The Effect Of Liangxue Jiedu Recipe And Indirubin On The Activation Of KC/??T Involved In The In Vitro And In Vivo Psoriasis Model CCL20

EXPERIMENT I:Expression and Role of Chemokine CCL20 in the Skin Lesions of PsoriasisObjective:To observe the expression and role of chemokine CC chemokine ligand 20(CCL20)in the Skin Lesions of Psoriasis.Methods:The expression of CCL20 in psoriasis patients and imiquimod induced psoriasis like lesions in mice by immunofluorescence;The mouse model of psoriasis was induced by adhesive tape stripping,after injected with recombinant mouse CCL20,The severity of psoriasis was assessed by the dermatologists using psoriasis area and severity index(PASI).The morphological changes of the skin tissues were observed under light microscope.The thickness of epidermis was measured.The mouse model of psoriasis was induced by imiquimod(IMQ),after subcutaneous injection of CCL20 antibody,the severity of psoriasis was assessed by PASI.The morphological changes of the skin tissues were observed under light microscope.Real-time PCR was used to detect the expression of CCL20 in the skin tissue samples.Results:The expression of CCL20 in psoriasis patients and mice were increased.Subcutaneous injection of recombinant mouse CCL20 united tape stripping group(CCL20 group)showed a large number of scales,erythema,infiltration and skin thickening.Compared with IMQ group,CCL20 antibody group had a lesser extent in erythema,scaling,infiltration,thickening of the epidermis and epidermal cell proliferation;the expression of CCL20was significantly lower than that in IMQ group.Conclusion:CCL20 was highly expressed in psoriatic lesions,recombinant mouse CCL20 can exacerbate the adhesive tape stripping induced psoriasis like lesions in mice,subcutaneous injection of CCL20 antibody had a therapeutic effect on IMQ mice.EXPERIMENT ?:Effect of Liangxue-Jiedu(LXJD)prescription on IL-17-Induced CCL20 Expression and secretion in Human KeratinocytesObjective:To observe the effect of Liangxue-Jiedu(LXJD)prescription,a Chinese medicine,on IL-17-Induced CC chemokine ligand 20(CCL20)expression and secretion in Human Keratinocytes(HaCaT cells)and to explore its mechanism.Methods:HaCaT cells were stimulated with IL-17(100 ng·mL-1)and incubated with different concentrations of LXJD prescription(8?g·mL-1,2?g·mL-1,0.5?g·mL-1).The effects of LXJD on viability of HaCaT cells were observed by MTT method.ELISA was used to detect CCL20 protein content in the cell supernatant.Western blot was used to detect the changes of intracellular CCL20 related protein(p-I?B?,p-P65).Real-time PCR was used to detect the expression of intracellular CCL20 mRNA,Piasy mRNA and trim32 mRNA.Results:IL-17 led to the upregulation of CCL20 expression in HaCaT cells.After incubation with LXJD,the expression of CCL20 were down-regulated at secretion(P<0.05),mRNA(P<0.05)and protein(P<0.05)levels significantly.The relative protein expression of NF-?B pathway on the phosphorylation level was significantly decreased(P<0.01).But the expression of related molecules Piasy mRNA and Trim32 mRNA had no significant effect.Conclusion:LXJD prescription can lead to the down-regulation of IL-17-induced CCL20 expression and secretion in HaCaT cells by activating NF-?B pathway,which is one of the mechanisms for the treatment of psoriasis.EXPERIMENT ?:Indirubin ameliorates imiquimod-induced psoriasis-like skin lesions in mice by inhibiting IL-17+ ?? T cell-mediated inflammatory responses via JAK3/STAT3 signaling pathwayObjectives:Indirubin(IR),a kind of bisindole compound,is extracted from the leaves of Indigo naturalis,a traditional Chinese herbal medicine that has been widely used in China to treat inflammatory and autoimmune diseases for years.Psoriasis is a chronic immune-mediated inflammatory skin disease in which ?? T cells play an important role.This study aimed to demonstrate the immunoregulation effect of indirubin in inflammatory process of psoriasis and explore the mechanism further.Methods:A mouse model of psoriasis was induced by administration of imiquimod.Mice were administered with 12.5,25 and 50 mg/kg indirubin,lmg/kg methotrexate(MTX),or normal saline intragastrically.Keratinocyte proliferation,inflammatory cell infiltration,cytokines mRNA levels,and JAK/STAT signaling pathway related proteins were examined.Results:Indirubin ameliorated keratinocyte proliferation,reduced infiltration of CD3+T cells,?? T cells,and CD1 lb in dermis to psoriatic lesions,altered ?? T+ cell and CCR6+?? T amounts in spleen and lymph node specimens,inhibited IL-1mRNA,IL-6mRNA,IL-23 P40 mRNA,IL-17AmRNA and IL-22mRNA levels expression and JAK3/STAT3 signaling in psoriatic lesions.Conclusion:Indirubin alleviates IMQ-induced psoriasis-like inflammation by reducing the infiltration of inflammatory cell and cytokine expression,It may inhibit IL-17+?? T cell mediated inflammatory responses via JAK3/STAT3 mediated signaling pathway.Our results indicate that indirubin is a promising drug in the treatment of psoriasis by intervening y8 T cell function.EXPERIMENT IV:Effect of Indirubin on CCL20 in Human KeratinocytesObjective:To observe the effect of indirubin on the expression and secretion of CCL20 in human epidermal keratinocytes and to explore its mechanism.Methods:HaCaT cells were stimulated with IL-17(100ng·mL-1)or IL-22(100ng·mL-1)and incubated with different concentrations of indirubin(16?M,8?M,2?M).The effects of indirubin on viability of HaCaT cells were observed by CCK-8 method.ELISA was used to detect CCL20 content in the cell supernatant.Real-time PCR was used to detect the expression of intracellular CCL20 mRNA.Results:IL-17A and IL-22 led to the upregulation of CCL20 expression in HaCaT cells.After incubation with indirubin,the expression of CCL20 were down-regulated at secretion(P<0.05),mRNA(P<0.05)levels significantly.Conclusion:Indirubin can lead to the down-regulation of CCL20 expression and secretion in HaCaT cells,which is one of the mechanisms for the treatment of psoriasis.EXPERIMENT V:Indirubin inhibits IL-17 secretion by ?? T cell in vitroObjectives:This study aimed to observe the effects of indirubin on T IL-17+?? T cell-mediated inflammatory responses in vitroMethods:Mouse spleen cells were incubated under ?? T cell conditions with indirubin(2?M)and cell viability was tested by CCK-8,secretion of IL-17A was measured by culture supernatant ELISA,activation of JAK3/STAT3 signaling pathway was detected by Western blotting and IL-17A and IFN-y expression was investigated by RT-PCR.Results:Indirubin inhibited the IL-17A expression and secretion,IFN-y expression of isolated ?? T cells,suppressed the JAK3/STAT3 signaling activation.Conclusion:Indirubin may inhibit IL-17+ ?? T cell mediated inflammatory responses via JAK3/STAT3 mediated signaling pathway.