Study On The Mechanism Of Dry Skin Itch And The Function Of Dangguiyinzi Decoction In Inhibiting Itching

Background and aimsItch is an unpleasant sensation which may cause desire to scratch associated with multiple skin diseases,such as dermatitis,eczema and psoriasis.It co?ld be also found in many system systemic diseases,such as cholestasis,diabetes and uremia.Pruritus affects the quality of people's life seriously,but the mechanism of it is not understood clearly,and many types of itch could not be treated very well in clinic.According to whether histamine participates in the itch formation,itch is divided into two types:histamine depend itch accounting for only a quarter and non-histamine-dependent itch accounting for three quarters in elinic.Recent studies have found that Mrgprs is closely related to the formation of non-histamine-dependent itch,which contains more than 50 subtypes.Among them,MrgprA3 is defined as an itch-specific receptor,and MrgprA3+ neurons in the DRG are defined as itch-specific neurons.Activation of this type of neurons produces only itch sensation without pain sensation.After this type of neuron is removed,it has a significant effect on itch and no effect on pain sensation.AEW model is a non-histamine-dependent itch model.The skin barrier function of the AEW model mice was destroyed,the hydration was weakened,the water loss was increased,the water content was reduced,and the symptoms of dry skin,desquamation,and itching appeared.It is also called chronic dry skin itch model.Currently,TRPA1 konwn as the downstream ion channel of MrgprA3 has been shown to be associated with the AEW model,but whether the itch-specific receptor-MrgprA3 and antother TRP channel which is coexpressed with MrgprA3 called TRPV4 have changed in this model,there is currently no relevant study.Unlike MrgprA3 i5 only expressed in DRG and TG,there are lots of TRPV4 expressions in the skin as well.Western medicine has no significant effect on the treatment of non-histamine-dependent itch,and the side effects are obvious.We try to screen drugs from Chinese medicine that can inhibit non-histamine-dependent itch.Our research on the literature related to pruritus in Chinese medicine found that Dangguiyinzi Decoction has been widely used as a classie Prescription for nourishing blood circuation,dispelling wind and relieving itching,especially for the treatment of pruritus with blood deficiency and wind-dryness type.However,it has not been studied the mechanism of inhibiting the itching at present.So our study is to explore whether MrgprA3 and TRPV4 are involved in the formation of the chronic dry skin itch and whether Dangguiyinzi could inhibit this type of itch.Experimental methods1 The AEW modeling was performed using WT mice,and after 3 days of modeling,CQ as the agonist of MrgprA3 was intradermally injected into the neck and back skin of the model area.We performed the same behavioral experiments using the MrgprA3EGFP-Cre;ROSA26tdTomat transgenic mice.2 Corrventional HE staining were used to observe the thickness of the epidermis of AEW model mice.3 Selecting MrgprA3EGFP-Cre;ROSA26tdTomato transgenic mice,detecting the changes of the density of the subcutaneous MrgprA3+ nerve fibers and the propotion of MrgprA3+ neurons in the DRG(C6-T10)before and after modeling and analyzing the expression of MrgprA3 at mRNA level in DRG.4 Calcium imaging technique was used to study whether the reaction ratio and intensity of DRG neurons responded to CQ stimulation before and after modeling were changed.5 AEW model mice were intraperitoneally injected with inhibitor HC067047 of TRPV4,and AEW modeling was performed with TRPV4-/-mice to investigate whether TRPV4 participated in the formation of AEW model at the behavioral level.6 Immunofluorescence and WB technique were used to analyze the expression of TRPV4 in DRG and skin of AEW model mice.7 Using WT mice to explore whether Dangguiyinzi could inhibit the scratch behavior of AEW model mice.8 The effect of Dangguiyinzi on skin epidermal thickness of AEW model mice was observed by HE staining.9 MrgprA3EGFP-Cre:ROSA26tdTomato transgenic mice were selected to examine the effects of Dangguiyinzi on the subcutaneous MrgprA3+ nerve fibers and MrgprA3+neurons in the DRG and analyze the expression of MrgprA3 at mRNA level in DRG.10 Immunofluorescence and WB were used to investigate effect of Dangguiyinzi on the propotion of TRPV4+ neurons and expression of TRPV4 protein in DRG and skin of AEW model mice.Experimental results1 AEW model mice were more sensitive to CQ stimulation.The behavioral results of MrgprA3EGFP-Cre;ROSA26tdTomato were consistent with that of WT mice.2 The thickness of epidermis was increased in the AEW model.3 The density of subcutaneous MrgprA3+ nerve fibers and the propotion of MrgprA3+neuron in the DRG of AEW model mice were significantly increased.The results of qPCR also showed that the MrgprA3 expression in DRG increased at the mRNA level.4 The reaction ratio of DRG neurons of AEW model mice to CQ stimulation was increased and had no change in intensity.5 The scratching behavior of AEW model mice could be inhibited by HC067047.The scratching behavior of AEW-modeled TRPV4-/-mice was significantly reduced relative to WT mice.6 TRPV4+ neurons were significantly increased in DRG neurons of AEW model mice,and the TRPV4 protein expression was also up-regulated.7 Dangguiyinzi could inhibit the scratch of AEW model.8 Dangguiyinzi could reduce the epidermal thickness of AEW model mice.9 Dangguiyinzi had no effect on the density of subcutaneous MrgprA3+ nerve fibers,the propotion of MrgprA3+ neuron and the expression of MrgprA3 at mRNA level in DRG.10 Dangguiyinzi could down-regulate the ratio of TRPV4+ neurons in DRG and the expression of TRPV4 protein in DRG and skin of AEW model mice.ConclusionMrgprA3 and TRPV4 are involved in the formation of the chronic dry skin itch.Dangguiyinzi can be used to inhibit itching of the chronic dry skin,which may be achieved by inhibiting TRPV4.