The Role And Mechanism Of TRPV3 In Skin Injury Caused By Metabolic Acidosis

Objective:Metabolic acidosis is a general term for a series of clinical symptoms associated with impairment of the cardiovascular,respiratory,gastrointestinal,and central nervous systems,as well as dryness and damage of the skin throughout the body.For example,diabetes patients may have skin infection and symptoms that are difficult to heal.When the condition worsens and acidosis occurs,the skin damage will further deteriorate,forming a vicious circle.If the treatment is not timely,it may cause ulceration,gangrene or even amputation.Therefore,it is of great significance to explore the specific mechanism of skin injury caused by metabolic acidosis.Tissue acidosis is mainly characterized by pain and inflammation,and transient receptor potential channels(TRP)play an important role in the regulation of both reactions.Transient receptor potential vanilloid(TRPV)is a member of the TRP family.Mammalian TRPV is composed of TRPV1-6.TRPV3 is mainly expressed in human skin tissue cells,mediates skin sensation,affects epidermal keratinocyte proliferation and differentiation,hair growth,participates in inflammatory response,and plays an important role in the maintenance of skin homeostasis and normal function.The ion channels are regulated by temperature,osmotic pressure,pH value and intracellular signaling molecules.In tissue acidosis,cells expressing TRPV3 are inevitably exposed to acidic pH conditions,so it is of great significance to study the role of acid in the regulation of TRPV3 channels.When pH is low,the presence of extra protons(H⁺)in the environment will induce the internal amino acids of the protein to obtain protons,that is,protonation modification.In general,protons regulate protein function by changing the conformation of glutamic acid(E),aspartic acid(D),or histidine(H)residues.Previous studies have suggested that acid can activate TRPV3 channels through intracellular S2 and S3 transmembrane helices,and can enhance the ligand activity of the non-selective TRPV channel agonist 2-APB by regulating the conformation,thus activating TRPV3 channels.However,the specific effects of protons on TRPV3 channels and through which protonated residues exert their functions remain to be further explored.To find out whether TRPV3 skin lesions involved in metabolic acidosis,besides the role of proton regulation TRPV3 channel,still need in metabolic acidosis skin damage model to detect the expression of TRPV3 changes and the skin lesions may trigger signal pathway,and clarify TRPV3 specific inhibitors for the treatment of metabolic acidosis skin damage effect,which will help reveal a variety of skin disorders,including metabolic acidosis,formation mechanism and therapeutic targets.Methods:Verify the specific effect of protons on the protonated residues of the TRPV3 channel.1.Protonated residues involved in proton regulation of TRPV3 function were verified by site-directed mutation.2.The intracellular Ca²⁺level was determined by fluorescence indicator Fluo-4/AM.3.TRPV3 current was recorded by whole cell patch clamp technique.4.The effects of site-directed mutations of E682,E689 and D727 on the C-terminal structure and free energy distribution of TRPV3 were analyzed by molecular dynamics simulation.Verify the mechanism of acidosis on TRPV3 in rat skin injury models and the therapeutic effect of forsythiaside B on rat skin lesions.1.Rat model of metabolic acidosis was established by feeding drinking water containing NH₄Cl,skin injury model was established by applying lactic acid and imiquimod cream,and forsythoside B was given or not given for treatment.2.Pathological changes of skin tissue were detected by HE staining.3.The mRNA and protein expression of TRPV3 were detected by Real-time PCR and Western blot.4.TGF-?levels in skin tissues were detected by ELISA.5.The expression of EGFR and p-EGFR protein was detected by Western blot.Results:Protonated residues are involved in inhibition and sensitization of TRPV3 channels1.Extracellular acid inhibits agonist-induced TRPV3 activation through aspartic acid residues(D641)in selective filters.2.Intracellular protons sensitize the channels by cytoplasmic C-terminal glutamate and aspartic acid residues(E682,E689 and D727).3.Neutralization of three C-terminal residues makes the channel sensitive to agonist stimulation in advance.4.The charge neutralization of the three C-terminal residues stabilizes the sensitization channel conformation and improves the probability of formation of?