| BackgroundFor a long time, SAP has been a topic issue for pancreas researchers. Especially, there is a shortage of understanding about SAP with disseminated intravascular coagulation (DIC). When there comes the complications, such as multiple organ dysfunction syndrome(MODS) led by thrombosis, apparent hemorrhage symptom, decreased platelets and fibrinogen decrease, prothrombin time extension, fibrin degradation product increase, and positive results of plasma protamine paracoagulation test, DIC has already developed to its middle-late stage, and has missed the best time of therapy. At the moment, the treatment becomes both difficult and complicated, and the curable rate is largely decreased. So, early diagnosis and protection are very important in the therapy of DIC.At present, study shows that blood coagulation activation and endothelium injury are cruxes in the formation of disseminated intravascular coagulation in patients who have suffered from severe acute pancreatitis, and endothelium injury plays an important role in the reaction mechanism of blood coagulation activation. The over-activation of leukocytes and the aggregation between the cells make the endothelium injured, which also results from mediators of inflammation, pancreatic enzyme, oxygen-derived free radicals, platelets activating factor, phospholipase and so on. The course of blood coagulation which is the main mechanism of disseminated intravascular coagulation is to be switched on by the means of anomal-expression of tissue factor (TF) after the endothelium injury.The coagulation process is caused by the activation of the endogenous and exogenous coagulation system. It is reported that the endogenous coagulation system is not the significant process of thrombosis, but the way of exogenous coagulation in the body. In the normal condition, the barrier of endothelium cell will separate the circulating blood from TF, however, the exogenous coagulation system occurs with TF release after the injury of blood vessel walls and other tissues. When the tissue destructs the barrier, TF can be combined withⅦ/Ⅶαfactors in the blood. It is the surface receptor of cellⅦfactor (FⅦ), and the assisting factors of FⅦand activatingⅦfactor (FⅦα) as well. The diffraction analysis of X-ray, as the receptor ofⅦ/Ⅶαfactor of TF extra cellular domain, indicates: the contacting areas of TF andⅦαfactor to theⅦα, from the bottom of extra cellular carboxyl terminus upward to the top of amino-terminal domain, are of great appetency; when forming compounds with FⅦ/FⅦα, it produces thrombin in the body within the existence of Ca2+ by activating X factor (FX)and IX factor (FIX), meanwhile starting the way of exogenous coagulation through the way of exogenous coagulation. While the production of thrombin is adjusted and controlled by the autocatalysis and feedback mechanism, through activatingⅤ,Ⅷfactors and platelet, a small amount of thrombin is produced in the early stage, which accelerating the activation of X factor and thrombin, prompting a large amount of thrombin in the local place, changing fibrinogen into fibrin, causing the blood concretion and forming the thrombus. It can be seen that the coagulation process is a series of reactions caused by the generating factors of TF which activates the thrombin. Thus, lowering the expression of TF can interdict the course of coagulation and, to an extent, control the incidence and development of DIC.The reports from animal models have showed that motherwort can not only improve body microcirculation, hemorheology, and anti-oxygen free radical, but also decrease the intracellular Ca2+ over-load. Meanwhile, the study indicates that motherwort is of obvious restraining effect to the formation of thrombosis, and the TF release of endothelial cells plays an important role in the course of thrombosis. It is reasonable to presume that motherwort may well have effect on the expression and transcription of TF by vascular endothelial cell. To explore the mechanism of motherwort on SAP with DIC, human vascular endothelial cells, HUVECs in vitro, was firstly established, and, the expression of TF induced by thrombin in HUVECs which were derived form subculture cells was observed. To analyze the expression of HUVECs TF in different concentration and time upon the effect of motherwort, seeking the necessary concentration and time for the greatest restraining effect of motherwort to the expression of HUVECs TF under the induction of thrombin; to further study the effect of motherwort to the expression of HUVECs TF and TF mRNA under such concentration and time induced by the thrombin. At last, in the rat model of SAP, whether motherwort has effect on pre-DIC was explored in the study.Objective1. To establish a human vascular endothelial cells (HUVECs) in vitro and to observe the effects of thrombin on the expression of tissue factor activity in HUVECs. 2. To approach relationship between motherwort in different concentration and diversity action time and expression of TF inducted by thrombin. Then to study the effects of motherwort on the expression and transcription of TF induced by thrombin.3. To explore the effect of motherwort on pre-DIC with a SAP rat model.Methods1. HUVECs collected by collagenase digestion of the interior of the umbilical vein from human umbilical cords were cultured using F12K supplemented with 10% fetal calf serum. Both primary and subcultured cells were examined for their identity with light microscopy, avidin-biotin-peroxidase complex (ABC), flow cytometer. HUVECs cultivated from each of the separated umbilical cord would be divided into two parts; 16 parts in 8 umbilical cords were randomized into two groups (A and B). Group A was cultivated with 25IU/ml of thrombin for 10 hours, while group B with phosphate buffered solution (PBS) for 10 hours. Immunofluorescence was used to test TF antigen in the flow cytometer in order to compare the TF expression of HUVECs under the effect of thrombin and PBS, and T-paired statistical measurement was employed.2. 