| Research background:Atherosderosis is a complicated disease caused by multy-factor.Endothelial cell damage and inflammatory mechanism play an important role during the occurrence and development of atherosderosis.Recent research shows that inflammation and activitation of immune system caused by non-infectious,even by infectious,run through the whole process of atherosderosis.However,it is not clear that the molecular mechanism relationship among inflammatory factor, immune reaction and AS.It is very important to deeply study inflammatory and immune mechanism of AS,and to approach how to block up invasion way of AS.The basic fuction of immune system includes discriminating exogenous antigen, inducing serial reaction of body to clear exogenous pathogenic material,keeping stable of body.It is known that there are innate immunity system and acquired immunity system resisting noxious substance of exterior and interior of body. Acquired immunity system recognize specifically distinctive antigenic determinant in the circumstance. Innate immunity system resists microorganism and noxious substance of body as the first t barrier shield and recognize high conservative antigen domain called PAMP.Recent research showes that Toller Receptor 4 is involved in occurrence and development of atherosderosis,which mediate congenital immunity and inflammatory reaction.TLR4 specifically binds self-ligand and activate a series of downstream cascade reaction in dependent or independent way of MyD88(marrow differentiation factor 88).The spike is transduced into cellular nucleus.It activates important immunogene transcription factors,such as nuclear factor-KB,activating protein-1, interferon regulatory factor,and induces interferon and cytokine ,such as interleukin(IL-1,IL-2),tumor necrosis factor-a(TNF-a), to synthesis and release. Finally it starts adaptive immunity in the way of promoting dendritic cell mature,and promote a series of immune incident aiming directly at different pathogenic microorganism.The research of TLR4 function during AS is started right now,and the indeed mechanisms of signal transduction(including TLR4,MyD88 dependent or MyD88 independent signal transduction)during the occurance and development of AS.The research provides a new point of view to study the pathogenesy of AS,and also provides new methods of preventing and curing AS.More and more inflammation correlation factors are discovered and the inflammation theory of AS is accepted generally,as AS is studied more and mor deeply.In the earlier period of AS,the most important beginning component element is adhesion and migration among WBC,MNC,LY and blood vessel endothelial cell.Many component elements ,including adhesion and migration of MNC and vascular endothelial cell aggregation of MP,migration and generation of smooth muscle cell, aggregation of extracellular matrix and so on, dependent on the function adhesion molecule such as VCAM-1,ICAM-1. It shows that cytokine strengthens adhesion function of monocyte-arterial smooth muscle cell in the way of VCAM-1 and ICAM-1 mechanism.It is proved that TNF-a can increase ICAM-1.As the independent risk factor leading to AS,ox-LDL mediates its biological fuction with the specific acceptor-LOX-1(lectin like ox-LDL receptor-1) combining, uptaking,degradating.TLR4and LOX-1 co-expressed in atheromatous plaque endothelialocyte.LPS is the specific ligand of TLR4.In the way of TLR4/NF-KB,LPS can up-regulate the expressionof LOX-1 and increase endothelial cell damage of oxLDL to accelerate atherosclerosis.So suppression of LOX-1,TNF-a and ICAM-1 expression an inhibit occurrence and development of atherosclerosis.In the aspect of treatment,it is showed that there are many medicine for anti- atherosclerosis. As one of the most effective medicine of atherosclerosis, statins have pleiotropic effects.It limits clinical application of statins that it has obvious adverse effect in long-term application and its mechanism of restrainning atherosclerosis hasn't illuminated clearly.The drug for atherosclerosis should restrain many links of atherosclerosis ,because the reason of atherosclerosis is very complicated. It provides abstract possibility for preventing and curing atherosclerosis that Traditional Chinese medicin has various chemical composition and extensive bioactivity.Its many good featurs have been paid more and more attention to for curing and preventing atherosclerosis, such as mutlti-pathway healing,multi-link healing, multi-traget healing,good stability and less adverse reaction.It is possible that we have further progress in the curing of atherosclerosis for the features of traditional Chinese medicine.Recently many traditional Chinese medicine have been used for preventing and curing atherosclerosis and other correlated disease effectively.But it haven't been studied that the therapeutic effect between traditional Chinese medicine and new type atherosclerosis medicine,and molecular biology mechanism of traditional Chinese medicine. Deeply study the effection of traditional Chinese medicine for bionomics features and regulation mechanism of TLR4 and downstream signal transduction iteral, for expression of LOX-1,TNF-a and ICAM-A.It is possible to develop idio-target and high-performance agonist drug to provide new incisivus of treatment and mechanism study of traditional Chinese medicine through prospective empirical study in cell and molecular level. Objective:To study the effect of the Chinese complex prescription towards TLR4, downstream MyD88 dependent and independent iter,and LOX-1,TNF-a,ICAM-1 expressing.To approach the mechanism of the Chinese complex prescription preventing and curing AS.Method:1. 