the biological superficial syndrome, patho-histomorphology and biochemistry function of vascular endothelium. Different kinds of dredge collaterals herbs including singal medicine-Radix Ginseng, compatibility of composition medicine-Siweitongluo,complex dredge collaterals medicine-Tongxinluo were used to treat the rats of complex model in this study. Compared with the effect of Benazepril, the effects of Herbs dredging collaterals on the complex model were observed.3. The function of NEI network in model rats with VED due to collateral-QI deficiency and the intervention research of Herbs dredging collateralsIn the complex models, Sympathoadrenomedullary systems including NE and E were detected by ELISA; hypothalamus-hypophysis cerebri-adrenal function axle including CRH and ACTH in the blood plasma, and CORT in the serum were detected by RIA; renin-angiotonin-aldosterone system including PRA, AngⅡand ALD in the blood plasma were detected by RIA ; and immune system including IL-1βand IL-2 in the serum were detected by RIA. The correlation between endothelial function system index (ET, NO, VWF) and the NEI network system correlative factors was analyzed with canonical correlative analysis method. At the same time, the effects of Herbs dredging collaterals were studied.4 The cell model was established by HUVEC cultivated with serum from rats of complex model, in which the expression of cytoskeleton protein and the effects of the Herbs dredging collaterals were investigated In the study, HUVEC cell strain was cultivated with serum from rats of complex model. The morphological character and distribution of cytoskeletal protein, F-actin, were observed by laser confocal microscopy, the contents of F-actin were observed by flow cytometry. The permeability of monoptychial endothelium was observed thorough BSA marked by Evans blue. The expression of F-actin, p38 as well as p-p38 protein in HUVEC cells were detected by Western blot. The changes of p38 MARK signal pathway were detected by Western blot. The intervention effects of three categories of Herbs dredging collaterals were observed. The experimental cells were divided into seven groups: (1) control group: fetal bovine serum with a final concentration of 20% was added to serum-free medium (F12K); (2) normal serum group: serum from normal rats with a final concentration of 20% was added to serum-free medium (F12K); (3) complex model serum group: serum from rats of the complex model with a final concentration of 20% was added to serum-free medium (F12K); (4) Tongxinluo group: Tongxinluo ultramicro-pulverization (final concentration: 1μg/ml) was added into serum-free medium (F12K) and incubated for 2h; then the serum from rats of the complex model with a final concentration of 20% was added; (5) SB203580 group: SB203580 (final concentration: 25μmol/L) was added into serum-free medium (F12K) and incubated for 1 h, then the serum from rats of the complex model with a final concentration of 20% was added; (6) Ginseng group: Ginseng (final concentration: 1μg / ml) was added into serum-free medium (F12K) and incubated for 2 h, then the serum from rat of the complex model with a final concentration of 20% was added; (7) the group of Herbs dredging collaterals : each kind of the four Herbs dredging collaterals (final concentration: 100μg/ ml) was added into serum-free medium (F12K) and incubated for 2 h, then the serum from rats of the complex model with a final concentration of 20% was added. The study of the cell culture was performed at time 2 h, 6h, 12h, and 24h after the adding of serum.Results: 1 The standards for TCM differentiation of syndroms of VED:Based on the entropy-based partition method for complex systems, the differentiation of symptoms and signs of the 400 cases of VED disease was analyzed by us. of The results were as following. TCM differentiation of syndroms of VED including stagnancy of collateral-QI, stagnancy of collateral-QI deficency, heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, yang asthenia, and so on. Combined the contribution scores of symptoms to its syndrom with the ROC curve, the threshold and the differentiation of symptoms and signs for classification of syndrome standard are concluded (the syndrome can be decided if the totle contribution scores of the every symptom exceed or equal the the threshold).Basic Syndromes:Syndrome with stagnancy of collateral-QI (5): chest distress 3, sigh 3, dysphoria 3, depressed emotion 3, stringy pulse 2; Syndrome of stagnancy of collateral-QI deficency (6): weakness 3, spiritlessness 2, short breath 2, palpitation 2, tiredness with talk 1, pale tongue 1, weak pulse 1. Accompanied Syndromes:Syndrome of heat retention (7): dry mouth 3, bitter taste of mouth 3, flushing 2, reddish urine 2, constipation 2, red tongue 1, yellowish fur 1, fast pulse 1; Syndrome of turbid-phlegm (5): chest distress and stomach gas pains 3, limb-exhausted 3, corpulent body 1, excessive phlegm 1, white and greasy fur 2, slippery pulse 1; Syndrome of heat-phlegm (4): cough and spit yellowish phlegm 2, bitter taste of mouth 2, yellow and greasy fur 1, slippery and fast pulse 1: Syndrome of blood stasis (1): dark tongue 1, unsmooth pulse 1; Syndrome of Yin deficiency (4): tidal fever fever and night sweating 2, dysphoria with feverish sensation in the chest, palms and soles 2, soreness and flaccidity in waist and knees 2, flushed cheeks 1, less or moss fur on the tongue 1, small and fast pulse 1; Syndrome of Yang deficiency(7): chilly 3, cold limbs 3, loose stool 2, urination taking long time and clear urine 1, pale and fat tongue 