Study On The Mechanism Of TRPV4 Participates In Acute And Chronic Itching And The Antipruritis Of Cimifugin

Experimental method:Part ?:The pathological slices of pruritic skin in clinical psoriasis patients were stained with HE to observe the epidermal hyperplasia and the expression of TRPV4 was detected by immunofluorescence staining experiments.Part ?:Behavioral Experiment:(1)Intradermal injection of itching source in the back of WT mice and TRPV4-/-mice to explore the pathway of TRPV4 involved in itching and the antipruritic effect of cimifugin.(2)Topical application of 5%imiquimod cream on the shaved back skin of mice for 5 consecutive days to induce a psoriasis model to explore the role of TRPV4 in chronic pruritus and the effect of cimifugin on psoriasis pruritus.Part ?:Mechanism Research:Real-time quantitative PCR technology,immunofluorescence staining experiment and Western Blot experiment were used to detect the expression of TRPV4 in the skin and DRG of the psoriasis model mice.Part ?:Cell Experiments.(1)Using HaCaT Cells,the effect of cimifugin on TRPV4 ion channel activity in human keratinocytes was studied by calcium imaging.(2)DRG neurons cultured in WT mice were used to study effect of cimifugin on TRPV4 ion channel activity in DRG of mice by calcium imaging technology.(3)Transfection of the plasmid of TRPV4 in HEK293 cells,and the use of calcium imaging technology to study whether cimifugin directly affects the TRPV4 ion channel.(4)TRPV4 plasmid was transfected in HEK293 cells,and the effect of Cimifugin on TRPV4 ion channel activity was studied by whole-cell patch clamp technology.Experimental results:(1)The biopsies from patients with psoriasis demonstrate characteristic features associated with skin barrier dysfunction and keratinocytes proliferation,the expression level of TRPV4 in human skin is closely related to the severity of psoriasis.(2)TRPV4 is required for itch and is involved in serotonin-dependent and histamine-dependent pruritus,but not with chloroquine-mediated pruritus.(3)TRPV4 is necessary for pruritic behavior in mice chronic model of psoriasis,the scratch behavior of TRPV4-/-mice was significantly reduced after psoriasis modeling.(4)In the mice chronic model of psoriasis,the expression of TRPV4 in the skin and DRG was increased.