开玄解毒核心方调节银屑病模型小鼠冷敏TRPM8神经元衍生信号改善银屑病皮损OA

Objective:To investigate the ameliorative effects of Kaixuan Jiedu core prescription(KXJD)on skin lesions in psoriasis-like mouse models under cold environment exposure,and to analyze its influences on transient receptor potential(TRP)channels and related neuroimmune regulatory factors.Methods:Thirty-six C57BL/6J mice were randomized into 6 groups,with 6 mice in each group.Two feeding conditions were set:Normal temperature and cold[simulating a cold environment at(10±0.5)℃,for 6 h daily].Mice were induced to develop psoriasis-like lesions by applying imiquimod externally.The model mice were allocated into model groups and KXJD(30.42 g·kg-1,continuous gavage for 5 days)groups.Normal mice were used as the control group.Specifically,mice were allocated into normal temperature,normal temperature model,normal temperature+KXJD,cold exposure control,cold exposure model,and cold exposure+KXJD groups.The pathological changes in skin lesions were observed by hematoxylin-eosin(HE)staining.The expression of cluster of differentiation(CD)3+T lymphocytes,CD11c+dendritic cells(DCs),phosphorylated extracellular signal-regulated kinase(p-ERK),and substance P(SP)were detected by immunofluorescence assay.The protein level of transient receptor potential cation channel subfamily M member 8(TRPM8)in the skin tissue was determined by Western blot.The expression of TRPM8,transient receptor potential cation channel subfamily V member 1(TRPV1),transient receptor potential cation channel subfamily A member 1(TRPA1),and transient receptor potential cation channel subfamily V member 2(TRPV2)at the protein and mRNA levels was determined by immunohistochemistry and Real-time PCR,respectively.The levels of calcitonin gene-related peptide(CGRP)and neuropeptide Y(NPY)in the serum were analyzed by enzyme-linked immunosorbent assay(ELISA).The enrichment analysis of differentially expressed genes(DEGs)and TRP pathway network construction were conducted based on the GEO database.The co-expression of TRPM8 and CGRP in the skin lesions was verified by immunofluorescence double labeling.Results:Both the normal temperature and cold exposure model groups showed typical psoriasis-like skin lesions.Compared with the normal temperature and cold exposure control groups,the model groups had excessive epidermal keratinization,thickened spinous layer,and inflammatory infiltration in the dermis,with increased pathological scores(P<0.05),increased infiltration of CD3+and CD11c+cells and expression of p-ERK and SP,upregulated mRNA levels of TRPM8,TRPA1,and TRPV2,downregulated mRNA level of TRPV1(P<0.05),and reduced content of CGRP and increased content of NPY in the serum.Compared with the normal temperature and cold exposure model groups,KXJD reduced the pathological manifestations and pathological scores of psoriasis-like skin lesions(P<0.05),and inhibited the infiltration of CD3+and CD11c+cells and the expression of p-ERK and SP.Gene enrichment analysis suggested that the DEGs of psoriasis were significantly enriched in the interleukin(IL)-17 signaling pathway and TRP channel inflammatory regulation.Compared with the normal temperature and cold exposure model groups,KXJD reversed the abnormal mRNA levels of genes related to the TRP channel subfamilies(P<0.05),increased the CGRP level,and decreased the NPY level.Immunofluorescence double labeling further confirmed that compared with the model groups,KXJD down-regulated the co-expression of TRPM8 and CGRP in the skin lesions.Conclusion:KXJD may ameliorate psoriasis-like skin lesions by downregulating the overexpressed cold-sensitive receptor TRPM8 in skin lesions and correcting the disorder of neuropeptide(such as SP and CGRP)release mediated by it,thereby inhibiting the IL-23/helper T cell 17(Th17)core inflammatory pathway,suppressing the infiltration of inflammatory cells and the activation of the ERK signaling pathway,and regulating the Xuanfu(sweat pore)-TRPM8-neuroimmune response axis.