The Study Of Psoriasis Mechanism Based On The State Of Blood

ObjectivePsoriasis is a refractory skin disease with multifarious pathogeny and complex mechanism. According to the different clinical manifestations, psoriasis is mainly divided into three syndromes in traditional Chinese medicine (TCM) theory:"Blood stasis", "Blood dryness" and "Blood heat". Xuan guowei, the National medical master, indicated that blood stasis is a pathological condition throughouting in psoriasis, and it is a important factor of effecting morbidity and prognosis. By observing the state of blood collected from the patients with blood stasis type of psoriasis and morphological characteristics of their red blood cells, We try to verify correlation between abnormal blood state and poriasis. Then the animal model of psoriasis with blood stasis and the splenectomy are established to discuss the mechanism of psoriasis and to lay the fondation for the treatment of psoriasis based on TCM theory and drug discovery.Methods1 Evaluation of blood state of patients with blood stasis type of psoriasis1.1 Collection of blood sample from patients with blood stasis type of psoriasisThe study collected venous blood from 50 patients with blood stasis type of psoriasis and 30 healthy volunteers according to inclusion and exclusion criteria and detected blood viscosity, erythrocyte aggregation index, erythrocyte rigidity index, hematocrit, cholesterol, triglycerides and high density lipoprotein cholesterol.1.2 Preparation of red blood smear and observation of red blood cells morbidityThe blood smear should be prepared within 4 hours and observed within 24 hours. Randomly select three different fields to observe under the upright microscope and record the number of poikilocyte, then calculated the rate of poikilocyte and rate of poikilocyte in microscope.1.3 Experiment separating red blood cell by the discontinuous percoll density gradient centrifugationThe whole blood was prepared into white red blood cell and then the white red blood cell collected from patients and volunteers were separated by the discontinuous percoll density gradient centrifugation. The percoll was confected into five different density layers in the glass tubes and the density layers successively were 1.0910g/mL,1.0985g/mL,1.1060g/mL, 1.1135g/mL,1.1210g/mL from top to bottom. Then the prepared blood cells were centrifuged and distributed into different layers similar densities. We measured the different layers’thickness by a caliper.2 Establishment and evaluation of animal model of psoriasis caused by blood stasis2.1 Establishment of animal model of psoriasis caused by blood stasisThe study established animal models in the method of congealing cold blood combined qi-deficiency and the method of qi-stagnacy.32 Kunming mouse were divided into four groups:Blank, Control, psoriasis with qi-stagnancy and blood stasis (PQSBS), psoriasis with congealing cold and qi-deficiency and blood stasis (PCQBS). To establish the blood stasis animal model, we used the way of swimming in ice water, hunger and bounding. After being barbered back, all groups were given 5% propranolol microemulsion except Blank.2.2 The evaluation of animal model of psoriasis caused by blood stasisWe observed and recorded the general conditions, then tested blood viscosity by rotary and lesions skin after executed on the 51h day. After PCQBS animal model successfully established, the spleen and thymus were minutely weighed on analytical balance and calculate for mouse spleen index and thymus index of each group. After being fixed in formalin solution, the skin tissue was maked into HE staining pathology.2.3 The change of inflammatory factors and energy metabolism capacity of animal model of psoriasis caused by blood stasis36 mouse were divided into three groups:Blank、Control、Congealing cold and qi-def iciency blood stasis (CQBS)、psoriasis with congealing cold and qi-def iciency blood (PCQBS). The CQBS and PCQBS were established model like title 2.1, but took the tissue one day in advance.The inflammatory factor just like Keratin-16, IL-6, IL-22, IL-23 in the skin of all groups were tested by the model of RT-PCR and ELISA.Gene and protein expression levels of vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF) in the skin of all groups were tested by the model of RT-PCR and ELISA.The exposed skin temperature of animals was detected in the infrared thermometers and compared after software processing. And the content and vitality of serum T-ATP enzyme, MAD, T-SOD, NO wer tested in kits. Take one half of spleen fixed by MDA and scan by transmission electron microscope to observe the ultrastructural changes like mitochondria and endoplasmic reticulum.3 The mechanism discussion about animal model of psoriasis caused by blood stasis3.1 Model establishment40 Kunming mouse were randomly divided into five groups:Blank, Control, Sham operation(SO), Splenectomy model (SM), Splenectomy and Propranolol (SP). SP and SM were performed partial splenectomy and SO were opened the abdominal cavity but did not resect spleen. Then SP, SO, Control were given 5%propranolol microemulsion 50uL and did nothing does not for the other two groups, and execute on the 4th day smearing.3.2 Model evaluationWe observed and recorded the general conditions and placed the skin tissue in formalin solution, then made HE staining pathology after being fixed, to observe the characteristics. Weitgh the thymus minutely on analytical balance and calculate for thymus index of each group.3.3 The mechanism discussion about animal model of psoriasis caused by splenectomyMouse skin tissue was tested using the model of RT-PCR on K-16, IL-6, VEGF, HIF, and using ELISA on L-6, IL-22, VEGF and HIF. Results1 Evaluation of blood state of patients with blood stasis type of psoriasis1.1 Change of blood state of patient with blood stasis type of psoriasisCompared with the normal reference values, erythrocyte rigidity index, whole blood viscosity (middle shear rate), whole blood viscosity (high shear rate), triglyceride of the patients with psoriasis was detected in higher value than the upper limit of normal. And whole blood viscosity (low shear rate), cholesterol were normal on the high side.1.2 Observation of red blood cells morbidityCompared with healthe volunteers there were red blood cells changed into poikilocyte showing acanthocyte, drop shape and irregular shape. The poikilocyte rate and microscopic poikilocyte rate