| Backgroud:Psoriasis is one of the most common chronic inflammatory proliferative skin disease, it’s recurrence and stubborn disease.it also can cause systemic damage. The main histopathological changes formed as angiogenesis, keratinocyte hyperplasia and inflammatory cell infiltration. Currently the etiology and pathogenesis of psoriasis is not yet entirely clear and there is no effective cure,but the serious disease can influence the patient’s quality of life, even the life cycle, psoriasis is one of the disease in today’s domestic and international focus on. Earlier studies have shown that formed new blood vessels in psoriatic lesions provide nutritional for epidermis cutin cell to over proliferation Inflammatory cells at the same time through the high permeability of blood vessel endothelium into diseased tissue.on the contrary, inflammatory cells infiltration to accelerate new angiogenesis, so it are mutually form a positive feedback effect and cause psoriasis disease break out repeatedly and aggravate.Thus it is one of the effective ways to treat psoriasis by interruptting the vicious circle.The present study found that dermal papillary vascular anomalies is first changes in thethree kinds of histopathological.Then inflammatory cells will migration and epidermal will proliferation and differentiation. VEGF is one of the most specific and strongest angiogenic factors directly. Research has pointed out:the expression of VEGF in the lesion area is higher than that of normal people.It is positively correlated in the degree of psoriasis illness severity and the concentration of VEGF in lesion area.The expression level of VEGF mRNA and protein is almost no expression in normal skin,while the expression level of them were significantly increased in the lesions of psoriasis patients. The expression levels of VEGF mRNA was also increased in normal skin of patients with protein and found the skin with psoriasis early pathological changes.The encoding of VEGF gene shift to the mouse,it’s skin can cause psoriasis change which is similar to psoriasis skin tissue in human and Koebner phenomenon.This further illustrate VEGF play an important role on the pathogenesis of psoriasis.Two receptors R1and R2of VEGF mRNA are expression in psoriasis activity. Studies have shown that the expression of VEGF mRNA and protein in patients with psoriasis skin lesions and peripheral blood is significantly higher than healthy people,And its expression and the quantity of the patient’s illness severity was significantly positive correlation. After system abdrugs,Psoriatic patients’Condition improved significantly and the expression of VEGF mRNA decreased obviously.Direct effect is to point to in vitro induced endothelial cell proliferation, migration and lumen. Such as G-CSF, GM-CSF, bFGF and VEGF, TGF-a,B and so on. These studies indicate that VEGF not only play an important role in the pathogenesis of psoriasis, but also can be used as an index of judging the severity of psoriasis patients. Detected the expression of VEGF in patients serum and/or skin lesions could be one of effectively way for the treatment of psoriasis.An important tool for the study of human disease etiology, pathogenesis, treatment is animal models, but psoriasis is rare in any non-human animals and naturally occurring cases are extremely rare, so it are not suitable for scientific research, therefore, psoriasis animals is particularly necessary to build the model. Therefore, this experiment included two aspects:First, the establishment of animal models of psoriasis, followed by real-time quantitative PCR was used to detect the expression of VEGF mRNA in skin and peripheral blood of effects of TGP on psoriasis mouseNow, psoriasis animal models include the following.1.1spontaneous animal modelSpontaneous animal model is not through artificial processing, experimental animals models is builed by the natural conditions such as Scaly skin of mutant mice, mutation hairless mice and so on. All of them can spontaneously produce similar skin and histopathological manifestations of human psoriasis. This model is mainly used in the study of the pathogenesis of psoriasis and drug screening.The spontaneous animal model including mutation genetic disease and tumor disease model of inbred line. But gene mutation probability is small, generally not spontaneous animal models of psoriasis and Inbred line survival rate is low,but still has some limitations.1.2induced animal modelsThrough some physical or chemical stimuli can induced animal models mainly(such as ultraviolet irradiation,chemical stimulants, besmear outside drugs, etc.) To speed up the animal epidermal