| Atopic dermatitis(AD)is the most common type of inflammatory skin disease,with severe chronic itching and significantly reducing the quality of life of patients.A large number of studies have shown that the destruction of the skin barrier is an important pathogenesis of atopic dermatitis,but the mechanism of barrier destruction is not fully understood at present.Most patients are not satisfied with the effect of existing treatment drugs and most patients have a poor prognosis.The effect of traditional Chinese medicine treatment on skin pruritus is significant effect,and there are a variety of effective antipruritic Chinese medicines,among which,Fructus Cnidii has an ideal anti-inflammatory and antipruritic effect,and osthole is the main active ingredient of Fructus Cnidii.This topic aims to reveal the mechanism of skin barrier destruction in patients with atopic dermatitis,and to find safe and effective therapeutic drugs among the active ingredients of antipruritic Chinese medicine.Objective:To investigate the damage mechanism of skin barrier in atopic dermatitis,evaluate the effect of the osthole in the treatment of atopic dermatitis and explore its treatment mechanism.Methods:1.Establish oxazolone-induced atopic dermatitis mouse chronic model.Abdominal sensitization of mice 5 days before stimulation and oxazolone stimulation was given to the neck of mice every 48 hours.The stimulus lasted four times.The mice were divided into a control group and a model group.The number of scratches of the mice was recorded on days 6,8,10,and 12(24 hours after stimulation),the mice were sacrificed on day 12,and the skins of the mice were collected.Mouse skin RNA was extracted and subjected to qRT-PCR to detect mRNA expression changes of tight junction related genes.2.Design the SiRNA to interfere with the expression of ZO-3 in HaCaT cells.The negative control cells were transfected with a non-functional SiRNA.Western blot and qRT-PCR were used to detect the expression of ZO-3 gene and protein in SiRNA transfected cells.The transmembrane resistance of human keratinocytes was measured after transfection.SiRNA transfection interfered with the expression of ZO-3 in human keratinocytes.Control group cells were transfected with a non-functional SiRNA,and the number of cells was detected by CCK8 kit.3.Establish a mouse model of chronic atopic dermatitis(method as above).The mice were divided into control group and model group(n=6).4.p-Akt agonist SC79 induced up-regulation of p-Akt in HaCaT cells,and the expression level of ZO-3 was detected by Western blot.5.Establishment of a mouse model of chronic atopic dermatitis induced by oxazolone(method as above),mice were divided into model group,treatment group p-Akt inhibitors(intraperitoneal injection,72 hours at a time),in the 12th day with a high-definition camera records will be executed in mice scratches,after the mice were killed,extracted skin total protein from modeling area,western blot detection ZO-3 and p-Akt expression changes,mice skin samples after fixed on frozen section and immunofluorescence staining.6.The ear swelling model of atopic dermatitis mice induced by oxazolidone was established.The right ear of the model mice was treated with high dose(0.9mg each mouse)and low dose(0.3mg each mouse)of osthole(for external use),and the left ear was treated with vehicle.The thickness of bilateral ears was measured every hour for 6 hours after treatment.7.Establish a mice model of atopic dermatitis(methods as above).The animals were divided into the model group,the high-dose group and the low-dose group.From day 5,osthole was treated twice daily(for external use,vehicle:80%acetone+20%olive oil,50 ?L),with a high dose of 0.9mg each mouse of osthole and a low dose of 0.3mg each mouse of osthole.The model group was treated with vehicle twice daily from day 5(for external use,vehicle:80%acetone+20%olive oil 50?L).On the 12th day,a high-definition camera was used to record the scratching behavior of the mice,and then the mice were sampled,qRT-PCRwas used to detect the changes of mRNA expression in the mouse model area.8.Western blot and immunofluorescence staining were used to detect skin protein expression in mouse modeling area.9.?Cells were divided into control group,low,medium and high dose osthole treatment group.Cells were seeded in 96-well plates.After cells were adhered,osthole was added(final concentration 10,20,40 ?M,0.1%DMSO in DMEM).Control The same concentration of solvent(0.1%DMSO in DMEM)was added to the group,and the cell number was detected by the CCK8 kit after 48 hours.?HaCaT cells were divided into control group,SC79 group,low,medium and high dose osthole treatment group.Cells were seeded in 12-well plates.After cells adhered to the wall,add osthole(final concentration 10,20,40 ?M,0.1%DMSO in DMEM),the control group and the SC79 group were added with the same concentration of vehicle(0.1%DMSO in DMEM),and 12 hours later,the agonist group and the low,medium,and