Effects Of Asiatic Acid On The Proliferation Of HaCaT Cells And Imiquimod-induced Psoriasis In Mice Model

Background:Asiatic acid is a kind of urane-type pentacyclic triterpene acid,which exists in the umbelliferous plant Asiatica.Asiatic acid is rich in resources and mainly distributed in the south of the Yangtze River Basin in China.It has been found that asiatic acid has the pharmacological activities of anti-tumor,anti-inflammatory,anti-oxidation and anti-depression.Psoriasis is a kind of chronic skin disease,its pathological manifestation includes the over-proliferation of keratinocyte cells and infiltration of inflammatory cells.HaCaT cell line is spontaneous immortalized human keratinocyte cells,which has been widely used in many skin diseases research.In this study,the effects and mechanism of asiatic acid on imidacretine-induced psoriasis mouse model and keratinocytes were studied.Methods:In this study,to investigate the effect of asiatic acid on skin lesion in vivo,the establishment of psoriasis mouse model was induced by imiquimod;the levels of IL-17,IL-23 and TNF-? in serum were measured to detect the release of inflammatory factors;the expression levels of PCNA,Bax and Bcl-2 protein in mouse skin lesions were investigated.The molecular mechanism of inhibiting TNF-?-induced HaCaT cell proliferation by asiatic acid in vitro was studied using the TNF-?-induced HaCaT cell proliferation model.TNF-? induced proliferational HaCaT cells were treated by different concentrations of asiatic acid for 24 h and the cell viability was detected by MTT assay;Flow cytometry was used to detect cell cycle;the nuclear translocation of NF-?B was detected by immunofluorescence staining;the expression of NF-?B and p-NF-?B were detected by Western blot.To study the effect of asiatic acid on HaCaT cell: MTT assay was used to detect the inhibittion of the cells;DCFH-DA staining was used to detect intracellular ROS production;JC-1 staining was used to detect mitochondrial membrane potential,and the content of ATP was detected by luciferase to analyze the function of mitochondria.Results: The appearance of the skin observation and tissue H & E staining showed that asiatic acid had the good effect on imidocormol-induced psoriasis-like symptoms in the skin model of psoriasis in mice.The expression of IL-17,IL-23 and TNF-? in the serum of mice were found to be related to the increase of IL-17,IL-23 and TNF-? expression in imidocormol-induced model of psoriasis in mice.After treatment with asiatic acid,it can significantly reduce the increase in TNF-?(P<0.01),while there was no significant effect on IL-17 and IL-23.The expression level of PCNA in the nucleus antigen was decreased,the expression of Bax was increased and the expression of Bcl-2 was decreased after treated with asiatic acid.Cell level studies have shown that asiatic acid could significantly inhibit TNF-?-induced HaCaT cell proliferation and block cell cycle in G2 phase.At the same time,asiatic acid could inhibit TNF-?-induced NF-?B phosphorylation and prevent its translocation into the nucleus.In addition,the treatment of asiatic acid could induced the increase of ROS production in HaCaT cells,promoted the collapse of mitochondrial membrane potential,inhibited the formation of intracellular ATP and finally induced the death of keratinocytes.Conclusions:Asiatic acid had a better effect on inhibiting the development of psoriasis in mice,manifested as inhibition of keratinocyte proliferation.At the cellular level,asiatic acid could inhibit the proliferation of HaCaT cells induced by TNF-? and could significantly damage the mitochondria of HaCaT cells and induce cell apoptosis.The mechanism may be related to the inhibition of TNF-?-induced NF-?B pathway activation by asiatic acid.