hyperplasia and induce the pathological changes which is similar to human psoriasis samples, but this model can only simulate the partial characteristic of psoriasis histopathological changes and there are bigger differences from human spontaneous psoriasis.such as GaylardeW built psoriasis sample model with propranolol guinea pigs,It can induces the formation of parakeratosis, hyperkeratosis and prickle cell layer hypertrophy which is pathological changes of patients with psoriasis.1.3allografts animal modelAllografts animal models is transplanting patients’psoriasis skin into animals’body surface to induce the animal models, this model can show the psoriasis appearance and tissue pathological changes in long time. It is widely used in severe combined immunodeficiency mice (SCID mice). But allografts animal models have limitations, because the model can only response to local skin changes, not on the whole simulation process of the pathogenesis of psoriasis, and construction process is relatively complex, so its application is limited。 1.4biomedical animal modelSuch as mouse tail scale model, this model was used to evaluate the influence of drug to the formation of psoriasis skin particles, As long as the drug can cause mouse tail particle layer to form, we argue that the drug is a treatment for psoriasis, another mouse vaginal epithelium model is mainly used for evaluation of inhibit excessive proliferation of the cells, Vanisi evaluated the effect of drugs on the inhibitory with mouse vaginal epithelium model1.5the transgenic animal modelTransgenic animal model is to exogenous genes into or endogenous gene deletion of animals with transgenic technology research. But the pathogenesis of psoriasis are related with multiple genes, so the model can reflect the local pathological physiology characteristic of psoriasis, can not fully express the factors associated with the onset of psoriasis..Due to various reasons, above all kinds of animal models, although all can show the characteristics of psoriasis in some places, but there is still not a single animal model can simulate the complete human aspects characteristics of psoriasis.Mi sinensis mott local regulator is a new kind of immune and non nucleoside class ring amine compounds, It mainly combine with toll-like receptor (TLR)-7,8which in the surface of monocytes, macrophages, B lymphocytes and dendritic cells and so on, inhibitory proteins1KB of phosphorylation NF-kB has change the struction, thus it separated from NF-kB, so NF-kB to activate. Activation of NF-kB though into the nucleus,wich induced transcription of target genes with related DNA base sequence.inflammatory markers such as interferons alpha, IFN-gamma, TNF alpha, IL-1, IL-6, IL-8, IL-12, g-csf and gm-csf, CCL4, CCL2, MIP-1, etc. These cytokines can acquired immune responses, For example IL-12include Thl type of cellular immunity, it play the role of antiviral and anticancer. IL-land IL-6induce initial T cells to Th17cells, so subsequent cascade constitute immune complex network system of autoimmune disease.Have been reported in succession, originally stable psoriasis patient appears the trend of increase, and even have appeared in the patients with a history of psoriasis psoriasis skin lesions, and the severity of the lesion and the doses or use time was significantly positive correlation in condyloma, mott melanoma or the disease such as bao warm disease were treated by the mi quetiapine. Such as Leslie give5%mi sinensis mulder cream regularly each day in BALB/c mice back skin, After five days he found typical of psoriatic lesions appearing on mice back skin. As the model is simple, the cost is less, we choose the model experiment.Vitamin A acid, immunosuppressants, biological preparation and so on are the main clinical drugs and methods in the treatment of psoriasis.On one hand it can achieve acertain effect, on the other hand there are some side effects and it can not suit the requirements of cure. Despite decades of psoriasis treatment has made great achievements, but there is no ideal therapeutic drugs and special way.Traditional Chinese medicine is a great treasure-house and have a few of side effects.Part of the traditional Chinese medicine (TCM) has confirmed in autoimmune diseases play a important role in the immune diease and the recurrence rate is low, in autoimmune diseases especially in psoriasis,It shows a unique application prospect,alba total glycosides (TGP), including the bitter, hydroxy paeoniflorin, peony flower peony glucoside, benzoyl, paeoniflorin and peony lactone glycosides.But glycosides accounted for more than90%. TCM believes that it has a nourishing