20 New Zealand male rabbits were divided into 4 groups in random:the first group were not feed with the Chinese complex prescription, the second were feed with high density of Chinese complex prescription,the third were middle density ,and the last group were low density.There were 5 rabbits in each group. They were all given gastric perfusion of normal saline and Chinese complex prescription everyday. In the 7th day, Blood was drawn from heart 2hs after gastric perfusion.Then the serum was separated and filtered aseptically by 0.22μm disposal filter and preserved in freezer -80℃.2. Human umbilical vein endotheliocyte was separated in the way of trypsin digestion from fresh umbilical core of health infant in asepsis environment.Then it was serially subcaltured in environment of 37℃,5% CO2 and accredited by factorⅧfos-related antigen.3. The LPS(1μg/ml)was used to stimulate human umbilical vein endothelial cell to activate TLR4 and downstream signal transduction iter.Different density of the Chinese complex prescription and astorvastatin was used to interfered in.Then the cells were collected in 24h.The method of Real time PCR and Western blot was used to respectively detect expressing of mRNA and protein of TLR4,MyD88,TRAF-6,TRAM,TRIF,NF-KB.4. The LPS(1μg/ml)was used to stimulate human umbilical vein endothelial cell to activate LOX-1,TNF-a,ICAM-1.Different density of the Chinese complex prescription and astorvastatin was used to interfered in.Then the cells were collected in 24h.The method of Real time PCR and Western blot was used to respectively detect expressing of mRNA and protein of LOX-1,TNF-a,ICAM-1.Result:1. The serum with medicine after the centrifugalization was atramineous and supernatant.20-25ml serum was obtained from 50ml blood.There were 4 groups of serum. The first group were not feed with the Chinese complex prescription, the second were feed with high density of Chinese complex prescription, the third were middle density ,and the last group were low density.2. The cells were arranged like monolayer road stone when they were cultured in confertus state.It is discover that majority cells was factorⅧfos-related antigen in the way of immunofluorescence staining of factorⅧfos-related antigen.3. Afer being stimulated by LPS in 24h,cells of pattern group have higher expressing level of mRNA and protein of TLR4 and the primary element of downstream signal transduction iter,such as Myd33,TRAF-6, NF-κB (compared with blank group, P<0.01);After being interfered in by medicine, each curing group has lower expressing level of mRNA and protein of TLR4,MyD88,TRAF-6, NF-κB(compared with pattern group, P<0.01);The 3 groups feed with Chinese complex prescription obviously restrain the expressing of mRNA and protein of TLR4,MyD88,TRAF-6, NF-κB(compared with pattern group, P<0.01 or P<0.05).Among them,the group feed with high density of Chinese complex prescription has the best restraining effect.4. Afer being stimulated by LPS in 24h, cells of pattern group have higher expressing level of mRNA of TLR4 and the primary downstream signal transduction iter, such as TRAM and TRIF (compared with blank group, P<0.01); After being feed with medicine, each curing group has no obviously lower expressing of mRNA TRAM and TRIF(compared with pattern group, P>0.05).5. Afer being stimulated by LPS in 24h,pattern group cells have obviously higher expressing of mRNA and protein of LOX-1,TNF-αand ICAM-1 (compared with blank group, P<0.01); After being feed with medicine, each curing group has obviously lower expressing of mRNA and protein of LOX-1,TNF-αand ICAM-1,among them the group feed with western medicine has the obvious effect(compared with pattern group, P<0.01); The 3 groups feed with Chinese complex prescription obviously restrain the expressing of mRNA and protein of LOX-1,TNF-αand ICAM-1 (compared with pattern group, P<0.01 or P<0.05).Among them,the group feed with high density of Chinese complex prescription has the best restraining effect.Conclusion:1. Medicine containing serum of different density could be obtained in above-mentioned way,which could be provided as the starting material for next extraorgan interfering experiment.2. Endothelial cells can be separated from human umbilical vein in the way of trypsin digestion.It is proved that they are the endothelial cells when detected by immunohistochemical method of human factorⅧfos-related antigen.3. LPS can activate mRNA and protein expressing of TLR4 and primary element of downstream MyD88 dependent signal transduction iter,such as MyD88,TRAF-6,NF-KB.The Chinese complex prescription can restrain mRNA and protein expressing of TLR4,MyD88,TRAF-6和NF-KB in 24h after being interfered in.That is illustrated the Chinese complex prescription can restrain mRNA and protein expressing of TLR4 and primary element of downstream MyD88 dependent signal transduction iter.4. LPS can activate mRNA and protein expressing of TLR4 and primary element of downstream MyD88 independent signal transduction iter,such as TRAM and TRIF. The Chinese complex prescription have no obvious restraining fuction of mRNA expressing of TRAM and TRIF in 24h after being interfered in. That is illustrated that the Chinese complex prescription have no restraining fuction of mRNA expressing of TLR4 and primary element of downstream MyD88 independent signal transduction iter.5. LPS can activate mRNA and protein expressing of LOX-1,TNF-αand ICAM-1. After being interfered in in 24h, the Chinese complex prescription can restrain mRNA and protein expressing of LOX-1,TNF-αand ICAM-1. It is illustrated that the Chinese complex prescription can restrain mRNA and protein expressing of LOX-1,TNF-αand ICAM-1,which maybe the mechanism of preventing and curing AS. |
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