Shikonin Ameliorates Inflammatory Response In Imiquimod-induced Psoriasis-like Mouse Model Via P38 MAPK Signaling Pathway

Objective: Psoriasis is a chronic recurrent autoimmune skin disease with squamous erythema or plaque as the main clinical manifestations.The prominent pathological features are inflammatory cell infiltration and keratinocyte hyperproliferation.The prevalence of psoriasis is estimated to be about 2-5% of the global population,severely affects the patients’ quality of life and psychological conditions.At present,the etiology and pathogenesis of psoriasis have not been fully elucidated,an increasing evidence indicates that MAPK signaling pathway plays a critical role in keratinocyte proliferation and differentiation and cellular regulation.MAPKs consist of three major members: Extracellular-signal-regulated kinase(ERKs),c-Jun N-terminal kinase(JNK),and p38 kinase(p38).p38 MAPK signaling pathway is most closely related to the inflammatory response.It is reported that the activation of MAPK signaling pathway promotes the release of proinflammation factors,such as interleukin-6(IL-6),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α),thereby increasing the severity of epidermis of lesioned skin from psoriasis patients.Shikonin is an active compound in Lithospermum,a traditional Chinese medicine,has anti-inflammatory,antioxidant and anti-tumor properties.The study demonstrates that shikonin may ameliorates D-galactose-induced neuroinflammation by down-regulating the expression of inflammatory factors through MAPK signaling pathway.In the preliminary study,we found that shikonin alleviates psoriasis inflammation by inhibiting keratinocyte proliferation and inducing apoptosis via suppressing JAK/STAT3 pathway.But there is no experimental evidence to prove whether shikonin is possible to treat psoriasis through MAPK signal pathway.Therefore,we established an imiquimod-induced psoriasis mouse model with the aim of exploring the functional role and regulatory mechanism of shikonin on psoriasis from the perspective of p38 MAPK pathway.Methods:BALB/C male mice aged 6-8 weeks were randomly divided into five groups: control group,imiquimod model group and low,medium and high dose shikonin groups,shaved back hair with an area of about 2×3cm,and then treatment group received a daily imiquimod cream on the shaved back while control animals were treated with Vaseline.The drugs were dispensed with edible oil,and the control group and model group were given matrix oil(1mg/kg/d containing 5% DMSO)by intragastric administration.The daily body weight of mice was recorded for 7 consecutive days,and a scoring system based on the clinical Psoriasis Area and Severity Index(PASI)was used to evaluated the skin inflammation on the backs of mice.The samples were collected 8 days after treatment,the Hematoxylin and eosin(HE)staining was used to observe the morphological changes in the tissues and the degree of inflammatory cell infiltration were observed under light microscope.Western Blot and Immunostaining was carried out to analyze the protein expressions of p38 and phospho-p38.Quantitative Realtime polymerase chain reaction(qRT-PCR)was performed to determine mRNA expression levels of IL-6,IL-1β and TNF-α.Results:1、The weight of the mice in the control group did not change significantly,but significantly decreased in the model group and shikonin treatment group,and the weight loss in the shikonin treatment group was lower than that in the model group(P<0.05 or 0.01).2、Shikonin significantly improved the appearance of psoriasis-like skin lesions in mice,and showed a dose-dependent relationship;the epidermis thickness and the inflammatory cell infiltration in the shikonin treatment group were larger than those in the control group and smaller than the model group(p<0.01).However,there was no significant difference in the thickness of the epidermis and the degree of inflammatory cell infiltration in the middle and high dose groups of shikonin(P>0.05).3、Western Blot and Immunostaining analysis demonstrated that the expression of phosphor-p38 in the shikonin treated was lower than that of model group(P<0.01 or0.001).4、The qRT-PCR results showed that the IL-6,IL-1β,and TNF-α mRNA expression in the model group were markedly elevated,and the shikonin treatment group was significantly decreased.With the treatment of increasing doses of shikonin,the mRNA expression level gradually decreased(P<0.01 or 0.001).Conclusion:Shikonin is capable of exhibiting anti-psoriasis activities in imiquimod-induced psoriasis-like mouse model.The mechanism may be related to the inhibition of the p38 MAPK signaling pathway,decreasing the release of the downstream inflammatory factors IL-6,IL-1β and TNF-α.