blood in the liver, slow the effect of the pain and the folding of the Yin khan, which is widely applied to the treatment of many diseases. Clinical application and pharmacological studies have found that the TGP has anti-inflammatory analgesic, regulating immunity, protect liver, vascular endothelial and less side effects, etc. which is widely applied to the treatment of many diseases, such as psoriasis, RA and SLE. In recent years, there are a number of studies suggest that multiple link of TGP is involved in autoimmune process:(1) As DNCB induced chronic dermatitis-wet creepy-crawlies mice model, the interleukin (IL)-2levels drop and IL-4levels increase, However Li Chuan use of the TGP treatment in mice model,he found that the level of IL-2can rise,but IL-4levels drop, its possible mechanism is the TGP can regulate secretion of Thl secretion of IL-2,keep balance in Th2and IL-4and inhibit Th2functions with raising the expression of Thl.(2) the TGP participated in the G protein-adenylate cyclase-cAMP signal transduction pathways and silk crack the original amp-activated protein kinase (MAPK) signal transduction pathway, and it through these two pathways regulating cell factors play a role.(3) the TGP can reduce the expression of the tumor necrosis factor (TNF)-a, IL-6, IL-8and interferon (IFN)-a. So TGP was used for the treatment of SLE and have apparent curative effect, ZhuYuHui etc used TGP,glucocorticoid and immunosuppressants in active treatment of SLE, the results showed that the use of the TGP can significantly reduce the dosage of the hormone.MARK play a extremely role in cell differentiation, proliferation, apoptosis, stress and a variety of physiological and pathological.such as inflammation and immune response, etc., It is an important signaling pathway in mammalian cells, it can deliver a variety of signal produced by extracellular to the nucleus,. Any link of the anomaly may result in abnormal cell proliferation and differentiation. MAPK signal transduction pathway includes MAPK kinase kinase, MAPK kinase and MAPK. The pathways of reaction is mainly tertiary enzyme-linked:first by activating factor to activate the MAPKKK, then the activated MAPKKK activate phosphorylation MAPKK, MAPKK induces MAPK and it can activate other series of protein kinases, such as cause cytoskeleton composition phosphorylation,make the nuclear transcription factors in activated by nuclear transfer in the nucleus, adjust the target genes of transcription factors and complete response to cell stimulation. Level3of enzyme reaction stimulating factor includes:cytokines, growth factors, hormones and a variety of stress stimuli. MAPK pathway is the core of MAPK and most of the substrate is within the nuclear transcription factors. MAPKS mainly includes three classic family way, extracellular regulating kinase1/2, p38MApK and Juk. P38is one of the sub-types of MAPK which play an important role in the Horny cells proliferation and differentiation.There are two main irritant inflammatory cytokines and environmental can activate P38.The activated P38is closely related to inflammatory factor TNF-a, IL-1, IL-6. Earlier studies have shown that p38MAPK plays a very important role in the process of the pathology of psoriasis. Studies have reported the TGP can activate p38MAPK and regulate anti-inflammatory effect of TNF-a, IL-1by activating p38MAPK, It suggests that the therapeutic effect of the TGP may adjust the signal path. Therefore, in order to further explore the mechanism of action of the TGP treatment of psoriasis, we chose the p38MAPK signaling pathway to study possible action mechanisms of the TGP.the influence of VEGF expression in patients with psoriasis or psoriatic animal model,the current domestic researcher may use ELISA, immunohistochemical method and computer image to analysis the expression of VEGF. The change of cytokines have close relationship with graft rejection, inflammation, autoimmune diseases.It is a kind of regulatory proteins. In the protein level,the testing needed to reach a certain amount of tissue samples, because the tissue samples is too small to meet. Even if the sensitivity of ELISA and other commonly used protein detection method is difficult to detect trace amounts of protein products. Although there are quantitative immunohistochemical method has the advantages of accurate, due to the tedious process of operation, long duration and the shortcomings of high technical requirements, It is limited in clinical application. So the application of quantitative PCR gene diagnosis becomes very meaningful.Real time quantitative PCR technology as an emerging technology, because of its remarkable