Study On The Mechanism Of CLDN5-induced Sensitive Skin

Sensitive skin(SS)refers to a high-responsive state of skin under physiological or pathological conditions.Up to 71%of people worldwide feel that their skin is sensitive.Its clinical manifestations mainly include subjective symptoms such as burning,tingling,itching and tightness,with or without objective signs such as erythema,telangiectasia and desquamation.Cosmetic ingredients,various environmental factors(air or temperature),psychological(such as stress)or hormone(menstrual cycle)factors can all contribute to the onset of SS.Although SS is not life-threatening,its unconscious symptoms still cause goreat physical and mental distress for patients.The occurrence and development of SS is a complicated process.So far,the pathogenesis of SS has not been fully clarified.It is currently believed that skin barrier destruction,neurological dysfunction,vascular hyperresponsiveness,and inflammatory response are all involved in the pathogenesis of SS.The team found that the expression of CLDN5 in facial skin lesions of SS patients with lactic acid>3 points and conscious sensitivity was reduced by sequencing.CLDN5 encoded claudin-5 is a tight junction protein,which forms a tight junction structure of the epidermal particle layer and regulates the skin’s permeability barrier.It may be related to the enhancement of the skin permeability of SS face and the increase of TEWL value.In addition,whether CLDN5 is associated with SS neurological dysfunction,vascular hyperresponsiveness,and inflammatory response is unknown,and further research is still needed.Objective:To investigate whether CLDN5 is involved in the pathogenesis of SS by mediating inflammatory response,neurological dysfunction,vasodilation and vascular hyperresponsiveness.Methods:Collect the paraffin-embedded facial skin of SS patients and normal people,and then immunohistochemically detect the expression of claudin-5 in the epidermis and blood vessels of facial skin of SS patients;CLDN5 and empty vector(NC)lentiviral shRNA were used to transfect human immortalized keratin A cell line(HaCat)was formed,and three HaCat cells from the CLDN5 knockdown group(sh-CLDN5)and the control group(sh-NC)were collected for transcriptome sequencing.Screening for increased expression and pro-inflammatory cytokines(IL-23A,IL-8,IL-1β)from differentially expressed mRNA,and using ELISA in HaCat cells to verify the IL-23A,IL-8,IL-1β Expression and Western Blot were used to verify NF-κB and MAPK related pathway proteins(IκBα,p65,JNK,ERK1/2,p38,etc.)and their phosphorylation.Q-PCR detects neurogenic inflammation-related molecules(SP,CGRP)and neurological dysfunction-related molecules(TRPV1,NGF).Then use CLDN5 and empty vector(NC)lentivirus shRNA to transfect human microvascular endothelial cell line(HMEC-1),collect control group(NC),CLDN5 knockdown group(cldn5-),CLDN5 knockdown and NO donor(SNP)group(SNP+sh-CLDN5),Q-PCR was used to detect the expression of VEGF and ET1;NO reagent was used to detect the expression of NO in the experimental group and the control group.Results:1.Immunohistochemical staining of the facial skin of SS patients and normal people showed that the expression of claudin-5 in the epidermal granular layer and vascular endothelium of SS tissue was significantly reduced compared with normal people.2.In epidermal cells(HaCat),there are 7 inflammatory cytokines that have different expressions in the cldn5-group and NC group transcriptome.Among them,5 are up-regulated,2 are down-regulated,and IL-23A is up-regulated and pro-inflammatory,IL-8,IL-1β.Western Blot showed that IL-23A increased in sh-CLDN5 group.ELISA verified that IL-8 in cldn5-group was higher than that in NC group,but there was no difference in IL-1β expression.Western Blot results showed that phosphorylated JNK,ERK1/2,p38,IκBα,and p65 all increased in the sh-CLDN5 group,and MAPK and NF-κB pathways were activated.3.In the human microvascular endothelial cell line(HMEC-1),compared with the NC group,the sh-CLDN5 group decreased NO expression,increased ET-1 expression,and decreased VEGF expression,and CLDN5 regulated ET-1 and VEGF by NO achieved.Conclusions:1.The expression of claudin-5 in the epidermis and dermal blood vessels of the SS is reduced,which is an important gene involved in the pathogenesis of SS.2.Down-regulation of CLDN5 in HaCat cells may participate in the process of early inflammation of SS by activating MAPK pathway and up-regulating IL-23A and IL-8.3.CLDN5 does not affect SS neurological dysfunction and neurogenic inflammation.4.CLDN5 endothelial cells up-regulate ET-1 by down-regulating NO,leading to endothelial dysfunction and causing vascular rigidity may be a potential mechanism of SS facial vasodilation.The decrease of VEGF may be to reduce the further vascular leakage caused by the decreased expression of CLDN5 And reduce angiogenesis.