Study On The Mechanism Of Qingxuedu Capsule Treating Psoriasis Vulgaris Via Spred2/Ras/ERK Pathway

[Objective]Through studies on the psoriasis mouse model and Ha Ca T cells,we explored the correlation between Spred2 and the Ras/ERK pathway,and verified the correlation between Spred2-NBR1 binding and the Ras/ERK pathway.By intervening in psoriasis mouse models with Qingxuedu Capsule,we investigated the possible mechanisms of treating psoriasis based on the Spred2/Ras/ERK signaling pathway,providing experimental evidence for treating psoriasis based on the toxin theory.[Method]1.Animal experiment of Spred2 overexpression: SPF-level BALB/C male mice were randomly divided into 6 groups.Three groups were induced to develop psoriasis model mice using chemical induction method IMQ,and vaseline group was given equal amount of vaseline cream intervention.The three model groups were the model group,empty virus II group,and overexpression II group,and the vaseline group was the normal group,empty I group,and overexpression I group,each with 8 mice.Two days before the experiment,empty I,II group,and overexpression I,II group were infected with empty virus and Spred2 overexpression adenovirus by micro-needle local infection.Saline intervention was administered to both the normal group and the model group.After 7 days of modeling,the mice were killed and sampled at the cervical dislocation site.During the experiment,the general condition,skin status,PASI score changes,etc.of the mice were observed.The tissue pathological changes of the mouse samples in each group were detected and counted,and the Western blot and RT-PCR experiments were used to observe the expression of Spred2 protein and m RNA in psoriasis-like mouse skin lesions and Ras/ERK pathway-related proteins and m RNA expression.2.Cell experiment of Spred2 overexpression: Ha Ca T cells were cultured in vitro and a psoriasis cell model was constructed using IL-22 and TNF-α.Four groups were set up: normal group,model group,empty group,and overexpression group.The control group did not receive any intervention,while the empty group and the overexpression group were infected with empty virus and Spred2 overexpression adenovirus,respectively.The expression of Spred2 protein m RNA was detected using PCR to confirm the successful establishment of the overexpression model.Cell proliferation and apoptosis were assessed by CCK8 and flow cytometry,respectively in each group.Besides,ELISA was employed to measure the levels of IL-6 and IL-1βexpression in all groups,and protein and m RNA expression levels of Spred2 and proteins related to the Ras/ERK pathway in each group were detected using Western blot and RT-PCR.In addition,co-immunoprecipitation was used to compare the Spred2-NBR1 binding status of cells in each group.3.Experimental intervention with traditional Chinese medicine: SPF-grade male BALB/C mice were divided into four groups using a randomization method,including the normal group,the model group,Compound Qingdai capsule group,and Qingxuedu capsule group.The IMQ was used to induce psoriasis mouse model,and the normal group was given vaseline intervention.The Compound Qingdai capsule group and the Qingxuedu capsule group were given Compound Qingdai capsule and Qingxuedu capsule respectively after modeling,The same volume of distilled water was orally administered to both the normal group and the model group.After 3 hours of topical application of IMQ,mice were given oral administration twice a day for 7days,and were euthanized for sampling after 7 days.During the experiment,Observations were made on the general condition,skin changes,and PASI scores of the mice in each group.After sampling,the histopathological changes of the mice in each group were examined,and Western blot and RT-PCR experiments were used to detect the expression of Spred2 protein and m RNA in each group,as well as the expression levels of Ras/ERK pathway-related proteins.Furthermore,detection was performed using immuno fluorescence confocal microscopy in order to detect the binding of Spred2 and NBR1 in each group.[Results]1.In the Spred2 overexpression animal experiment,compared to the empty vector I group and the normal group the Spred2 protein and m RNA levels were significantly elevated in the overexpression I group( < 0.001);no significant differences were observed in the levels of Spred2 protein and m RNA.between the empty vector I group and the overexpression I group(>0.05);the PASI score and Baker score were significantly decreased in the overexpression II group(<0.01),compared with the empty vector II group and the model group.Meantime,the skin pathological changes were milder than those in the model group and the empty vector II group,indicating that Spred2 overexpression can alleviate skin pathological changes and related symptoms of psoriasis to a certain extent.Relative to the model group,no significant difference was detected in Raf1 m RNA,Ras m RNA,ERK1 m RNA,ERK2 m RNA,VEGFA m RNA expression,as well as the protein expression in the empty vector II group(> 0.05);however,the Raf1 m RNA,Ras m RNA,ERK1 m RNA,ERK2 m RNA,VEGFA m RNA expression,and protein expression,compared with the model group,were greatly diminished in the overexpression II group( < 0.001),further