advantages, not only has been widely applied in various fields of molecular biology, but also began to used in clinical as a diagnostic tool. Its application scope includes:amount of DNA, RNA and virus load of quantitative analysis, DNA polymorphism analysis, gene mutation, and other fields. Wellmann are pointed out real time quantitative PCR technology are used to analysis of VEGF in different spliceosome than other traditional methods.because it has many advantages than semi-quantitative competitive rt-pcr.such as specificity, sensitivity, reduce the cross contamination and more rapid, more accurate, and suitable for a large sample of synchronous quantitative, etc. So we believe it will be a very promising tool in the study of VEGF in diagnosis and treatment of psoriasis.TGP will play an important role in the treatment of psoriasis, because it can reduce inflammation and relieve the illness, but the mechanism is still not clear, whether the TGP inhibiting the expression and secretion of VEGF mRNA and regulating signaling pathways to cure of psoriasis. To explore the TGP possible mechanism for the treatment of psoriasis. This study test the back skin tissue and peripheral blood of VEGF mRNA expression by real-time fluorescent quantitative PCR in normal BALB/c mice and TGP dosing psoriasis mice and analysis the correlation of the TGP treatment and VEGF mRNA.If we can further understand the mechanism of action of TGP in psoriasis, we will provide more evidence for the application of TGP in clinic.Objective:1. To establish animal models of psoriasis.2. Research the influence of TGP in expression of VEGF mRNA in BALB/c mice psoriasis skin tissue and peripheral blood.To discuss the TGP treatment psoriasis may work targets and mechanisms.Methods:1. The animal buildingIn microbiology, immunology and other trials, fur often need to get rid of the mice, for experimental research. At present the methods of Mice unhairing are to cut off and depilatory hair removal. The two methods not only trouble, also easy to cause skin damage in mice and unhairing not clearly. Therefore,we have adopted a new method of shaving:the buzzer and electric razor shearing, it can avoid the above shortcomings.60BALB/c mice were randomly divided into6groups, each group of10:blank group, positive control group, A, B, C and D groups. After electric shavers with5%mi sinensis mulder cream daub on every mouse back skin except the blank group to establish animal models of psoriasis.2. Building evaluationCompared with normal control group, after1d the model group with IMQ daub, the skin appears erythema; on2-3d have scales,4-5d is the most serious;6d skin became bit better,7-8d again and continue to thickening of the skin. Pathological section:skin became hyperkeratosis and parakeratosis, stratum spinosum hypertrophy and horny layer which is similar to human histological changes in patients with psoriasis.3dosing methodAccording to "Human and animal body surface area reduced equivalent dose ratio table"and the TGP goods medicine PaFuLin regular agent (60mg/kg,1200-1800d), and referring to the past research, the conclusion lavage administration respectively.Blank group:normal saline lavage,0.2ml/10g once a dayPositive control group:normal saline lavage,0.2ml/10g once a dayFor each group A:modeling and give the TGP suspension50mg/kg to fill the stomach Once a dayFor each group B:modeling and give the TGP suspension100mg/kg to fill the stomach Once a dayFor each group C:modeling and give the TGP suspension200mg/kg to fill the stomach Once a dayAfter8day, we killed all of above animals and collect back bare skin tissue and blood in mice.Group D:modeling after8days, continue to use the mi sinensis5%mulder on the bare skin of mice’s back and give the TGP suspension100mg/kg. Only to fill the stomach,Once a day,As long as her lesions dissipated we killed all and collect back bare skin tissue and blood in mice.Real-time fluorescent quantitative PCR method is used respectively to detect the expression VEGF mRNA in kin tissue and peripheral blood of every type.4. Make tissue section and HE staining4%neutral formalin fixed skin tissue in mice after15h, water flushing group2hours.(1) Surface of dehydration and transparency:six hours and50%ethanol,70%ethanol,85%ethanol,95%ethanol and three hours.Dehydration of ethanol gradient every2hours, xylene transparent2hours. (2) Diction and paraffin embedding:liquid paraffin in60incubator for4hours, then the embedding.(3) slice:5microns thickness slice.