The Effect And Mechanism Of Shikonin On Inflammatory Cytokine,Apoptosis And Cell Cycle In TNF-α Treated HaCaT Cells

IntroductionPsoriasis is a chronic,inflammatory disease,and breaks out repeatedly.It can cause the serious impact on the lives of patients,even cause anxiety,depression and other mental symptoms,but the pathogenesis is still unclear.Therefore,the study of the pathogenesis of psoriasis is of great significance for the treatment and improvement of patients’quality of life.The characteristic pathologic features of psoriasis include the interaction of various immune cells and chemokines,the proliferation of keratinocytes,abnormal differentiation and the increase of epidermal angiogenesis.In the network of cytokines with complex pathogenesis of psoriasis,pro-inflammatory cytokine TNF-αplays an important role in the formation and development of psoriasis lesions.TNF-αis closely related to the inflammatory infiltration of psoriasis lesions and excessive proliferation of keratinocytes.The concentration of psoriatic lesions in psoriatic lesions was significantly higher than normal skin or normal skin.After treatment or skin lesions disappeared,the concentration of TNF-αalso decreased.TNF-αis mainly secreted by activated T cells,keratinocytes,macrophages,and Langhans cells,and can activate activated T cells,macrophages,and keratinocytes to secrete more IL-6,IL-1,IL-8 and NF-κB and cell adhesion molecules,thereby expanding the spread of immune signals,promote excessive proliferation of keratinocytes,affecting T cell activation,chemotaxis,and promote the inflammatory response of psoriasis.Clinically,TNF-αreceptor antagonists can effectively treat psoriasis,and they also show that TNF-αplays an important role in the pathogenesis of psoriasis.At present,the commonly used treatment methods mainly include local application of hormones,the application of immunosuppressive agents and even biological agents.But these immune inhibitors or biological agents have more contraindications,application scope is narrow,so make scenario,whether the Shikonin has caused the inhibition of TNF-αpathological role in physiological processes,so as to find an efficient psoriasis treatment has a wide application range.For this,we observed the Shikonin of TNF-αinduced inflammation and apoptosis of HaCaT cells cycle process,the influence of at the same time observe the alkannin influence on the NF-κB pathway,explore the mechanism of action of shikonin treatment of psoriasis.Shikonin,originated from Chinese herb Lithospermum,was first recorded in the"Shen Nong’s Herbal Classics".It is a perennial herb,a traditional Chinese medicinal material in China,and its roots are used as medicine.Its efficacy is numerous:cooling blood,blood circulation,detoxification,rash,etc.Clinically it is used for the treatment of measles,eczema,erysipelas,burns and other diseases.The active ingredients of Lithospermum are mainly derived from the roots and exhibit various biological activities such as anti-inflammatory,anti-tumor and immune regulation.The mechanism of action of Lithospermum on psoriasis is not clear.This study investigated the mechanism of Lithospermum treatment of psoriasis.Using TNF-αto simulate the inflammatory environment of a psoriasis,the effects of shikonin on TNF-α-induced HaCaT cell-associated inflammatory factors and apoptotic cycle were observed.Simultaneously,we observed the effects of shikonin on NF-κB pathway to investigate the mechanism of shikonin in the treatment of psoriasis.Methods1.Cell cultureThe study used HaCaT cells（cells from Department of Dermatology Laboratory of China Medical University）as research objects,and cultured RPMI 1640 medium containing 10%fetal bovine serum,100U/ml penicillin and streptomycin 100U/ml,and cultured in a incubator containing 5%CO2 and 37℃.2.Determination of cell proliferation activity by MTSHaCaT cells were inoculated to 96 orifice plates with 4×10⁴/ml,after 24 hours of culture,adding different