128 samples of HUVECs were divides into four groups, which were treated by thrombin 25IU/ml for 10 hours, then HUVEC were incubated with different concentration motherwort (2.5μg/ml, 5.0μg/ml, 7.5μg/ml and 10.0μg/ml). And each group was divided into four subgroups A, B, C and D, which were incubated for different time (6 hours, 12 hours, 24 hours, 36 hours). The expression of TF induced by thrombin in HUVECs was detected in different motherwort concentration and diversity action time by a flow cytometer with immumofluorescence method. 3. The freeze-thawing HUVECs were divided into three groups: the first as control group (group C), the second as thrombin group (group B) and the third as thrombin+Motherwort group (group A). HUVECs in group A was treated by thrombin 25IU/ml for 10 hours, then incubated with motherwort 5.0μg/ml for 24 hours, while the group B was treated by thrombin 25IU/ml for 10 hours, then by phosphate buffered solution for24 hours. The group C was treated by phosphate buffered solution unsteadily. The transcription of TF induced by thrombin in HUVECs was examined by RT-PCR technique, and active factorⅦwas detected automatically by hemagglutination meter. The results were analyzed by using one-way ANOVA.4. A total of 75 Wistar rats were randomized into 5 groups, the control group (C), SAP group (S) (n=15), DDFA treated group (D), motherwort treated group (M) and DDFA+Motherwort treated group (DM) (n=15). The rats in group SO (n=15) were only punctured without injection. The model of SAP was established by retrograde injection the pancreas- bile duct of 5% sodium taurocholate solution. Then, saline was injected into the right jugular vein in group S (n=15), DDFA was injected into jugular vein in group D (n=15), and motherwort was injected into jugular vein in group M (n=15) while DDFA+Motherwort were injected into jugular vein in group DM (n=15). The experimental rats were killed at the 6, 12 and 24 hour after establishment of models, antithrombin-Ⅲ(AT-Ⅲ), plasma Prothrombin fragment (F1+2), a plasma Prothrombin time (PT) and TF were determined, for analyzing the information of blood coagulation in rat model, serum AMY were determined, for analyzing the effect ofmotherwort on them in rat model, The results were analyzed by using one-way ANOVA, while the pancreas were observed by light microscopy. Results1. HUVECs grew with high survival rate and expressed confluent monolayer of polynonal cells at 7th day under light microscopy. The expression rates of CD31 is 90.42%, while CD105 96.56%, evaluated by flow cytometer. There were viridis fluorescent light in intracytoplasm of HUVECs but no fluorescence in the control by fluorescent microscopy. T-paired statistical analysis was employed to test the expression ofHUVECs TF on the effect ofthrombin and PBS.2. The different concentration of motherwort had different effects on the expression of TF induced by thrombin in HUVECs (P＜0.01), so did different action time of motherwort (P＜0.01). But it was no reason that there were interactions between two factors (P＞0.05).3. 3-group comparison of TF expression: A group, B group, and C group. F measures are 408.07 (P＜0.01) with clear difference; 3-group comparison of brightness ratio of TFmRNA/GAPDHmRNA, F measures are 48.81 (P＜0.01) with clear difference. Meanwhile, there is a clear difference (P＜0.01), under an in-between comparison; B group to C group, and A group.4. The serum amylase contents in different groups of rats, 6h, 12h, 24h, F measures are 86.61,114.20,241.54 respectively, average P measures are＜0.01 with the statistical significance of diversity between the groups (P＜0.01), There is a clear difference between the groups according to SNK test(P＜0.01).The TF comparison of plasma in different groups, 6h, 12h, 24h, F measures are 99.98, 193.66, 77.80 respectively, average P measures＜0.01 with the statistical significance. According to SNK test, there is an in-between difference (P＜0.01) in 6h and 12h groups, the expressions of TF take turns as follows: SAP group＞DDFAgroup＞M group＞DDFAM group, and SO group; the expressions of 24h TF as follows: SAP group＞DDFA group＞M group＞DDFAM group, and SO group. The comparison of plasma F1+2 in different groups, 6h, 12h, 24h, F measures are 37.44, 36.98, 113.53 respectively, average P measures＜0.01. There is a clear difference between the groups according to SNK test: F1+2 concentration of 6h and 12h: SAP group and DDFA group＞M group＞DDFAM group and SO group; F1+2 concentration of 24h: SAP group and DDFA group＞M group＞DDFAM group and SO group.The comparison of plasma AT-Ⅲin different groups, 6h, 12h, 24h, F measures are 11.94, 14.80, 37.26 respectively, average P measures＜0.01. There is an obvious difference between the groups according to SNK test: the activation of 6h AT-Ⅲ: SAP group and DDFA group＞M group and DDFAM group and SO group.The comparison of plasma PT in different groups, 6h, 12h, 24h, F measures are 0.82(P＞0.05), 1.75(P＞0.05), 56.91 (P＜0.01 ) respectively, with no statistical significance in group 6h and 12h. There is an obvious difference between the groups in 24h group according to SNK test: PT in SAP group is longer than that of the DDFA group, and PT in the latter is longer than that of the DDFAM, M, and SO group.Conclusions1. The purity HUVECs are cultured with F12K solution. This method is able to provide experimental material. The thrombin can induce the expression of TF antigen, while there is a little expression of TF antigen in HUVECs with phosphate buffered solution.2. The effect of thrombin on the expression of TF in HUVECs can be inhibited by motherwort, and the depressive effect is connected with different concentration of motherwort and action time of motherwort. The experimental result also indicated that the TF expression of HUVECs induced by motherwort to thrombin was realized through the restraining transcription of HUVECsTF mRNA.3.Though motherwort alone cannot prevent the inflammation process in SAP rat, Motherwort+DDFA can decrease the expression of TF and lead to down-regulation of F1+2 and AT-Ⅲof blood coagulation system, extension of PT, which intervene SAP with DIC.
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