(4) HE staining:dimethyl benzene dewaxing20minutes, in turn from anhydrous ethanol,95%,85%,75%ethanol, steamedDistillation water hydration every2to5minutes, wood grain-eosin staining and dehydration, transparent, neutral gum sealing piece.Optical microscopic observation in mice back skin tissue pathological changes. HE staining slice,10(eyepiece) in optical microscope><20(objective) under observation.Results:1. The general situationMedication before each group during the experiment in mice feeding and hair is normal, the ease; With the passage of time, except the blank group mice,the others gradually appear mental burnout, xi nuzzles, feeding and weight loss. But there is no obvious hair slip, but Hair luster than previous.With Mi sinensis mott daub back skin,the mice appear fretted and resistance, and became violent aggressive, one mice of C group was killed by other mice. Mice with IMQ were Hair luster, after the TGP treatment in Group D, as her lesions subsided, mice spirit become better and food intake will increase. In the process of the experimental groups of mice, eye, ear, nose and mouth are not seen abnormal secretions; Shit no powder, no exception of urine, urine clear.2. The changes of skin tissue in miceCCompared with normal control group, after1d the model group with IMQ daub, the skin appears erythema; on2～3d have scales,4～5d is the most serious;6d skin became bit better,7～8d again and continue to thickening of the skin. Pathological section:skin became hyperkeratosis and parakeratosis, stratum spinosum hypertrophy and horny layer which is similar to human histological changes in patients with psoriasis.Group D mice’s lesions on7completely faded with100mg/kg, total glycosides lavage treatment,its HE staining slice:skin epidermis and the dermis is roughly normal, not seen corneous layer thickening, hyperkeratosis and Angle is not congruent.3. The expression of VEGF mRNA in skin tissue and peripheral blood in miceAfter8days, The expression level of VEGF mRNA in skin tissue of positive control group is obviously higher than that of blank group (P=0.004),The expression level of VEGF mRNA in peripheral blood is obviously higher than that of blank group, difference was statistically significant (P=0.000). Skin tissues:The expression level of VEGF mRNA in B, C and D group are less than positive group, the difference was statistically significant (P<0.05); and the three groups compared with the blank group, the difference had no statistical significance (P)0.05); The expression level of VEGF mRNA in group A is higher than the blank group, the difference was statistically significant (P<0.05), there was no statistically significant difference between group A and positive group (P)0.05). Peripheral blood:The expression level of VEGF mRNA in A, B, C and D group respectively compared with positive group and the blank group, there were no statistically significant difference.4. The correlation analysisThere are negatively relation between the concentration of the root of herbaceous peony total glycosides and skin tissue VEGF mRNA expression quantity is (Kendall’s tau_b:r=0.312, p=0.036; Spearman’s rho:r=0.383, p=0.040) but the correlation is no significant r<0.50; There are no relation between the expression of VEGF mRNA in peripheral blood (Kendall’s tau_b:r=0.012, p=0.936; Spearman’s rho:r=0.012, p=0.012)Conclusion:1. Mi sinensis treated mouse dorsal skin, appear similar to human psoriasis-like changes. By HE staining, mouse dorsal skin cutin layer thickening parakeratosis, granular layer thinning, stratum spinosum hypertrophy, epidermal ridge to extend the histologic changes, this is consistent with previous findings, description mi sinensis stimulates the dorsal skin of mice to establish the model of psoriasis. By real-time fluorescent quantitative PCR detection:the mi sinensis upregulates the expression of mouse skin tissue and VEGF mRNA in peripheral blood. 2. After TGP treatment can reduce the amount of VEGF mRNA in psoriasis mouse skin tissue, and slow disease progression, however, no significant effect on the expression of VEGF mRNA in peripheral blood. Therefore, we speculate that the therapeutic effect of TGP to psoriasis may be related to reduced VEGF mRNA expression in mouse skin tissue.3. The concentration of the TGP and skin tissue and peripheral blood of VEGF mRNA correlation analysis, found no significant correlation between the two.4. This topic will help understand the role of VEGF in the pathogenesis of psoriasis and the impact of TGP to them, for further research in the future, for the development of psoriasis in inflammatory signal transduction pathway associated, potential drug targets for anti-psoriasis drugs provide a theoretical basis for the clinical application. |
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