high-dose osthole treatment group were added with SC79(final concentration 20 ?M,0.1%DMSO in DMEM),the control group was added with the same concentration of vehicle(0.1%DMSO in DMEM).Total protein was extracted after 48h.Results:1.Skin lesions were scored on the 12th day.The degree of skin damage in the model group was significantly higher than that in the control group.Scratching behavior of mice recorded on days 6-12 showed that oxazolone-induced atopic dermatitis model mice were accompanied by severe itching.Results showed that compared with the control group,the expression levels of tight-chain protein mRNA in the skin of model mice Cldn-1,Cldn-6,Cldn-8,Cldn-9,Cldn-23,ZO-3 and Ocln were significantly down-regulated,and Cldn-3,5,7,15,and JAM-2 were significantly up-regulated.Immunofluorescence results showed that:ZO-3 protein is mainly located on the epidermal cell membrane.Compared with the control group,the expression level of ZO-3 protein and mRNA in the skin of model mice was obviously down-regulated.2.ZO-3 plays an important role in the formation of tight junctions of human keratinocytes.ZO-3 SiRNA can effectively reduce ZO-3 mRNA and protein expression levels.Decreased ZO-3 expression levels can significantly reduce the transmembrane resistance of human keratinocytes.The number of cells in the ZO-3 SiRNA transfected group was significantly more than that in the negative control group.3.The expression of p-Akt in the skin of mice with atopic dermatitis was up-regulated.Compared with the control group,there was no change in the expression of p-Akt in the skin of mice in the model group.4.The expression of ZO-3 in HaCaT cells is regulated by the PI3K-Akt signaling pathway.SC79 can effectively induce up-regulation of p-Akt expression in HaCaT cells,and down-regulation of ZO-3 expression.5.The PI3K-Akt signaling pathway is involved in the regulation of ZO-3 expression and skin itching in atopic dermatitis model mice.LY294002 effectively inhibited the up-regulation of p-Akt expression in the skin of mice with atopic dermatitis model,and the inhibitor treatment significantly reduced the scratching behavior of mice.The ZO-3 fluorescence signal of the mouse skin in the inhibitor treatment group was significantly higher than that in the model group.6.Osthole can effectively relieve oxazolone-induced ear swelling in mice.Osthole can effectively alleviate oxazolone-induced ear swelling in mice.At the 2,3,4,5,and 6 hours after treatment,the thickness of the right ear of the mice treated with high dose was significantly lower than that of the left Ear thickness,at 3 h after treatment,the thickness of the right ear was significantly lower in mice given low-dose treatment than in the untreated left ear.7.Osthole can protect the skin barrier and effectively alleviate the itching symptoms of atopic dermatitis.Osthole treatment can significantly decreased skin lesions in mice in dose-dependent.On day 10,12,the number of scratching osthole high dose treated mice were significantly lower than the model group,on day 10 of scratching osthole low dose treated mice.The frequency was significantly lower than the model group.Compared with the model group,the mRNA expression of Cldn6,Cldn8,ZO-1,ZO-2,ZO-3,JAM-1,JAM-3,MUPP-1 in the skin of mice treated with high-dose osthole was significantly up-regulated.The mRNA expression of Cldn8,ZO-1,and JAM-3 was significantly up-regulated in the skin of mice treated with low-dose osthole.8.Osthole can regulate the Akt/ZO-3 pathway.The treatment of osthole could significantly decrease the expression level of p-Akt protein in the skin of model mice and it was concentration-dependent.The results of immunofluorescence showed that the fluorescence signal of ZO-3 protein in the skin of mice treated with high-dose osthole was significantly stronger than that in the model group.9.Osthole regulates the expression of ZO-3 protein on HaCaT cells through the Akt signaling pathway.10?M osthole acted on HaCaT cells for 48h without affecting cell proliferation,while 20?M and 40?M osthole could inhibit cell proliferation.The expression level of p-Akt in the agonist group was significantly higher than that in the control group,10?M,20?M,and 40?M of osthole significantly inhibited the up-regulation of p-Akt expression induced by SC79,and 20?M,40?M of osthole significantly inhibited the proliferation of HaCaT cells for 48 hours.The expression level of ZO-3 in the agonist group was significantly lower than that in the control group,and 10?M of osthole could inhibit the down-regulation of ZO-3 expression in HaCaT cells.Conclusions:?Down-regulation of ZO-3 expression is an important mechanism for the pathogenesis of atopic dermatitis.?The expression of ZO-3 is regulated by Akt signaling pathway.?Osthole can regulate the expression of ZO-3 through the Akt signaling pathway and inhibit the skin barrier destruction and epidermal hyperplasia in atopic dermatitis. |
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