1, sunken, infrequent and feeble pulse 1.The results of retrospective and prospective tests showed that the sensitivity, specificity and agreement rate of various syndromes were all more than 85%, which means that the threshold of differentiation of syndroms for diagnosis of syndromes have a better diagnosis efficacy. Bases on the correlation analysis among the syndromes , more intension association between the stagnancy of collateral-QI deficency and the heat accumulation, blood stasis, yin asthenia, yang asthenia was observed; and the more intension association between stagnancy of collateral-QI deficency and phlegmatic hygrosis, blood stasis, yin asthenia, yang asthenia was observed the intension association between phlegmatic hygrosis and phlegmatic heat, phlegmatic hygrosis and heat accumulation, heat accumulation and yang asthenia was also observed. Based on the correlation analysis and the distribution rule about the pattern of syndromes, the results were that Syndrome of stagnancy of collateral-QI deficiency accounted for 45.3%, Syndrome of stagnancy of collateral-QI accounted for 55.8%, Syndrome of phlegmatic hygrosis accounted for 17.8%, Syndrome of phlegmatic heat accounted for 22%, Syndrome of blood stasis1 accounted for 3.5%, Syndrome of yin asthenia accounted for 38.3%, Syndrome of yang asthenia accounted for 11.8%. All the above indicated that there were two basic patterns of syndromes, the stagnancy of collateral-QI deficency and stagnancy of collateral-QI, in TCM differentiation of syndromes of VED, in which the heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, and yang asthenia can exist.2 Establishment of the model and the interventing effects of Herbs dredging collateralsModel evaluation was made after the establishment of the model. (1) Biological appearance rats in complex model group were tired with movement, depressed, limb-twisted, moved slowly, and with whitish and dry tail and nose. Compared with the control group, the biological appearance score was increased significantly (P<0.01); both heart rate and respiratory rate were increased significantly (P<0.01) , whereas, climbing-time was decreased significantly (P<0.01). (2) Morphology character of the endothelial cell of thoracic aorta was observed. The results by light microscope showed that endothelial cell swelling, uneven distribution, density increasing, local endothelial cells disappearing, part of endangium thickening, inflammatory cell infiltration in endangium, fracture of inner elastic plate were observed in the complex model group. The results by electron microscope showed that the majority cristae and membrane in mitochondria of endothelial cell were fused or even disappeared, the rough endoplasmic reticulum degranulated obviously and distended to the shape of oval or circle, cytoplasm and cellular nucleus were dropsy, part membrane collapsed and content released from the cell to outside. (3) The results by immunohistochemical staining showed that both ET1 and iNOS were increased significantly, and eNOS was decreased significantly in pathological model group and complex model group. (4) The index about vascular endothelial function. Compared with the control group, the plasma ET, plasma vWF of rats in pathological model group and complex model group were increased significantly (P<0.01, P<0.05), and serum NO content was decreased significantly (P<0.01). In this study, the complex model that combined the disease of collateral-QI deficiency syndrome and the pathological character of VED was made.The effects of Herbs dredging collaterals were studied. (1) The syndromes were improved by the Herbs observed. Compared with the complex model group, all of the biological score, the heart rate, and the respiratory rate were improved at different levels after intervention by the Herbs (P<0.01). The effect of Tongxinluo group was the best. However, no significant effect of benazepril was observed on the syndromes (P>0.05). (2) The morphological character of the endothelial cells of thoracic aorta was observed. Compared with the complex model group, the results of light microscopic examination showed that the pathological damages of intima of all the medication groups were significantly reduced, and the ultrastructural damages of aortic endothelial cells in medication groups were also significantly reduced. (3) Compared with the complex model group, the results by immunohistochemical staining showed that the expression of iNOS and ET1 were increased significantly, and the expression of eNOS was decreased significantly of each medication group. Based on the morphological character of blood vessel endothelium , the effects of Tongxinluo and benazepril were better than those of Ginseng and the four dredge collaterals Herbs. (4) The index of vascular endothelial function was observed. Compared with the complex model group, the plasma ET and plasma vWF were significantly decreased; the level of NO of serum were significantly increased in Tongxinluo group and benazepril group (P<0.01, P<0.05), there were no significant differences between the two groups ( P>0.05); ET were significantly decreased (P<0.01, P<0.05), NO and vWF had no significant differences in Ginseng and Siweitongluo groups. Above all, the effects of Tongxinluo group and benazepril group were better.3 The changes of NEI network in model rats with VED due to stagnancy of collateral-QI deficiency and the intervention effects of Herbs dredging collateralsThe changes of NEI network in model rats with VED due to stagnancy of collateral-QI deficiency were observed. The function