concentrations（0-8μM）of Shikonin.They were cultured for 24h,48h and 72h respectively,to observe the effect of Shikonin on the proliferation activity of HaCaT cells.3.The expression of IL-1 beta,IL-6 and IL-8 mRNA.RT-PCR was used to determine the expression of Shikonin on the expression of IL-1beta,IL-6 and IL-8 mRNA in HaCaT cells induced by TNF-α.Total RNA was extracted by using mi RNeasy mini kit and reverse-transcribed with Prime Script RT reagent kit.The c DNA obtained was used as a template for subsequent PCR amplification and relatice quantitatine result was analyzed with 2^-ΔΔCT.4.Detection of expression of IL-6 and IL-8 by ELISAELISA was used to determine the expression of Shikonin on the secretion of IL-6and IL-8 in TNF-α-induced HaCaT cells.The cells were seeded in 6-well plates for24h,and added drug.At 2h,6h,the secretion of IL-6 and IL-8 was analyzed by measureing the absorbance at 450nm/540nm using a spectrophotometer.5.Detection of Apoptosis and cycle by Flow CytometryFlow cytometry was used to detect the effect of Shikonin on the apoptosis and cycle of TNF-αinduced HaCaT.The cells were seeded in 6-well plates and FITC Annexin V kit and PI were used to detect cell cycle and apoptosis,respectively.6.Detection of related protein expression of Apoptosis and NF-κB pathwayThe Western blot was used to detect the effect of Shikonin on the influence of peroxin on the NF-κBp-p65,NF-κBp65,p IκB,IκB,bcl-2,caspase3 in TNF-αinduced HaCaT.Total protein wsa extracted ad the conentration was measured with BCA method.The bands were visualized with ECL Western Blotting Sbstrate.7.Lentivirus transfected HaCaT cells,and inhibited HMGB1 expression.Use lentivirus transfection method to establish HMGB1 silent stable transfection cell line.Slow virus transfection experiments,in strict accordance with the instructions to find transfection best conditions,and then a formal experiment,infected HaCaT cells,and then use puro screening,to establish stable transfection cell line.8.Imiquimod was used to induce psoriasis like mice model,and HE staining was used to observe the effect of shikonin on the thickness of the epidermis.Imiquimod was used on the mouse back skin for once a day.On the third day,The mouse received medium and different concentrations of shikonin by intraperitoneal injection.And on the 6^thday and the 9^thday,took the skin respectively.After HE staining,we observed the changes of mice skin thickness.9.statistical analysisStatistical analysis was performed using Graph Pad Prism and SPSS 16.0.Quantitative results were expressed as mean±SD,and t-test.A p value of less than 0.05 was considered statistically significant.Results1.MTS AssayWith different concentrations（0,0.25,0.5,1,2,4,8μM）of shikonin in cultured HaCaT cells 24h,48h,72h,shikonin concentration less than or equal to 1μM,the proliferative activity of HaCaT cells had no obvious change.The proliferation activity of HaCaT cells was significantly decreased when the concentration of shikonin was greater than 2μM.2.Detection of inflammatory cytokine expression levels.The effect of shikonin on the expression of cytokines in HaCaT cells induced by TNF-αwas demonstrated by q RT-PCR and ELISA.Experiment is divided into seven groups:control,TNF-α,TNF-α+DMX（2μM）,TNF-α+MTX（0.2μM）,TNF-α+SHI（1μM）,TNF-α+SHI（2μM）,TNF-α+SHI（4μM）.（1）The effect of shikonin on IL-8 in TNF-α-induced HaCaT cellsa.The TNF-αgroup compared with control group,IL-8 mRNA level increased obviously（P<0.05）.Adding different concentrations of shikonin,IL-8 mRNA level significantly decreased（P<0.05）.Positive control group:dexamethasone group and methotrexate group also can decrease the IL-8 mRNA level（P<0.05）.b.The TNF-αgroup compared with control group,IL-8 level increased obviously（P<0.05）.Adding different concentrations of shikonin,IL-8 levels significantly decreased.Positive control group:dexamethasone group and methotrexate group also can decrease the level of IL-8（P<0.05）.