Study On The Effect Of Shikonin On NF-κB Signaling Pathway And Related Inflammatory Factors In Imiquimod-Induced Inflammation Model Of Psoriasis In Mice

Introduction: Psoriasis is a chronic inflammatory skin disease,which mainly manifests as hyperkeratosis and hypokeratosis,massive infiltration of inflammatory cells,and dermal angiogenesis.The exact pathogenesis is not yet clear,but it is generally believed to be related to the excessive proliferation of keratinocytes and the massive release of inflammatory factors after the immune response,among which NF-κB plays an important regulatory role.NF-κB is one of psoriasis susceptible gene loci,which is a transcription factor composed of five members: p50,p52,Rel A(p65),Rel B and c-Rel.p65 is currently the main research subunit.NF-κB can regulate cell proliferation and differentiation,and is a key way to regulate various cellular processes including proliferation and inflammation.It has been found to be activated in psoriasis skin lesions and participate in the pathogenesis of psoriasis.Shikonin,a natural naphthoquinone derivative isolated from the traditional herb comfrey,has anti-inflammatory,anti-tumor and wound healing effects.It has long been used to treat burns,infections,ulcers,tumors and psoriasis.Experiments have shown that shikonin can effectively inhibit the activation of NF-κB in colitis.However,whether shikonin can improve the inflammatory changes in psoriasis by inhibiting the NF-κB pathway is still unclear.Here,we observed the effect of shikonin on the expression of inflammatory factors such as IL-6,IL-1βand TNF-αin the inflammation model of psoriasis through in vivo experiments,and explored its possible mechanism of action through the NF-κB pathway.Methods:BALB/c mice were randomly divided into blank control group,imiquimod(IMQ)model group,shikonin low-dose [5mg/kg/d],middle-dose [7.5mg/kg/d] and high-dose [10mg/kg/d] group.Continuously shave the hair on the 3.0cm X2.0cm area on the back of the mouse and apply 62.5mg of 5% IMQ cream for 7 days,and apply the same dose of petrolatum to the blank control group.The shikonin group was given daily by gavage Drugs(the drugs of each group are dissolved in edible oil),and the blank control group and the IMQ model group were given edible oil containing 5% DMSO [1mg/kg/d] by gavage.Take pictures of the skin lesions daily for PASI score.On the 8th day,the mice were sacrificed and the skin tissues were collected.The histological changes of the skin lesions were observed by hematoxylin and eosin(H&E)staining.Immunohistochemistry(IHC)to detect the expression of NF-κB(p65)protein in skin lesions.Western blotting detects the level of NF-κB(p65)protein,and real-time q PCR(RT-q PCR)was used to detect the expression of inflammatory factors IL-6,IL-1βand TNF-αin the skin lesions.Results:1.Pathological changes in the skin lesions of mice in each groupCompared with the IMQ model group,the shikonin drug group can reduce the inflammatory reaction on the back of mice to varying degrees,improve skin erythema,scale and thickening,and the shikonin high-dose group has more obvious therapeutic effects.The PASI score results showed that the shikonin group was significantly lower than the IMQ model group,and the difference was statistically significant(P<0.05).HE staining200 times microscope showed that the IMQ model group was significantly thicker than the blank control group,with increased inflammatory cell infiltration,and local Munro microabscesses.Compared with the IMQ model group,the shikonin drug group became thinner and less inflammatory cell infiltration.The above results suggest that shikonin can relieve imiquimod-induced skin inflammation in mice with psoriasis.2.The expression level of NF-κB(p65)protein in the skin lesions of mice in each group2.1 After detection by IHC and observation under a 200 x optical microscope,compared with the control group,the expression level of NF-κB(p65)protein in the skin tissue of the IMQ model group was significantly increased(P < 0.05);Compared with the IMQ model group,The expression level of NF-κB(p65)protein in the skin tissue of mice in the shikonin drug group decreased in a dose-dependent manner(P<0.05).The above results suggest that shikonin has an inhibitory effect on the expression of NF-κB(p65)protein in the skin lesions of imiquimod-induced psoriasis mice,and the inhibitory effect of shikonin high-dose group was more obvious.2.2 After the WB test,compared with the blank control group,the level of NF-κB(p65)in the skin of the IMQ model group increased.After the addition of shikonin,the middle-dose and high-dose shikonin can significantly inhibit NF-κB(p65)expression,the difference was statistically significant(P<0.05).The above results suggest that shikonin has an inhibitory effect on the expression of NF-κB(p65)protein in the skin lesions of mice with psoriasis induced by imiquimod.3.The m RNA expression of IL-6,IL-1β and TNF-α in the skin tissue of each group of miceCompared with the blank control group,the expression of IL-6,IL-1βand TNF-αm RNA in the skin tissues of the IMQ model group increased significantly(P<0.05).Compared with the IMQ model group,the expression of IL-6,IL-1βand TNF-αm RNA in the skin tissues of the mice in the shikonin drug group showed a decreasing trend with the increase of the drug dose,and the differences were statistically significant(P< 0.05).Conclusion: Shikonin can down-regulate the expression of NF-κB(p65)protein and IL-6,IL-1β,and TNF-α m RNA,it can reduce inflammation and improve the clinical symptoms in mice with psoriasis.This provides new ideas for the study of the mechanism of shikonin in the treatment of psoriasis.