of sympathoadrenomedullary system was revealed by the level of NE and E. Compared with the control group, the level of NE in serum of pathological model group and complex model group were increased significantly (P<0.01, P <0.05), the level of E was increased in complex model group (P<0.01), but no significant change was observed in pathological model group. The function of HPAA was revealed by the level of CRH, ACTH and CORT. All of the CRH, ACTH and CORT of complex model group were decreased significantly (P<0.01, P<0.05), however, only CRH of the pathological model group was decreased significantly (P<0.01). The function of RAAS was revealed by the level of PRA, AngII, and Ald. All of the PRA, AngII, and Ald of complex model group were increased significantly (P<0.01, P<0.05), however, no significant change was observed in the pathological model group (P>0.05). In the complex model group, immunological cytokines IL-1βwas increased (P<0.01), while IL-2 was decreased (P<0.05). Neither IL-1βnor IL-2 had significant change in the pathological model group (P>0.05). Compared with the pathological model group, ACTH was decreased significantly (P <0.01), whereas, PRA and AngII were increased (P <0.01), and no significant difference of Ald was observed (P>0.05) in the complex model group. Compared with the pathological model group, the level of IL-1βwas increased significantly (P<0.05). All the above showed that the disturbance of NEI network existed in the rats of VED due to stagnancy of collateral-QI deficiency, and the symdrom factors, collateral-QI deficiency, had an important effect on the disturbance of NEI network.The canonical correlation analysis between vascular endothelium functional parameter and correlation factor of NEI network was undertaken. The first canonical correlation coefficiency was of statistical significance (P<0.01), which indicates that the two systems are closely related to each other. The relevance of the two systems is mainly reflected by the relationship between NO, VWF and ACTH, NE, CORT, IL-2, as well as CRH. All the obove showed that there was close interrelation between the imbalance of NEI network and VED.The effects of Herbs dredging collaterals was studied. Compared with the complex model group, the levels of NE and E in serum of all treated groups were decreased in different degrees (P<0.01, P<0.05), except for that the NE had no ignificant differences in benazepri group (P >0.05).Compared with the complex model group, CRH, ACTH and CORT of Tongxinluo group were increased significantly (P<0.05), ACTH WAS increased in Ginseng group, and CORT was increased significantly in siweitongluo group (P<0.05), benazepri had no significant effect. Compared with the complex model group, except for PRA of benazepril group, all of the PRA, AngII, and Ald weredecreased significantly to some extent in Tongxinluo group and benazepril group (P<0.01, P<0.05).. AngII of Ginseng group was decreased significantly (P<0.05). Compared with the complex model group, IL-1βwas decreased significantly in all treated groups (P<0.01), there was no statistical difference among these groups (P>0.05); the level of IL-2 was increased significantly in siweitongluo group and Tongxinluo group (P<0.05), but no significant difference was observed in Ginseng group and benazepril group (P>0.05). It can be conclued that Tongxinluo was more efficient to adjust the function of NEI network.4 The establishment of Cell model of HUVEC cultured by serum from rats of complex model, and observation the expression of cytoskeleton protein by the HUVEC and the effects of the Herbs dredging collateralsThe cytoskeletal protein, F-actin, was observed. In the control group, the F-actin surrounded endothelial cells and formed pykno-periphery. Compared with the control group, there was no significant difference of F-actin at 2h time point; Depolymerization and redistribution of F-actin in endothelial cells were observed in the complex model serum group, which reached peak at 6 h time point. The F-actin depolymerized obviously, F-actin within the cytoplasm was increased obviously, and a lot of stress fibers were observed., Then, the depolymerization and redistribution slightly reduced at 12 h time point, and gradually restored at 24 h time point.The content of F-actin was detected by flow cytometry. Compared with the control group, there was no significant change of F-actin content of normal serum group at each time point; F-actin content of complex model serum group was decreased significantly at 6h and 12h time points (P<0.05 ,P<0.01), However, no significant changes of F-actin in complex model serum group at 2h and 24h time points (P>0.05).The permeability of monolayer HUVEC was detected.. Compared with the control group, the permeability of endothelial cells of complex model serum group was increased slightly at 2h time point. The permeability was significantly increased (p<0.01) at 6h and 12h time points, and then declining trend was observed at 24h time point.. The expression of F-actin, P38, and p-p38 protein in HUVEC was obsertved. Compared with the control group and normal serum group, the expression of F-actin protein was significantly downregulated in complex model serum group (p<0.05), and P-P38 significantly increased (p <0.01),and no obvious change of p38 was observed.The effects of Herbs dredging collaterals were studied. The changes of shape and distribution of F-actin were observed. Compared with the complex model group, the shape and distribution of F-actin in endothelial cells of all medication groups and SB203580 group were