| [Objective]Through clinical experiments,explore the differences in the expression of Spred protein in psoriasis patients and normal tissues,and clarify the correlation between Spred protein and psoriasis;at the same time,through experiments on imiquimod-induced psoriasis mice,it is clear that the psoriasis is Correlation between Spred-1 protein and Ras/ERK signaling pathway,and based on Spred-1/Ras/ERK signaling pathway to study the possible mechanism of Qingxuedu Granules mixture in the treatment of psoriasis.[Method]1.Include an appropriate number of healthy person and patients with psoriasis vulgaris.The healthy person took skin tissue about 0.5cm×0.5cm in size.Psoriasis patients took tissues of the same size in the lesion area,near-lesion area and non-lesion area.All samples were detected by tissue immunofluorescence to observe the expression location and abundance of Spred-1,Spred-2 and Spred-3 in health person and psoriasis patients.2.There are 6 groups of mice in Overexpression group Ⅰ,Overexpression group Ⅱ,Empty virus group Ⅰ,Empty virus group Ⅱ,Normal group,Model group.Each group have 8 mice.Mice in Overexpression group I and II,Empty virus group I and II were injected with 100 μ l corresponding virus via tail vein,and model group and normal group were injected with 100 μ l normal saline.After 2 days of virus injection,the mice in the model group,the Empty virus group I and the Over expression group I were made by IMQ.The mice in the Overexpression group II,the Empty virus group II and the Normal group were treated with Vaseline for 7consecutive days,and samples were sacrificed after the 8th day.Observe the changes of mouse PASI scores during the experiment.Observe the expression of Spred-1protein and Ras/ERK pathway related proteins in the skin lesion area,near lesion area,and non-lesion area in psoriasis-like mice through WB experiments and RT-PCRexperiments3.32 male mice were randomly divided into Qingxuedu Granules group,Compound Qingdai capsule group,Model group,and Normal group.Each group have8 mice.The model of Qingxuedu granule group,Model group and Compound Qingdai capsule group was established by IMQ,while the Normal group was treated with Vaseline.The mice in the Model group and Normal group were intragastrically perfused with d H2 O,while those in the qingxuedu granule group and Compound Qingdai capsule group were intragastrically administered with corresponding drugs.The experiment continued for 7 days,and was executed on the 8th day.Observe the changes of PASI scores in mice during the experiment.Observe the expression of Spred-1 protein and Ras/ERK pathway related proteins in the skin lesion area,near lesion area,and non-lesion area in psoriasis-like mice through WB experiments and RT-PCR experiments.[Result]1.In the part of clinical experiment,Spred-1 and Spred-2 protein is expressed in the epidermis and dermis of normal people,however,spred-1 was mainly concentrated in basal layer,spinous layer and lower part of granular layer,and spred-2protein was mainly located in spinous layer,granular layer and cuticle layer.Spred-3protein is expressed in the spinous layer,subgranular layer and stratum corneum.At the same time,the expression of Spred-1,Spred-2 and Spred-3 in the three areas of psoriasis decreased compared with normal people,and the average fluorescence density value also decreased(P<0.05).2.In animal experiments,Spred-1 protein and m RNA in the Overexpression group Ⅱ increased(P<0.001).This indicates that Spred-1 overexpressing adenovirus can be expressed normally in mice.Spred-1 protein and m RNA in the Empty virus group II did not change(P>0.05).This indicates that the Empty virus has no effect on the Spred-1 protein.At the same time,the skin pathology of the three groups of mice was similar,and the average epidermal thickness between the groups was not change(P>0.05),indicating that Spred-1 overexpression of adenovirus and empty virus cannot produce spontaneous skin lesions and it has no obvious effect on normal skin pathology.3.In the Spred-1 overexpression experiment,mice in the Model group showed a little