（2）The effect of shikonin on IL-6 in TNF-α-induced HaCaT cellsa.TNF-αgroup compared with control group,IL-6 mRNA level significantly increased（P<0.05）.Adding different concentrations of shikonin,IL-6 mRNA level significantly decreased（P<0.05）.Positive control group,dexamethasone group and methotrexate group also can decrease the IL-6 mRNA level（P<0.05）.b.TNF-αgroup compared with control group,IL-6 levels significantly higher（P<0.05）.Adding different concentration of shikonin,IL-6 levels significantly decreased（P<0.05）.Positive control group:dexamethasone group and methotrexate group also can decrease the level of IL-6（P<0.05）.（3）The effect of shikonin on IL-1βin TNF-α-induced HaCaT cellsa.TNF-αgroup compared with control group,IL-1βmRNA level significantly increased（P<0.05）.Adding different concentrations of shikonin,IL-1βmRNA level significantly decreased（P<0.05）.Positive control group:dexamethasone group（2μm）2 h,6 h,IL-1βmRNA level significantly decreased（P<0.05）.Methotrexate group（0.2μM）had no obvious inhibitory effect on 2 h（P>0.05）,IL-1βmRNA level significantly decreased on 6h（P<0.05）.3.Apoptosis cycle detection and related gene protein detection.（1）The effects of shikonin on the apoptosis of TNF-αinduced HaCaT cells were verified by flow cytometry and Western blotting.Compared with TNF-α-induced HaCaT cells,the apoptosis rate of HaCaT cells induced by methotrexate and shikonin was increased（P<0.05）,while the apoptosis rate of HaCaT cells induced by dexamethasone did not increase significantly（P>0.05）.Compared with TNF-α-induced HaCaT cells,pro-caspase-3 and Bcl-2 decreased in shikonin group,but there was no significant change in pro-caspase-3 and Bcl-2 in the dexamethasone group and methotrexate group.（2）The effect of shikonin on TNF-α-induced HaCaT cells cycle was verified by flow cytometry and Western blotting.Compared with TNF-α-induced HaCaT cell cycle,there was no significant change in cell cycle of shikonin group（P>0.05）,but the proportion of GO/G1 phase cells in methotrexate group was significantly higher than TNF-α-induced HaCaT cells（P<0.05）.There was no significant change in the dexamethasone group.4.Detection of related protein expression in NF-κB pathway.The effect of shikonin on NF-κB pathway in TNF-α-induced HaCaT cell was verified by western blot.In TNF-α-induced HaCaT cell,NF-κBp-p65,p-IκB increased,IκB decreased（P<0.05）.After the addition of shikonin,compared with TNF-α-induced HaCaT cells,NF-κBp-p65,p-IκB decreased,IκB increased（P<0.05）.5.The effect of shikonin on the expression of HMGB1 in TNF-α-induced HaCaT cells and the function of HMGB1.The effect of shikonin on the expression of HMGB1 in TNF-α-induced HaCaT cells was verified by Western blot.It can reduce the expression of HMGB1.After the silencing of the HMGB1 gene of HaCaT cells,the expression of HMGB1 protein decreased obviously,and the level of IL-6 and IL-8mRNA decreased significantly（P<0.05）.6.Effect of shikonin on the skin lesions of psoriasis like mice induced by imiquimod.Imiquimod was used on the mouse back skin for once a day.On the third day,The mouse received medium and different concentrations of shikonin by intraperitoneal injection.And on the 3^thday and the 6^thday treated with shikonin,took the skin respectively.After HE staining,we observed the changes of mice skin thickness.The results showed that shikonin alleviated the red spot scale on the back of the mice,and the thickness of the back skin became thinner,P<0.05.Conclusions1.Shikonin can inhibit the proliferation of HaCaT cells.2.Shikonin inhibits the expression of cytokines IL-1β,IL-6,IL-8 in TNF-α-induced HaCaT cells.3.Shikonin can promote the apoptosis of TNF-α-induced HaCaT cells and has no obvious effect on its cycle.4.Shikonin can inhibit the NF-κB pathway in TNF-α-induced HaCaT cells through inhibiting the expression of HMGB1,then inhibit the expression of inflammatory cytokine,and induce the apoptosis.5.Shikonin improved the skin lesions in psoriasis like mice induced by imiquimod.