indicating that increased Spred2 protein expression can inhibit m RNA expression of the Ras/ERK pathway.2.In the Spred2 overexpression cell experiment,compared with the normal and empty vector groups,The expression levels of Spred2 protein and m RNA were significantly higher in the overexpression group compared to the model group(<0.01),although no significant changes in Spred2 levels between the normal and empty vector groups(>0.05),indicating that the Spred2 overexpression adenovirus can express normally in HaCaT cells.The CCK8 proliferation results showed that the cell proliferation was distinctly decelerated(<0.01).On the contrary,when compared with the model group and there was a significant increase in the rate of apoptosis(<0.01).This suggests that overexpression of Spred2 protein can impede the proliferation and facilitate apoptosis of Ha Ca T cells related to psoriasis.Compared to the model group,the overexpression group had significantly lower IL-1β and IL-6 protein levels(<0.01),The results suggest that upregulating Spred2 expression can suppress the expression of inflammatory factors in Ha Ca T cells related to psoriasis.The model group showed a significant increase in Ras/ERK pathway related protein and m RNA levels whlie compared to the normal group(<0.01).By contrast,the overexpression group had significantly decreased levels of Raf1 m RNA,Ras m RNA,ERK1 m RNA,ERK2 m RNA,VEGFA m RNA expression,and protein expression(<0.001),while compared with the model group.Further demonstrating that increased Spred2 protein expression can inhibit Ras/ERK pathway-related m RNA expression.Immunoprecipitation results showed that compared with the empty vector group,the overexpression group had stronger binding between Spred2 and NBR1,with stronger protein expression.This result indicates that overexpression of Spred2 protein can enhance the binding of Spred2 and NBR1 in psoriasis Ha Ca T cells and strengthen the inhibitory effect on the Ras/ERK pathway.3.During the experiment involving Chinese medicine intervention,when compared to the model group,the PASI score decreased significantly in the Qingdai and Qingxuedu groups( < 0.01),and the Baker score decreased( < 0.01).Meanwhile,the improvement in redness,skin thickening,scaling,and other symptoms in the Qingxuedu group was better than that in the Qingdai group,with a lower PASI score( < 0.01)and a lower Baker score( < 0.05),indicating that Qingxuedu capsule had a better effect on improving the symptoms and pathological degree of psoriasis in model mice than Qingdai capsule.In comparison to the model group and Qingdai group,the content of Spred2 protein and m RNA in the Qingxuedu group was significantly increased(<0.01),while there was no statistical significance in the changes of Spred2 protein and m RNA expression in the Qingdai group compared with the model group(>0.05).This indicates that Qingdai capsules do not participate in the pathogenesis of psoriasis by regulating Spred2 protein,while Qingxuedu capsule may treat psoriasis by regulating Spred2 protein.In comparison to the normal group,the expression of p-ERK,ERK,p-Ras,Ras,p-Raf1,Raf1,and VEGFA proteins in the model group has declined significantly(< 0.001).Different from the model group,the level of p-ERK,ERK,p-Ras,Ras,p-Raf1,Raf1,and VEGFA proteins decreased in the Qingdai and Qingxuedu groups(<0.001),and the content of related proteins in the Qingxuedu group was less than that in the Qingdai group;In the same way,significant differences occurred in p-ERK,p-Ras,p-Raf1,and VEGFA(<0.01).There was a significantly reduce in the binding of Spred2 and NBR1 in the model group compared to the normal group(<0.001).Different from the model group,the cumulative optical density values(IOD)of Spred2 protein and NBR1 protein in the Qingdai group were slightly increased,instead the change was not obvious(>0.05);Divergent from the model and Qingdai groups,the IOD values of Spred2 protein and NBR1 protein in the Qingxuedu group were remarkably upgraded( < 0.01).This indicates that the compound Qingdai capsule cannot promote the binding of Spred2 protein and NBR1 protein,while Qingxuedu capsule may enhance the Ras/ERK pathway inhibitory effect by enhancing the ability of Spred2 binding to NBR1,thus enhancing the therapeutic effect on psoriasis.[Conclusion ]1.The expression of Spred2 protein was found to be decreased in the skin tissue of psoriasis mouse models,while the expression of Ras/ERK pathway-related proteins and m RNA increased.Nonetheless,the expression of these pathway-related proteins and m RNA can be inhibited by overexpression of Spred2,leading to the alleviation of symptoms and improvement of the pathological manifestations associated with psoriasis in the mouse models.2.In a cell model of psoriasis,overexpression of Spred2 can inhibit Ha Ca T cell proliferation,promote cell apoptosis,reduce the expression of inflammatory factors,enhance the binding with NBR1,and inhibit the expression of Ras/ERK pathway-related proteins and m RNA.3.Possible mechanism underlying the therapeutic effect of Qingxiedu capsule on psoriasis was found to be through enhancing Spred2 protein expression,strengthening the binding between Spred2 and NBR1,and inhibiting the expression of Ras/ERK pathway-related proteins.