| Atherosclerosis (AS) is the principal pathogenic foundation of cardio-cerebrovascular diseases, and endothelial dysfunction is the initiating factor of AS and involves in the whole process.Platelet,as one of the the most important blood components, plays an important role in AS immunity, inflammation, thrombosis and other pathologic stage by releasing many bioactive substances or membrane glycoprotein, and mediates interaction between platelet and endothelial. Vessels Collateral theorysystem is establishedheritedfrom "XUEMAI" in Nei Jing, and a new concept of "Vessels Collateral-vascular system disease" is put forward, Ying-Wei Theory is the core theory.Based on the correlation Ying QI withvascular endothelial, Ying Xue with blood components, this study explores the endothelial cell injury induced by platelet activation and the intervention effect of TONGXINLUO,which will enrich and develop Vessels Collateral theory in the prevention and control of AS.Objective:Under the guidance of Ying Wei theory of context theory,this study is to discuss the role of Ying Xue which can cause abnormal of Vessels Collateral-vascular system in AS and the effect of tongluo durgs. Platelets in the blood as the object,this study to explore the effect of platelet activation on endothelial cell injury by in vivo animal and isolated cell experiments, at the same time to study the effect of tongxinluo on inhibition of platelet actication and protection mechanism on endothelial cell.Methods:(1) 70 New Zealand rabbits were randomly divided into normal group, model group, TONGXINLUO low, medium and high dose group, Atorvastatin and Aspirin group,10 in each group.Except the normal group,the rest of groups were feed with high fat forage, at the same time, the treatment groups were given corresponding drugs, once per day, sequentially lavaged 12 w. At the end time, fasting was necessary for 12h after the last giving drugs. Automatic biochemical analyzer was to test total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C);Enzyme-linked immunosorbent (ELISA) was used to detect the tumor necrosis factor-a (TNF-a), interleukin 1 beta (IL-1 beta), high-sensitivity c-reactive protein (hs-CRP);Oil red O staining and HE staining was to observe the aorta lipid deposition and the changes of endometrial pathological.Automatic blood cell analyzer was to test platelet parameters; Electron microscopy was to observe platelet ultrastructure; Flow cytometry instrument was to observe Ca2+ changes in platelet intracellular;ELIS A method was to detect platelet secretion factors,such as platelet factor 4 (PF4), soluble CD62P (sCD62P); ELISA also was to detect the level of serum soluble CD40L(sCD40L) of 0,2,4,8,12 weeks;Immunohistochemical was to detect the expression of CD40L and CD40 in aorta; Western blot was used to detect protein expression of CD40L, CD40, caspase3, Bax and Bcl-2 in aortic, and RT-PCR to detect CD40L, CD40 mRNA of aortic.(2) Human umbilical vein endothelial cells (HUVEC) was cultured, and blood fromhealthy people was collected and separated to acquire platelets. ①Resting platelet suspension, activated platelet suspension and tongxinluo suspension, each other cultivated with the endothelial cells. Dynamic living cell imaging system was used to observe platelet aggregation, adhesion, and release process.to observethe effect of activated platelet on endothelial cell apoptosis, and the process.of mitochondrial membrane potential Flow cytometry instrument was to detect platelet membrane CD40L, CD62P expression; ELISA was to detect the expression of sCD40L of platelet.② Further, CD40L monoclonal antibody was used to block CD40L from activated platelets supernatant fluid combined with CD40 of endothelial cells, the experimental group was divided to the normal group, ADP negative control group, resting platelets group, platelet activation group, tongxinluo group, CD40L ligand monoclonal antibody. MTS was used to test endothelial cell survival activity, fluorescence to detect endothelial cells mitochondrial membrane potential. ③For the purpose to validate the role of CD40L in the endothelial cell apoptosis, we used recombinant soluble CD40L (rshCD40L) to induce endothelial cell damage model, the experimental group was divided to normal group, the rshCD40L model group, tongxinluo low, medium and high dose group. The change of endothelial cell survival was detected by MTS and Western blot was to detect endothelial cells ERK1/2 related signaling pathways ERK1/2, p-ERK1/2, CyclinD, C-Myc, Cytc, Bax, Bcl-2, Caspase3 protein expression.④ Then, using plasmid transfection technology to silence the gene expression of ERK1/2, the experimental group was individ to the normal group, siRNA Control group, the siRNA group, Tongxinluo group, Tongxinluo+siRNA. Western blot was used to detect ERK1/2 signal path about p-ERK1/2, CyclinD, C-Myc, Cytc of endothelial cells.Results:(1) Animal experiments:①The intimal was thicken in the mold group by ahigh-fat feeding, the level of serum TG, TC, LDL-C was significantly higher than the normal group, the level of TNF-α、IL-1β、hs-CRP was higher too.Compared with the normal group, apoptosis protein of Caspase3, Bax significantly raised in the model group, antiapoptotic proteins of Bcl-2 significantly lowered.TONGXINLUO couldinhibit intimal hyperplasia,reduce the level of serum lipid and the level of TNF-α、IL-1β、hs-CRP.TONGXINLUO also could reducethe expression of Caspase3, Bax and increase Bcl-2.②In the model group the shape of platelet changed, the levels of sCD40L、PF4、sCD62P increased,and intracellular calcium ion significantly increased too.CD40L, CD40 protein and mRNA increased by the results of Immunohistochemical, Western blot and RT-PCR. TONGXINLUO couldimprove the shape of platelet, significantly reduced the level of sCD40L,PF4,sCD62P and intracellular calcium ion.TONGXINLUO could also reduce the expression of CD40L and CD40.(2)Cell experiments:①The platelet happened aggregation, adhesion and releasing reaction after activated, increased the level of CD62P, CD40L, sCD40L, reduced the levelof NO of endothelialand rised ET-1, at the same time reduced the activity of endothelial cell survival, drived down mitochondrial membrane potential.②CD40L/CD40played an important role in mitochondrial apoptosispathway of endothelial cellsby inhibiting the activity of ERK1/2, increasedthe protein expression of Cytc, caspase3, Bax, and reduced CyclinD, Bcl-2 (3)TONGXINLUOcould inhibitedthe expression of CD62P,CD40L, sCD40L, at the same time reduce the ability of activated platelets adhered toendothelial cell.TONGXINLUO may be activating ERK1/2 signal pathway to inhibit the apoptosis of endothelial cellsinduced byCD40L/CD40 of activated platelet.Conclusion:TONGXINLUO has a protective effect of endothelial cell injury induced by platelets activated, inhibitsthe activation of platelet, reducesthe adhesion ability of platelet to endothelial cell, lowers activity of CD40L/CD40. TONGXINLUOinhibits endothelial cell apoptosis induced by CD40/CD40L, its mechanism may be related to activate ERK1/2 activity, reducprotein expressionof Cytc, C-Myc,and raise CyclinD. |
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