Effects And Mechanisms Of Qingshi Antipruritic Ointment On TNF-α/IFN-γ-induced Keratinocyte Inflammation Mode

Atopic dermatitis,which belongs to the category of "ruxuan","taichuang" and"siwanfeng" in traditional Chinese medicine,is a chronic,recurrent,inflammatory skin disease mainly manifested as chronic eczema-like rash accompanied by dry skin and itching.The pathogenesis of this disease is complicated,the prevalence rate continues to rise,and the existing treatment methods have limitations,which brings a huge physical and mental burden to patients.Studies have found that the increase in epidermal nerve fibers is closely related to this disease.The growth of epidermal nerve fibers is bidirectionally regulated by semaphorin 3A and nerve growth factor,and other related molecules are also involved.By regulating the levels of Sema3A/NGF and related molecules,the growth of epidermal nerve fibers can be reduced and pruritus symptoms can be improved.Qingshi Zhiyang ointment is a preparation of my hospital.It is improved from the experienced prescription of a famous old Chinese medicine doctor.Our team’s early clinical observations found that the application of Qingshi Zhiyang ointment in the treatment of eczema,atopic dermatitis,neurodermatitis and other inflammatory skin diseases has a significant effect.Animal experiments have found that Qingshi Zhiyang ointment can improve atopic dermatitis by increasing the levels of microRNA-145 and semaphorin 3 A,and reducing the levels of interleukin-31,nerve growth factor,and neurotrophic factor 4.However,there is still a lack of research on the treatment of skin inflammatory cells with Qingshi Zhiyang ointment.Therefore,in this study,different concentrations of Qingshi Zhiyang ointment were used to intervene the inflammation model of human immortalized keratinocytes and human epidermal keratinocytes induced by tumor necrosis factor-α and interferon-γ,to observe the expression changes of Sema3A/NGF and related molecules;-and to explore the anti-inflammatory and anti-itch mechanism of Qingshi Zhiyang ointment.Specifically,it includes the following parts:Objective:To investigate the effect and mechanism of Qingshi Zhiyang ointment on HaCaT and human epidermal keratinocytes induced by TNF-α/IFN-γ.Methods:HaCaT and human epidermal keratinocytes were divided into blank group,model group and experimental group,respectively.The blank group was not given any intervention measures;the model group was stimulated with TNF-α(1Ong/mL)+IFN-γ(10ng/mL)for 6h to induce inflammation model;the experimental group was cultured in 0.0001,0.001,0.01,0.1g/mL Qingshi Zhiyang ointment containing medium on the basis of modeling,and according to the above concentrations was divided into 4 subgroups.The effect of Qingshi Zhiyang ointment on cell viability was determined by cell counting kit-8,miR-145 was quantified by real-time quantitative polymerase chain reaction,Sema3A protein expression was detected by western blotting,the levels of IL-31,NGF and NT-4 in the cell culture supernatant was determined by enzyme-linked immunosorbent assay.Results:Different concentrations of Qingshi Zhiyang ointment had no significant inhibition on cell proliferation.HaCaT:Compared with the blank group,the expression of miR-145 in the model group was low,the expression of Sema3A protein was high,and the levels of IL-31,NGF and NT-4 in the cell culture supernatant were high(P<0.05).Compared with the model group,the expression of miR-145 in the 0.001g/mL group,0.01g/mL group and 0.1g/mL group were all increased(P<0.05);the expression of Sema3A protein was all decreased(P<0.05);the levels of IL-31,NGF and NT-4 in the cell culture supernatant of the 0.0001g/mL group,0.001g/mL group,0.01g/mL group and 0.1g/mL group gradually decreased,and the differences among the latter three groups were statistically significant(P<0.05).Human epidermal keratinocytes:Compared with the blank group,the expression of miR-145 in the model group was low,the expression of Sema3A protein was high,and the levels of IL-31,NGF and NT-4 in the cell culture supernatant were high(P<0.05).Compared with the model group,the expression of miR-145 in the 0.01g/mL group,0.1g/mL group was high(P<0.05);the expression of Sema3A protein in the 0.001g/mL group,0.01g/mL and 0.1g/mL group was low(P<0.05);the levels of IL-31,NGF and NT-4 in the cell culture supernatant of the 0.01g/mL group and 0.1g/mL group were low(P<0.05),and the levels of IL-31 and NGF in the cell culture supernatant of the 0.0001g/mL group and 0.001g/mL group were low(P<0.05).Conclusion:Qingshi Zhiyang ointment can significantly improve the inflammation of HaCaT and human epidermal keratinocytes,and its mechanism may be through regulating the levels of Sema3A/NGF and related molecules.