improved to various extents at 6h and 12h time points. The effects of Tongxinluo group and SB203580 group were better. Compared with the complex model group, the F-actin contents at 6h and 12h time points of treated groups were increased to varying degrees (P<0.01) except siweitongluo group at 12h time point (P>0.05). Compared with the complex model group, the permeability of monolayer HUVEC of intervention groups was decreased significantly at 6h and 12h time points (p<0.01). Compared with the complex model group, the expression of F-actin protein of the three Herbs dredging collaterals groups and SB203580 group were increased significantly(P<0.05,P<0.01), the expression of p-p38 protein of tongxinluo group and SB203580 group were decreased significantly(P<0.05)at 6h and 12h time points. The function of EC was improved by every herb dredging collaterals. Tongxinluo was the best.Above all, the serum from rats of complex model group induced the redistributing of F-actin and downregulation of F-actin content in HUVEC, and increased permeability of monolayer endothelial cell. p38 MAPK signaling pathway maybe involved in the pathological process. The Herbs dredging collaterals could reaggregate the F-actin and increase its content and reduce endothelial permeability. The protective effects of tongxinluo on endothelial cells may be shown through inhibiting p38 MAPK signaling pathway. Conclusion:1 It is the first time that the standards for Classification of TCM differentiation of symptoms and signs of VED syndromes have been established.In China, it is the first time that the entropy-based partition method for complex systems has been applied to establish the standards for TCM differentiation of syndromes by the investigating method of clinical epidemiology. The results of the study showed that the method could be applied to the research on the standards for TCM differentiation of syndroms, which provides new ideas and approaches for the standardization and normalization of TCM syndromes. The quantitative standards for TCM differentiation of syndromes of VED were established. The disease of VED included two basic syndromes, stagnancy of collateral-QI as well as stagnancy of collateral-QI deficiency, at the same time, accompanied potentially by the heat accumulation, phlegmatic hygrosis, phlegmatic heat, blood stasis, yin asthenia, and yang asthenia. Stagnancy of collateral-QI and stagnancy of collateral-QI deficiency are important in VED.2 The rat complex model combined VED with collateral-QI deficiency was establishedAccording to the TCM standards established, under the guidance of TCM theory of"over-strain consuming essence"and"hunger damaging essence", rats in our study were given homomethionin diet to establish VED models, then"Basic Diet combined loaded Swimming"was applied to evoke the syndrome of collateral-QI deficiency. As a result, The combined disease and syndrome animal models with VED due to stagnancy of collateral-QI deficiency were successfully established, which offered experiment model for the study of pathophysiology mechanisms of the VED.3. The relationship between the disturbance of the function of NEI network and the disfunction of blood vessel endothelium in rats of complex model combined VED with collateral-QI deficiency. The abnormal changes of NEI network in complex model rats was observed, which showed the hyperfunction of sympathoadrenomedullary system and RAAS, inhibition of HPAA, and the upregulation of IL-1βand the dwnregulation of IL-2. All the above suggested disturbance or disorder of NEI network. The syndrome factors, stagnancy of collateral-QI deficiency, played a key or special role in disturbance of NEI network of complex model. The canonical correlation analysis shows that there is a correlation between VEC disfunction and NEI network, embodied in the reltion between NO, VWF and ACTH, NE, CORT, IL-2, and CRH. It is suggested that the disturbance of NEI network may be one of the pathophysiologic mechanisms of VED developing under the state of stagnancy of collateral-QI deficiency.4. It is the first time the VED cell model of stagnancy of collateral-QI deficiency was establishedThe HUVEC was cultured by serum from rats of complex model, and the effect of syndrome combined VED and collateral-QI deficiency on endothelial cell was imitated. The result showed that the content of F-actin in complex model was decreased significantly, depolymerization and redistribution of F-actin in endothelial cells were observed, and the permeability of endothelial cells of complex group was increased significantly. The above changes maybe connected with p38 MAPK signaling pathway.5. The effect of Herbs dredging collaterals on regulating the VED Different kinds of Herbs dredging collaterals can improve the stagnancy of collateral-QI deficiency symptom of complex rat model, which lightened the damage to the shape of EC, adjusted the EC correlation factor, and improved the immunologic derangement in adrenergic nerve-adrenal medulla system, HPAA, and RAAS obviously. Tongxinlu, the complex prescription, was the best. All the above showed the superiorityof the integrating of different kinds of Herbs dredging collaterals in protecting the endothelium.Tongxinluo, the complex dredge collaterals medicine, improved the distribution of F-actin, increased the content of F-actin, and decreased the permeability of monolayer cell in the experient in vitro. The mechanism of Tongxinluo protecting endothelium maybe related to the p38 MAPK signaling pathway inhibited. |
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