of psoriatic patients were significantly increased.1.3 Experiment separating red blood cell by the discontinuous percoll density gradient centrifugationRed blood cells in psoriatic patients were mainly distributed in the level of high density with high degree of aging, and in the healthy volunteers were mainly distributed in the low density level with low degree of aging.2 Establishment and evaluation of animal model of psoriasis caused by blood stasis2.1 Establishment of animal model of psoriasis caused by blood stasis2.1.1 General conditionThe mouse’weight reduced in the PCQBS and PQSBS with dull fur, less dynamic, dark skin color and even showed dark purple.2.1.2 Change of blood viscosityThere were no significant difference in whole blood viscosity between Blank and Control, and compared PCQBS, PQSBS with Blank and Control in whole blood viscosity, there were significantly increased, under low shear rate conditions more obvious. Between PCQBS and PQSBS, the former increased more significantly.2.1.3 Pathological featuresThe pathological section of PCQBS and PQSBS showed that thickened spinous layer, decreased granular layer cells, inflammatory cell infiltration, etc.2.1.4 Change of spleen and thymus index affected by modelingThere are no significant difference between Blank and Control neither index of spleen nor thymus. The PCQBS and PQSBS index of thymus is increased in varying degrees. On the contrary, spleen index was decreased.2.2 The change of inflammatory factors and energy metabolism capacity of animal model of psoriasis caused by blood stasisThe expression level of inflammatory factor in skin tissue RT-PCR result showed that there were no significant difference between Blank and Control in K-16, IL-6, IL-22 and IL-23. Compared with Blank K-16, IL-6, IL-22, IL-23 in the CQBS skin were indifferent. The expression level of K-16, IL-6, IL-22 and IL-23 of PCQBS skin tissue were significantly higher than CQBS and Blank.The ELISA result showed that there are no significant difference between Blank and Control in IL-6, IL-22 and IL-23. Compared with Blank IL-6, IL-22, IL-23 in the CQBS skin had no difference. The expression level of IL-6, IL-22 and IL-23 of PCQBS skin tissue were significantly higher than CQBS and Blank with strong similar tendency of human psoriasis.RT-PCR test and ELISA test showed the same tendency among the different groups:there were no significant difference between Blank and Control in VEGF and HIF. Compared with Blank, HIF increased but VEGF had no difference in the CQBS. Compared with CQBS, VEGF increased but HIF had no difference in the PCQBS.Infrared thermometer test results showed that skin temperature had no difference between Blank and Control. Compared with CQBS the skin temperature of PCQBS increased significantly.The results of T-ATP, MDA and T-SOD content test showed that there were no significant difference between Blank and Control in T-ATP, MDA and T-SOD. Compared with Blank, MDA increased significantly in the CQBS serum, but T-ATP and T-SOD had no difference. Compared with CQBS, content of T-SOD reduced, T-ATP increased but MDA had no difference in PCQBS serum.The level of serum NO test showed us the NO level of Control was significantly higher than Blank, the level of CQBS had no difference than Blank and the serum level of PCQBS was significantly higher than CQBS and Control.On the Transmission electron microscope (TEM) scan the Blank and Control had normal spleen ultrastructure. The scan of CQBS and PCQBS had irregular cell shape and vacuolated mitochondria, the free ribosome also could be catched in the cytoplasm.3 The mechanism discussion about animal model of psoriasis caused by blood stasis3.1 The change of thymus index affected by splenectomyThere were no difference between Blank and Control. Compared with Blank the SO, SM had no significant difference. But the thymus index of SP was higher than SO and SM.3.2 The change of appearance and tissue pathology affected by splenectomyThe mouse skin was smooth and color-normal among Blank, SO and SM. There was normal skin color and slightly rough surface among Control. The mouse skin looked red, dying, rough and felt hard, further more the dander cover on the partial surface.The pathological section of Blank, SO, SM showed normal pathology, but the SP showed that thickened spinous layer, granular layer cells decreased or abnormal arrangement, inflammatory cell infiltration, etc.3.3 The K-16, IL-6, VEGF HIF expression level of on gene and protein in the model of splenectomyRT-PCR test and ELISA test showed the same tendency among the different groups:there are no significant difference between Blank and Control. Compared with Blank, K-16, IL-6, HIF, VEGF kept unchanged in SO and SM. Compared with SO and SM, all the four factors significantly increased on the expression.3.4 Change of serum NO level affected by modelingThe level of serum NO test showed that the NO level of Blank and Control was indifferent and level of the SO, SM also had no difference. Compared with SO and SM, the level of SP increased significantly. Conclusion1. In whole blood samples of psoriasis patients, the related indicators of the blood status are abnormal, the number, frequency of poikilocyte increases, it indicates the red blood cells age of psoriasic patients also increase. According to above, the incidence of psoriasis is closely related to the state of the blood.2. We can establish the animal model of psoriasis with blood stasis by introducing psoriasis on the state of blood stasis.The model is consistent with the basic pathological features of psoriasic patients belong to blood stasis type, and it can be used to study the pathogenesis of psoriasis, mechanism of Chinese medicine treatment and drug discovery, etc.3. The onset and progression of blood stasis psoriasis is closely related to the increase in expression of inflammatory cytokines and keratin, local changes in energy metabolism, oxidative stress, etc.4. Pathological conditions of congealing cold and qi-deficiency and blood stasis can damage mitochondria and other organelles of spleen, then influence structure and function of the spleen. After the structure and function of the spleen is limited the normal physiological function of the immune system and red blood cells is inhibited, which is the common link of the occurrence of blood stasis disease and psoriasis.The link may be one of the pathogenesis of psoriasis of blood stasis type.