erythema and scales from the 4-5th days,followed by increased erythema,infiltration and thickening of the skin lesions,and scaly layers.The most severe symptoms appeared on the 7-9th day.Compared with the Model group,the PASI score,the average skin thickness and the pathological Baker score of the Empty virus group I were similar(P>0.05),indicating that the empty virus in vivo injection has no effect on the psoriasis skin lesions and pathology after IMQ external use.The overall severity of skin lesions in the Overexpression group Ⅰ was less than that of Model group and the Empty virus group Ⅰ(P<0.05).The average epidermal thickness and pathological Baker score of the Overexpression group I were lower(P<0.05),the epidermal process was reduced,the thickening of the stratum corneum was relieved,and the thickening of the spinous cell layer was reduced,indicating Spred-1overexpression can reduce the psoriasis-like skin lesions induced by IMQ to a certain extent.4.The Overexpression group I p-Ras,p-Raf1,p-ERK1/2,VEGF protein and Ras,Raf1,ERK1/2,VEGF m RNA expression is lower(P<0.001),indicating that the increased expression of Spred-1 protein inhibits the expression of Ras/ERK pathway related proteins and m RNA.The protein and m RNA expressions of Ras,p-Ras,Raf1,p-Raf1,ERK1/2,p-ERK1/2,VEGF in the Empty virus group I did not change much(P>0.05).It shows that the empty virus has no effect on the expression of m RNA in the Ras/ERK pathway.5.Compared with the model group,the symptoms of erythema,thickness in the Qingxuedu granule group and the Compound Qingdai capsule group were lighter,the PASI score decreased,and the average skin thickness and pathological Baker score decreased Obviously(P<0.05),it shows that Qingxuedu Granule Mixture and Compound Indigo Naturalis Capsule have certain therapeutic effect on IMQ-induced psoriasis-like mice.Comparing the Qingxuedu Granules group and the compound Qingdai capsule group,we found that the mice in the Qingxuedu Granules group recovered faster from erythema,infiltration and scales.The PASI score,the average skin thickness of the skin and the pathological Baker score were also lower(P<0.05).The above results indicate that the Qingxuedu Granules mixture has better effect on the skin lesions of psoriasis-like mice than the compound Qingdai capsule.6.In the traditional Chinese medicine intervention experiment,we used Qingxuedu Granules to treat psoriasis-like mice,and found that the expression of Spred-1 protein in the skin lesions,near-lesion areas,and non-lesion areas wereincreased after treatment.The protein expression of p-Ras,p-Raf1,p-ERK1/2,VEGF were decreased after treatment(P<0.001).These results indicate that Qingxuedu Granules Mixture can achieve the purpose of treating psoriasis by intervening the Spred-1/Ras/ERK pathway.There was no statistically significant change in Spred-1protein in the three areas after Compound Qingdai capsule treatment of IMQ-induced psoriasis-like mice(P>0.05).The protein expression of p-Ras,p-Raf1,p-ERK1/2,VEGF and the m RNA expression of Ras,Raf1,ERK1/2,VEGF in the lesion area were decreased after Compound Qingdai capsule treatment(P < 0.001),indicating that the compound Qingdai capsule does not target Spred-1 protein to regulate the Ras/ERK pathway to treat psoriasis,but through other means Regulate Ras/ERK pathway.[Conclusion]1.Spred-1,Spred-2,and Spred-3 proteins are all expressed in normal human epidermis,but their expressions are slightly different in each layer;at the same time,the expression of the three proteins in the three regions of psoriasis is consistent with the expression of normal people.However,the average fluorescence density value decreases.2.In the skin tissues of psoriasis-like mice,the expression of Spred-1 protein decreased,and the expression of Ras/ERK pathway related proteins and m RNA increased;Spred-1 overexpression can inhibit the expression of Ras/ERK pathway related proteins and relieve psoriasis Mice symptoms.3.Qingxuedu Granules Mixture can treat psoriasis by regulating Spred-1/Ras/ERK/VEGF pathway. |
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