The Study Of The Mechanism Of PSORI-CM02 And Paeoniflorin In The Treatment Of Psoriasis Based On Dendritic Cells

ObjectivePSORI-CM02 comes from master of national physician professor Guowei Xuan's prescription for the treatment of psoriasis,and professor Chuanjian Lu's team constantly optimized the prescription.Previous studies have shown that PSORI-CM02 has significant clinical efficacy,and anti-inflammatory,anti-proliferation,immune regulation in animal and cell experiment.However,the effect of PSORI-CM02 on dendritic cells(DCs),the initiation of immunopathology in psoriasis,has not been studied.Paeoniflorin is one of the main active components of PSORI-CM02.In this study,the effects of PSORI-CM02 and paeoniflorin on the phenotype and function of DCs in vivo and in vitro were observed in the animal model of psoriasis induced by imiquimod(IMQ)and bone marrow derived dendritic cells(BMDCs)cultured in vitro.To further clarify the mechanism of PSORI-CM02 and its main active ingredient paeoniflorin in the treatment of psoriasis.Methods1.Establish the mouse model of psoriasis and observe the therapeutic effect of PSORI-CM02 on psoriatic mice:Balb/c mice were randomly divided into 5 groups-normal control group,IMQ group,methotrexate(MIX)group,PSORI-CM02 low dose group,and PSORI-CM02 high dose group(Or 0.5%and 2%paeoniflorin group).IMQ ointment was applied to the back skin for 7 days for psoriasis modeling.During the modeling process,the body weight,erythema,scales,infiltration and PASI score of mice were measured and observed every other day.IMQ ointment was applied on the back skin for 7 days for psoriasis modeling,and the body weight,erythema,scales,infiltration and psoriasis areas and severity index(PASI))of mice were measured and evaluated every other day during the modeling process.On the 7th day,samples were taken and spleen index and HE staining were detected.2.Expression of DCs in skin and lymph nodes and T cells in spleen of mice with psoriasis:Siglech protein immunohistochemical staining was performed on the skin.Flow cytometry was used to detect the expression of CD83,CD86 and MHCII on the surface of CDllc+and CD207+DCs,as well as the proportions of CD207+CD103+,CD207+CCR7+DCs in lymph nodes,and the expression of CD4+IFN-?+,CD4+T-bet+,CD4+IL-17a+and CD4+ROR ? T+cells in spleen.3.Culture and identification of mouse BMDCs:GM-CSF and IL-4 induced mouse bone marrow mononuclear cells were cultured for 6 to 7 days,and the proportion of CDllc+cells was identified by flow detection.4.Proliferation and morphology of BMDCs:CCK8 was used to detect the effects of PSORI-CM02,LiPoPolysaccharide(LPS)and IMQ on BMDCs proliferation.BMDCs with PSORI-CM02,LPS and IMQ intervention were observed and photographed under inverted phase contrast microscope.5.Antigen uptake in BMDCs:PSORI-CM02 and OVA-FITC intervene BMDCs,and the proportion of FITC positive cells in BMDCs was detected by flow analysis.6.BMDCs maturation:Flow cytometry was used to detect the expression of CD83,CD86 and MHCII on the surface of BMDCs with PSORI-CM02 or paeoniflorin pre-treatment stimulated by LPS or IMQ.7.Inflammatory and chemokine expression in BMDCs:RT-PCR was performed to detect the expressions of IL-12A,IFN-?,CCL20,TNF,IL-17F and IL-23 in BMDCs stimulated by LPS treated with psori-com02,and the expressions of IL-12A,IFN-? and IL-23 in IMQ stimulated BMDCs treated with PSORI-CM02,and the expressions of IL-1?,IL-6,IL-12A,IL-23,IFN-? and TNF in IMQ stimulated BMDCs treated with paeoniflorin.Flow cytometry was performed to detect the expressions of IL-12p35 in BMDCs stimulated by LPS after the advance intervention of PSORI-CM02.8.BMDCs regulates the differentiation of HelPer T cells(Th):Naive CD4+T cells in spleen cells were separated by magnetic beads.Drugs and modeling agents were used to interfere with the co-culture system(BMDCs and Naive CD4+T cells were co-cultured in a ratio of 1:9).Flow cytometry was used to detect the proportions of Thl cells(CD4+IFN-y+,CD4+T-bet+cells)and Treg cells(CD4+CD25+,CD4+CD152+cells).9.Expression of signaling pathway protein in BMDCs:the expressions of p-Stat3(Ser727),p-Stat3(Tyr705),Stat3,p-P38 MAPK and P38 MAPK in BMDCs pre-intervened by PSORI-CM02 and stimulated by LPS were detected by western-blot.Results1.Effect of PSORI-CM02 on IMQ-induced psoriatic mice and DCsA.The therapeutic effect of PSORI-CM02 on psoriatic miceEffects on the improvement of