Regulation Of MiR-215-5p On Epidermal Cell Proliferation And The Mechanism Of Ma Fang Xi Jiao Di Huang Decoction In The Treatment Of Psoriasis

Objective:Observe the effect and regulation of miR-215-5p on epidermal cell proliferation;establish a psoriasis mouse model,observe the effect of miR-215-5p on psoriasis mouse model,to observe the effect of Ma fang xi jiao di huang decoction on the expression of miR-215-5p,oxidative stress of skin tissue,inflammatory factors and related signaling pathways after intervention in psoriasis mouse model,to explore the mechanism of action of Ma fang xi jiao di huang decoction in the treatment of psoriasis,and provide a reference for clinical medication.Methods:1.Treat HaCaT cells with 50 ng / ml IL-6,IL-22,IL-17 A,and use qRT-PCR to detect the miR-215-5p expression changes;transfected miR-215-5p mimic into HaCaT cells,use qRT-PCR detection of miR-215-5p expression changes,CCK-8 experiment to determine cell proliferation,flow cytometry to detect cell cycle,Edu staining method to determine cell proliferation;bioinformatics software predicts miR-215-5p and DYRK1 A 3’UTR has two complementary binding sites,site1 and site2,using the luciferase reporter system to identify the targeting relationship between the two;transfected miR-215-5p mimic into HaCaT cells,qRT-PCR method to determine the expression of DYRK1 A mRNA,Western blot detection of DYRK1 A,EGFR、AKT、p-AKT、ERK、p-ERK protein expression changes;IMQ induced construction of psoriasis mouse model,subcutaneous injection of miR-215-5p agomir,observe mouse skin lesions,PASI score,qRT-PCR method to measure miR-215-5p expression changes,HE staining to observe skin tissue pathological changes,immunohistochemistry to determine Ki67 expression in skin tissues,qRT-PCR detection of DYRK1 A mRNA expression changes,Western blot detection of DYRK1 A,EGFR,AKT,p-AKT,ERK,p-ERK protein expression;2.IMQ induced the construction of a psoriasis mouse model,given Ma fang xi jiao di huang decoction by intragastric treatment,measuring the body weight of each group and observing the skin lesions,performing PASI score,measuring the thickness of the outer edge of the auricle,using the HE staining method observe the pathological changes of skin,and determine the expressio level of miR-215-5p in skin tissue by qRT-PCR method;3.IMQ induced the construction of a psoriasis mouse model,and given Ma fang xi jiao di huang decoction by intragastric treatment.The skin tissue was taken and tested for GSH,CAT,MDA,SOD content or activity by GSH assay kit,CAT assay kit,MDA assay kit,SOD assay kit;4.IMQ induced the construction of a psoriasis mouse model,given Ma fang xi jiao di huang decoction by intragastric treatment,skin tissue was taken,and qRT-PCR was used to detect TNF-α,IL-6,IL-1β,IL-17 A,IL-22 mRNA Level,ELISA method to determine the content of TNF-α,IL-6,IL-1β,IL-17 A,IL-22,Western blot detection of IκB,p-IκB,p65,p-p65,JNK,p-JNK,ERK,p-ERK,p38,p-p38 protein levels.Results:1.The expression level of miR-215-5p in HaCaT cells after IL-6,IL-22 and IL-17 A treatment was reduced.The expression level of miR-215-5p in HaCaT cells after miR-215-5p mimic transfection increased,the cell proliferation capacity decreased,the proportion of G1 phase cells increased,and the proportion of S phase decreased.miR-215-5p and DYRK1 A were in a targeted relationship with each other.After transfected with miR-215-5p mimic,the expression levels of DYRK1 A mRNA and protein in HaCaT cells decreased,the expression levels of EGFR,p-AKT,and p-ERK proteins also decreased,and the expression levels of AKT and ERK proteins did not change significantly.The IMQ psoriasis mouse model was successfully replicated.The skin lesions were obvious,the scales increased,the PASI score increased,the epidermal spine cell layer in the skin tissue was significantly thickened,there was significant inflammatory cell infiltration,the number of Ki67 positive cells increased,DYRK1 A mRNA and protein levels increased,EGFR,p-AKT,p-ERK protein levels also increased,AKT,ERK protein expression levels did not change significantly;psoriasis model mice after subcutaneous injection of miR-215-5p agomir had obvious lesions improved,PASI score decreased,the thickness of epidermal spine cell layer in skin tissue decreased,inflammatory cell infiltration decreased,the number of Ki67 positive cells decreased,DYRK1 A mRNA and protein levels decreased,EGFR,p-AKT,p-ERK protein levels also decreased,AKT and ERK protein expression levels did not change significantly.2.Psoriasis model mice had significantly reduced body weight,obvious erythema and scale on the skin surface,increased PASI score,increased thickness of the outer edge of the auricle,increased thickness of skin tissue epidermis,increased inflammatory cell infiltration,and miR-the expression level of 215-5p decreased;the weight of psoriasis model mice treated with Ma fang xi jiao di huang decoction after oral administration increased,the erythema and scales of the skin surface of the mice improved,the PASI score decreased,the thickness of the outer edge of the auricle decreased,and the skin tissue epidermis both thickness and inflammatory cell infiltration were significantly improved,and the expression level of miR-215-5p in skin tissue increased.3.SOD activity in skin tissue of psoriasis model mice decreased,MDA content increased,GSH content decreased,CAT activity decreased;psoriasis model mice after intragastric treatment of Ma fang xi jiao di huang decoction,SOD activity increased,MDA content decreased,GSH content increased,CAT activity increased.4.TNF-α,IL-6,IL-1β,IL-17 A,IL-22 mRNA and content in skin tissue of psoriasis model mice are increased,p-IκB,p-p65,p-JNK,p-ERK and p-p38 protein levels also increased,and IκB protein levels decreased;psoriasis model mice after intragastric treatment of Ma fang xi jiao di huang decoction,TNF-α,IL-6,IL-1β,IL-17 A,IL-22 mRNA and content of 17 A and IL-22 decreased,and the protein levels of p-IκB,p-p65,p-JNK,p-ERK,and p-p38 also decreased,and the level of IκB protein increased.Conclusions:1.The high expression of miR-215-5p could inhibit DYRK1 A and affect the activation of EGFR,AKT and ERK signaling pathways,further inhibit epidermal cell proliferation and improve the pathological status of psoriasis model mice.miR-215-5p may be a protective factor for psoriasis.2.Ma fang xi jiao di huang decoction could improve the pathological state of psoriasis model mice and improve the quality of life.3.Ma fang xi jiao di huang decoction might play a role in treating psoriasis by up-regulating the expression of miR-215-5p,targeting the inhibition of DYRK1 A and affect the activation of EGFR,AKT and ERK signaling pathways for the treatment of psoriasis.4.Ma fang xi jiao di huang decoction might inhibit the activation of MAPK,NF-κB signaling pathways,and reduce the levels of inflammatory factors such as TNF-α,IL-6,IL-1β,IL-17 A,IL-22 play a role in the treatment of psoriasis;5.Ma fang xi jiao di huang decoction could improve the oxidative stress of psoriasis and play a role in treating psoriasis.