| Itching is an unpleasant scratching reaction.Abnormal itching not only affects the body’s mentality,but also seriously affects normal life.A recent epidemiological study in Europe showed that the prevalence of pruritus in patients with skin diseases was 54.4%,the prevalence of acute pruritus in the general population was 8.4%,and the prevalence of chronic pruritus was 13.5%in the general population.The lifetime prevalence of chronic itching is 22%,which indicates that more than one-fifth of people have experienced chronic itching once in their lifetime.The specific mechanism of chronic pruritus is still unclear.Antihistamines commonly used to relieve pruritus are not effective in some patients with pruritus,indicating that in addition to histamine,the pathogenesis of pruritus involves many mediators and signal pathways.Therefore,it is of great significance to study the pathogenesis of pruritus,especially chronic pruritus,and find effective antipruritic ingredients.As the "first responder" of the immune system,mast cells act as regulators of the immune system and participate in the immune response by releasing chemical mediators of immune defense and absorbing other harmful substances.In recent years,studies have found that mast cells play an important role in a variety of skin diseases.It has been reported that some skin diseases are mast cell-dependent,such as atopic dermatitis(AD)and allergic contact dermatitis(ACD),But its specific mechanism has not yet been clarified.MRGPRB2/X2 is a receptor on mast cells that was reported only in 2015.Some studies have shown that MRGPRB2 deficiency reduces itching in a variety of preclinical models of allergic contact dermatitis,but its specific mechanism needs to be further studied.Curcumin is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae family.It has a variety of biological activities,including antioxidant,anti-inflammatory and anti-cancer properties.In recent years,some studies have reported that curcumin can inhibit the acute itching caused by compound 48/80,which indicates that curcumin may have the potential to relieve itching,but the role of curcumin in chronic itching and its mechanism of action have not yet been elucidated.This subject mainly observes that MRGPRB2 receptors in mast cells are closely related to the production of itching in allergic contact dermatitis,and establish a mouse acute and chronic model to further explore the role of mast cells in itching,and observe curcumin’s effects on it.The antipruritic effect of the mediated pruritus model provides potential antipruritic drugs for the clinic.Objective:(1)Explore the relationship between mast cells and pruritus in the compound 48/80 acute model and ACD chronic model(2)Explore the relationship between curcumin and mast cells(3)Whether curcumin can inhibit the 48/80 acute model and ACD Scratching behavior of mice in chronic models.(4)The molecular and cellular mechanisms of curcumin inhibiting itching.method:(1)Wild type(Wide type)WT mice(20-25 g)and mast cell knockout mice(KIT-HOW)sash mice were injected subcutaneously with compound 48/80(50 ug/mice)to observe whether mast cells participate in compound 48/80 Itching.(2)Use WT mice and sash mice to establish ACD models to evaluate the role of mast cells in ACD models.(3)Using hematoxylin-Eosin(HE)and toluidine blue(Toloniumchloride)staining to evaluate the changes in the thickness of the mouse epidermis,the number of mast cells,and the degree of inflammatory cell infiltration before and after the ACD model.(4)To study whether curcumin can inhibit the activation of mouse peritoneal mast cells caused by compound 48/80 by calcium imaging technology and whole-cell patch clamp technology.(5)Using QPCR technology to analyze the mRNA levels of mouse peritoneal mast cells(in vitro experiments)and ACD model site skin samples,respectively,to observe whether high concentrations of curcumin can affect the expression of related inflammatory factors.(6)Western Blot experiment to detect the phosphorylation level of mouse peritoneal mast cells(in vitro experiment)and the skin nuclear factor-κB(NF-κB)at the ACD model site.(7)The immunofluorescence staining experiment further observes the phosphorylation level of NF-κB in the skin of the ACD model site.(8)Transfect the plasmid of MRGPRX2/B2 in HEK293 cells,and use calcium imaging technology to explore the relationship between curcumin and MRGPRX2/B2.Result:(1)After mast cell knockout,the scratching behavior of compound 48/80 in acute model and ACD chronic model is significantly reduced.Mast cell knockout can also improve the thickening of epidermis and inflammatory cell infiltration in the modeling area of ACD model mice.(2)Curcumin can alleviate the itching behavior in compound 48/80 acute model and ACD chronic model.HE staining found that the curcumin-administered group could reduce the infiltration of inflammatory cells in the ACD model site,and the toluidine blue staining results showed that the model can significantly reduce the increase in mast cells caused by the model after administration.(3)Incubating mouse peritoneal mast cells with curcumin for one hour in advance can inhibit the influx of calcium ions and the production of inward and outward currents caused by compound 48/80.(4)Curcumin can inhibit the phosphorylation of NF-κB-P65 in the skin of ACD model mice.After curcumin was administered,the expression of TNF-α and IL-6 in the skin and cells were down-regulated(mRNA).(5)The establishment of an ACD model in B2mut mice(MRGPRB2 receptor mutant mice on mast cells)found that MRGPRB2 receptor mutant mice can significantly alleviate the scratching behavior in the ACD model.(6)Curcumin can inhibit the influx of calcium ions and the production of inward and outward currents in mouse peritoneal mast cells caused by MRGPRB2 receptor agonist compound 48/80.Curcumin can inhibit the phosphorylation of NF-κB in mouse peritoneal mast cells and the expression(mRNA)of TNF-α and IL-6 downstream of NF-κB caused by the MRGPRB2 receptor agonist compound 48/80.(7)Curcumin can inhibit the influx of calcium ions in mouse peritoneal mast cells caused by MRGPRB2 receptor agonist PAMP9-20.The increase in phosphorylation level of NF-κB and the expression of inflammatory factors in mouse peritoneal mast cells caused by PAMP9-20 can be inhibited by curcumin.(8)Curcumin can inhibit the influx of calcium ions in MRGPRX2/B2-HEK cells caused by MRGPRB2 receptor agonists PAMP9-20 and compond 48/80.The administration of PKC agonists and inhibitors can respectively increase or decrease MRGPRB2 receptors.Calcium influx caused by body agonists.(9)Research conclusion:Curcumin reduces the secretion of inflammatory factors TNF-α and IL-6 by inhibiting the MRGPRX2/B2-PKC-NF-κB signaling pathway,and then inhibits the scratching behavior in allergic inflammation. |
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