animal skin lesions:IMQ ointment can increase PASI score and induce mice psoriasis model.Low and high doses of PSORI-CM02 and MTX can improve psoriasis-like lesions and reduce PASI score.Effects on animal weight:the body weight of the normal control group increased gradually during the 7-day experiment.The body weight of IMQ group,MTX group and low and high dose PSORI-CM02 group showed different degrees of decline compared with that of normal control group.Effects on spleen index:the spleen index of IMQ group was significantly higher than that of normal control group.The spleen index of MTX group was significantly lower than that of IMQ group.Compared with IMQ group,low and high dose PSORI-CM02 group showed declining trend,but there was no statistical difference.Effects on skin histopathology:the results of HE staining showed that the back skin of IMQ group mice presented the histological morphology of psoriasis.Histological morphology of psoriatic lesions in MTX group and PSORI-CM02 low and high dose group were improved.The skin thickness of IMQ group was significantly thicker than that of normal control group,and the skin thickness of MTX group and PSORI-CM02 low and high dose group were significantly thinner than that of IMQ group.B.PSORI-CM02 affected the distribution of DCs in IMQ-induced psoriatic miceDistribution of Plasmacytoid dendritic cells(PDCs)in the skin:the results of immunohistochemical staining of skin PDCs(Siglech positive staining area)showed that the expression of Siglech in IMQ group was significantly higher than that in normal control group,while the expression of Siglech in PSORI-CM02 low and high dose group and MTX group was significantly lower than that in IMQ group.Distribution of migratory DCs in draining lymph nodes:flow cytometry results showed that the proportion of CD207+CD103+migrating dermal dendritic cells(mDDCs)in IMQ group was significantly higher than that in the normal control group,and the proportion of CD207+CD103+mDDCs in the PSORI-CM02 low and high dose group and MTX group was significantly lower than that in IMQ group,with statistically significant differences.The expression of chemokine receptor 7(CCR7)on the surface of CD207+langerhans cells(LCs)in IMQ group significantly increased compared with that in normal control group.The expression of CCR7 on the surface of LCs in the PSORI-CM02 low and high dose groups and the MTX group decreased compared with that in IMQ group,and the differences were statistically significant.C.PSORI-CM02 inhibited the maturation of DCs in IMQ-induced psoriatic miceThe results of flow cytometry for LCs maturation:the proportions of CD207+CD83+,CD207+CD86+and CD207+MHCII+cells in the lymph nodes of IMQ group significantly increased compared with that of normal control group.The proportions of three crowds of LCs in low and high dose PSORI-CM02 and MTX group were significantly decreased compared with that in IMQ group.The maturation results of CD11c+DCs detected by flow cytometry showed that:the expression of CD83+,CD86+and MHCII+on the surface of CD11c+DCs in the lymph node cells of IMQ group was significantly increased compared with that of normal control group.The proportions of three crowds of DCs showed a decreasing trend compared with IMQ group after the intervention of low and high dose of PSORI-CM02 and MTX.D.PSORI-CM02 could affect the differentiation of T cells induced by DCs inIMQ-induced psoriatic miceThe differentiation results of Th cells in spleen detected by flow cytometry:the proportions of CD4+IFN-?+,CD4+T-bet+?CD4+IL-17A+,CD4+ROR ? t+cells significantly increased compared with normal control group.Compared with IMQ group,the proportions of four crowds of cells in PSORI-CM02 low dose and high dose group and MTX group decreased significantly.The expression of cytokine IL-12 in DCs of lymph nodes detected by flow cytometry:the proportion of CD11c+IL-12p35+cells significantly increased compared with control group.The proportion of cells in low and high doses of PSORI-CM02 and MTX group showed significant decreasing trends.2.Effect of PSORI-CM02 on BMDCs function and its mechanismA.Effect of PSORI-CM02 on morphology and proliferation of BMDCsObserve cell morphology by microscope and identify cell purity by Flow cytometry:the cells' volumes increase and the surfaces of cells form protuberance.LPS and IMQ stimulation can increase and lengthen the protuberances,and PSORI-CM02 