-helix in the linker between the S6 transmembrane segment and the Trp domain.The mechanism of TRPV3 in metabolic acidosis rat skin injury model and the therapeutic effect of forsythoside B.1.The results of epidermal observation showed that lactic acid treatment and imiquimod treatment could induce the symptoms of skin injury significantly,and the effects of both treatments were the most serious,and the injury degree of metabolic acidosis rats was higher than that of normal rats.2.HE staining results showed that lactic acid treatment and imiquimod treatment could significantly induce pathological symptoms of skin injury,such as less or thickened keratin and inflammation,and the effects of both treatments were the most serious,and the injury degree of metabolic acidosis rats was higher than that of normal rats.3.Real-time PCR and Western blot results showed that the expression of TRPV3mRNA and protein in the skin of metabolic acidosis rats was significantly higher than that of normal rats;However,in normal rats and in acidosis rats,lactic acid and imiquimod treatments could significantly up-regulate the expression of TRPV3mRNA and protein,and the expression of TRPV3 mRNA and protein in metabolic acidosis+lactic acid+imiquimod group was higher than that in other groups.4.ELISA results showed that the skin TGF-?level of metabolic acidosis rats was significantly higher than that of normal rats;However,in normal rats and acidosis rats,both lactic acid and imiquimod treatments could significantly upregulate the level of TGF-?,and the TGF-?level in metabolic acidosis+lactic acid+imiquimod group was higher than that in other groups.5.Western blot results showed that the p-EGFR protein level of metabolic acidosis rats was significantly higher than that of normal rats,but the change of EGFR protein was not significant;In normal rats,imiquimod treatment could significantly up-regulate the levels of p-EGFR and EGFR protein,and the levels of p-EGFR and EGFR protein in lactic acid and imiquimod combined treatment group were higher than those in other groups.In metabolic acidosis rats,the levels of p-EGFR and EGFR protein were significantly up-regulated by both lactic acid and imiquimod treatment,and the levels of p-EGFR and EGFR protein in metabolic acidosis+lactic acid+imiquimod group were higher than those in other groups.6.The results of epidermal observation showed that forsythoside B could effectively inhibit the symptoms of skin injury in normal rat model and metabolic acidosis model.7.The results of HE staining showed that forsythoside B could effectively alleviate the lesions in the normal rat skin injury model and the metabolic acidosis skin injury model.8.Real-time PCR and Western blot results showed that forsythoside B could significantly effect the expression of TRPV3 mRNA and protein in normal rat skin injury model and metabolic acidosis skin injury rats.9.ELISA results showed that forsythoside B could significantly down-regulate the level of TGF-?in normal rat skin injury model and metabolic acidosis skin injury model.10.Western blot results showed that forsythoside B could significantly down-regulate p-EGFR and EGFR protein levels in normal rat skin injury model and metabolic acidosis skin injury model.Conclusion:The protons inhibited the TRPV3 function by acting on the lateral selective screen of the TRPV3 channel,but promoted the transition of the closed state channel from the natural unsensitized state to the sensitized configuration by acting on the medial cell of the TRPV3 channel.This novel TRPV3 proton sensing mechanism provides new insights into the role of TRPV3 in the regulation of epidermal barrier permeability and in cutaneous lesions in the context of tissue acidosis.In metabolic acidosis skin injury model,TRPV3 expression was significantly up-regulated,and TRPV3 may promote skin injury by activating TGF-?/EGFR signaling.The TRPV3specific inhibitor forsythoside B can down-regulate the expression of TRPV3 mRNA and protein in the damaged skin tissues,and block the TGF-?/EGFR signal,and finally improve the skin damage and pathological changes of metabolic acidosis.