can inhibit the formation of cell protuberances.The ratio of CD11c+cells was 88.4%.Detection of PSORI-CM02 intervention on BMDCs proliferation by CCK8:4 and 8 mg/mL PSORI-CM02 drastically inhibited the proliferation of BMDCs,0.25 to 2 mg/mL PSORI-CM02 slightly inhibited the proliferation of BMDCs,0.0625 and 0.125 mg/mL PSORI-CM02 had no effect on the proliferation of BMDCs,and the difference was not statistically significant.LPS significantly promoted BMDCs proliferation,while 0.5 to 2 mg/mL PSORI-CM02 significantly inhibited LPS-induced BMDCs proliferation.IMQ significantly inhibited BMDCs proliferation,while 0.5 to 2 mg/mL PSORI-CM02 further significantly inhibited BMDCs proliferation on the basis of IMQ intervention.B.PSORI-CM02 inhibited the maturation and antigen uptake of BMDCsDetection of the maturation of BMDCs by flow cytometry:the results of BMDCs maturation by flow cytometry showed that the expression of CD83,CD86 and MHCII on the surface of BMDCs in LPS group significantly increased compared with blank control group;PSORI-CM02 at various concentrations significantly inhibited the expression of CD83 and CD86,but significantly promoted the expression of MHCII.The expression of three crowds of BMDCs in IMQ group increased significantly compared with blank control group;PSORI-CM02 at 0.5 to 2mg/mL concentrations significantly inhibited the expression of CD83,CD86 and MHCII.Detection of BMDCs antigen uptake by flow cytometry:compared with blank control group,the proportion of FITC positive cells in the OVA-FITC single intervention group increased significantly,and the proportion of FITC positive cells were significantly inhibited by different concentrations of PSORI-CM02.C.Effect of PSORI-CM02 on inflammatory cytokines in BMDCsDetection of the gene expression of cytokines in BMDCs by RT-PCR:compared with blank control group,the expressions of IL-12A,IFN-y,CCL20,TNF,IL-17F and IL-23 of LPS group significantly increased.Except for TNF and IL-17F in 0.5 mg/mL PSORI-CM02 group,the expressions of IL-12A,IFN-?,CCL20,TNF and IL-17F were down-regulated significantly by PSORI-CM02 at various concentrations.However,PSORI-CM02 had no inhibit.ory effect on the abnormal increase of IL-23 expression,and the difference was not statistically significant.Compared with blank control group,the expressions of IL-12A,IFN-? and IL-23 in BMDCs of IMQ group increased significantly,and the expressions of IL-12 and IFN-y were significantly inhibited by PSORI-CM02 at various concentrations.The expression of IL-23 was inhibited by 2mg/mL PSORI-CM02,and the difference was not statistically significant.However,the expression of IL-23 was promoted significantly by 0.5 and lmg/mL PSORI-CM02.Detection of intracellular protein in BMDCs by flow cytometry:compared with blank control group,the proportions of CDllc+IL-12p35+and CDllc+IFN-y+cells in LPS group increased,the proportions of two crowds of cells in 0.5 to 2 mg/mL PSORI-CM02 group significantly down-regulatedD.PSORI-CM02 affect BMDCs regulation of T cell differentiationDetection of intracellular proteins in CD4+T cells by flow cytometry:after LPS stimulated BMDCs and Naive CD4+T cells,the proportions of CD4+IFN-?+and CD4+T-bet+cells increased significantly.The proportions of two crowds of cells in 1 and 2 mg/mL PSORI-CM02 groups showed decreasing trends compared with LPS group,and the differences were statistically significant.E.PSORI-CM02 inhibited P38 and Stat3 signaling pathways in BMDCsDetection of the signal pathway in BMDCs by western-blot:compared with blank control group,the expression of p-P38 significantly increased in LPS group.The expressions of p-P38 and p-Stat3(Tyr705)were down-regulated in dose-dependent manner at different concentrations of PSORI-CM02,and the differences were statistically significant.3.Effects of paeoniflorin on psoriasis and DCsA.The therapeutic effect and mechanism of paeoniflorin on IMQ-induced psoriatic miceImprovement of animal skin lesions:symptoms of psoriasis lesions in mice induced by IMQ in MTX group,0.5%and 2%paeoniflorin group were improved.The PAST score of IMQ group was significantly higher than normal control group.Compared with IMQ group,PASI score of psoriasis lesions in 0.5%and 2%paeoniflorin groups and MIX group decreased significantly.Results of HE staining:the lesions in IMQ group showed histopathological manifestations of psoriasis,while those in MTX group and 0.5%and 2%paeoniflorin group were alleviated.Animal weight results:During the experiment,the body weight of mice in normal control group showed a gradually increasing trend.Compared with normal control group,the weight of mice in IMQ group,MTX group,0.5%and 2%paeoniflorin group showed different degrees of decline,in which the weight of the MTX group and IMQ group decreased significantly,while 0.5%and 2%paeoniflorin alleviated the weight loss caused by IMQ.Animal spleen index results:The spleen index of IMQ group was significantly higher than normal control group.The spleen index of MTX group was lower than IMQ group,and the difference was statistically significant.Compared with IMQ group,0.5%and 2%paeoniflorin group showed downward trends,but the differences were not statistically significant.Detection of CD11c+DCs maturation in lymph nodes by flow cytometry:Compared with normal control group,the expression of CD83,CD86 and MHCII on the surface of CD11c+DCs in IMQ group was significantly increased,and the differences were statistically significant.0.5%and 2%paeoniflorin could not inhibit the expression of the three crowds of DCs,and the differences were not statistically significant,but the expression of the three crowds of DCs in the MTX group showed downward trends.B.Effects of paeoniflorin on BMDCs maturation,secretion of cytokines and regulation of T cell differentiationDetection of BMDCs maturation by flow cytometry:The experiment was divided into 6 groups-control group,IMQ group and paeoniflorin groups(5,20,50,100 M).The expression of CD83 and CD86 on the surface of BMDCs in IMQ group was significantly increased compared with blank control group.The expression of CD83 and CD86 on the surface of BMDCs were not inhibited by paeoniflorin at different concentrations,and the difference was not statistically significant.Detection of inflammatory cytokines in BMDCs by RT-PCR:The experiment was divided into four groups-blank control group,IMQ group and paeoniflorin groups(50,100 M).Compared with blank control group,IMQ group showed increased expression of IL-1??IL-6?IL-12A?IL-23?IFN-y and TNF,with statistical difference.Compared with IMQ group,5 and 100 M paeoniflorin could not inhibit the expression of the above cytokines,and even promoted the expression of inflammatory factors IL-10?IL-6?IL-23?IFN-? and TNF,with statistically significant differences.Detection of BMDCs and Naive CD4+T cells co-culture system by flow cytometry:The experiment was divided into 5 groups-blank control group,LPS group and paeoniflorin groups(5,20,100 M).The proportions of CD4+CD25+and CD4+CD152+cells in LPS stimulated co-culture system decreased,and the difference was statistically significant.The proportions of two crowds of cells in 5,20 and 100 M paeoniflorin groups showed increasing trends,and the difference were statistically significant.Conclusion1.PSORI-CM02 have therapeutic effect on IMQ-induced psoriatic mice,inhibit the infiltration of PDCs in the skin of psoriatic mice,affect the maturity and distribution migration of DCs in lymph nodes,and inhibit the differentiation of Thl and Thl7 cells in the spleen of psoriasis model mice.2.PSORI-CM02 could inhibit the antigen uptake,maturation and inflammatory factor secretion of BMDCs in vitro.Moreover,PSORI-CM02 could inhibit the differentiation of CD4+Naive T cells into Th1 cells in the co-culture system of BMDCs and Naive CD4+T cells.We speculate that PSORI-CM02 plays a role in the treatment of psoriasis by affecting the function of DCs,and intervening the differentiation and exerting effect of Th1 cells.3.PSORI-CM02 could inhibit the expression of p-Stat3 and p-p38 MAPK proteins in LPS-stimulated BMDCs.We speculated that PSORI-CM02 could inhibit a series of downstream inflammatory processes in psoriasis by inhibiting the STAT3 and P38 MAPK signaling pathways in DCs during the pathogenesis of psoriasis.4.Paeoniflorin has therapeutic effect on IMQ-induced psoriasis in mice,but cannot inhibit the maturation of DCs in lymph nodes5.Paeoniflorin could not inhibit maturation and the expression of inflammatory factors of BMDCs in vitro experiments,but it could regulate the differentiation of CD4+ Naive T cells into Treg cells in the co-culture system of BMDCs and Naive CD4+T cells.We speculate that paeoniflorin may play a role in the treatment of psoriasis by affecting DCs and thereby promoting the differentiation of Treg cells.