| ObjectiveIn this study,the essential targets and compounds of YXYH acting on psoriasis were generated via network pharmacology methods,and the possible pathways of YXYH acting on psoriasis were further predicted through network analysis.The psoriatic cell model was constructed to verify the effect of YXYH on Ha Ca T cell mediated by TNF-?,then construct the VDR over-expression plasmid,and detect whether VDR can regulate VDR/VEGF signal pathway and its mechanism.Finally,animal model of psoriasis was constructed to explore whether YXYH can regulate the immune disorders of psoriasis by intervening in the VDR/VEGF signaling pathway,and whether it is dose-dependent.Methods1.Obtain the corresponding compound composition of YXYH through the TCMSP database and literature.The YXYH drug-target-disease network was obtained after the relevant targets corresponding to the components of YXYH and the targets corresponding to psoriasis were retrieved in relative databases;all targets are sorted by degrees to construct a drug-target-disease core network;GO-BP and KEGG database were used for enrichment analysis.2.25 SD rats were randomly divided into 2 groups and given saline and YXYH by gavage to obtain saline serum and YXYH serum;Ha Ca T cells were mediated by 25?g/L TNF-? to construct psoriasis cell model.After making sure the best drug-containing serum concentration,set cells to blank,model,control and YXYH group and use CCK-8 to detect cell viability and reproductive inhibition rate,ELISA to detect the expression levels of inflammatory cytokines and Annexin-V-FIFC and PI to determine cell apoptosis in each group.VDR overexpression plasmid was constructed and transfected into Ha Ca T cells mediated by TNF-?.Set cells to control,OE-NC and OE-VDR group,q PCR and WB to detect the expression level of VDR.Set cells to blank,model,YXYH,model/OE-NC,YXYH/OE-NC,model/OE-VDR,YXYH/OE-VDR group,CCK-8 to detect cell viability and survival rate,q PCR to detect gene expression level of VDR,VEGF,RAR,RXR and WB to detect VDR,VEGF protein expressions in the posterior four groups.3.30 BALB/c mice were randomly divided into control,model,YXYH-L,YXYH-M,YXYH-H group after adaptive feeding.Except for the control group,the other groups were smeared on the skin with 62.5mg Imiquimod ointment everyday,YXYH-L,YXYH-M,YXYH-H groups were given YXYH with dosage of 20.34g/kg/d,40.69g/kg/d and81.38g/kg/d at the same time.The general condition,weight,back thickness and PASI score of each group were observed during the intervention period.The peripheral blood,spleen tissue and back skin lesions were observed after mice sacrifice to detect the distribution of peripheral blood T lymphocytes,the quality of spleen and spleen swelling index,the expression levels of VDR,VEGF,RAR,RXR and the pathological changes of the back skin lesions.Results1.A total of 148 active ingredients,193 verified targets and 215 predicted targets corresponding to the YXYH ingredient were obtained,and 390 targets for psoriasis were obtained.YXYH drug-target-disease network includes 59 corresponding compounds and 43 related targets,plus the drug-target-disease core network contains 18 core compounds and15 core targets.12 main pathways were observed and among which the VEGF metabolism pathway and corticosteroid reflection signal pathway were marked as cross-talk pathways.2.25% was finally selected as the best drug-containing serum concentration.3.Compared with the control group,cell viability of the model group was increased(P<0.01)and the proliferation inhibitory rate was decreased(P<0.01);Compared with the model group,cell viability of the YXYH group was decreased(P < 0.01)and the proliferation inhibitory rate was increased(P<0.01).4.Compared with the control group,the expression of IL-4 was decreased(P<0.01)and the expression of TNF-?,IFN-? were increased(P < 0.01);Compared with the model group,the expression of IL-4 was increased(P<0.01)and the expression of TNF-?,IFN-?were decreased(P<0.01).5.Compared with the control group,the proportion of normal cells was increased(P<0.01)and the proportion of apoptotic cells was decreased(P < 0.01);Compared with the model group,the proportion of normal cells was decreased and the proportion of apoptotic cells was increased(P<0.01).6.VDR gene had been successfully cloned into the pc DNA3.0 eukaryotic vector.7.Compared with the control group,there were no difference of VDR expression in gene and protein levels in OE-NC group(P>0.05);Compared with OE-NC group,VDR expression in both gene and protein level were increased in OE-VDR group(P<0.01).8.Compared with the model group,cell viability of YXYH group was decreased(P<0.01)while cell viability of OE-NC group was without any difference(P>0.05);Compared with model/OE-NC group,cell viability in model/OE-VDR was decreased(P <0.05);Compared with the model/OE-VDR,cell viability of YXYH/OE-VDR was decreased(P<0.05)while cell proliferation inhibition rate was increased(P<0.05).9.Compared with model group,gene expressions of VDR,RAR and RXR in YXYH group were increased(P<0.05,P<0.01,P<0.01)while gene expression of VEGF was decreased(P<0.05);Compared with the model/OE-NC group,gene expressions of VDR,RAR and RXR in model/OE-VDR group were increased(P<0.01,P<0.05,P<0.01)while gene expression of VEGF was decreased(P<0.05);Compared with the model/OE-VDR group,gene expressions of VDR,RAR and RXR in YXYH/OE-VDR group were increased(P<0.05,P<0.05,P<0.01)while gene expression of VEGF was decreased(P<0.01).10.Compared with the model/OE-NC group,protein expression of VDR in model/OE-VDR group was increased(P <0.05)while protein expression of VEGF was decreased(P<0.05);Compared with the model/OE-VDR group,protein expression of VDR in YXYH/OE-VDR group was increased(P<0.01)while protein expression of VEGF was decreased(P<0.05).11.During intervention period,all mice in each group could move,eat,drink normally,and there was no difference in the body weight on D0 and D3(P>0.05).On the 7th day,compared with control group,weight of mice in model and YXYH-L group were decreased(P < 0.05);compared with model group,body weight were with no difference in YXYH-L,YXYH-M and YXYH-H groups.12.Back skin lesions of the all mice were normal before modeling and there was no difference in the thickness of back lesions and the PASI score of mice in each group(P>0.05).On the 7th day,the back of mice in model group showed typical appearance of psoriasis.Compared with model group,PASI scores in YXYH-L,YXYH-M,YXYH-H groups were decreased consequently(P<0.01,P<0.05).13.There was no abnormality in the back lesions of mice in control group via HE stain.The stratum corneum of model group showed full-thickened epidermis with large number of incomplete keratinocytes and spines.The dermal papilla layer was slightly extended,and inflammatory infiltrating cells were visible.Skin lesions of the mice in YXYH-L group showed the similarities between the model group.14.Compared with control group,spleen quality and spleen swelling index in model group were increased(P < 0.01);Compared with model group,spleen quality and spleen swelling index of the mice in YXYH-L,YXYH-M,YXYH-H groups were decreased consequently(P<0.01).15.Compared with control group,gene and protein expression of VDR,RAR,RXR were decreased(P<0.01)while gene and protein expression of VEGF were increased(P<0.01)in model group;Compared with model group,gene and protein expression of VDR,RAR,RXR were increased(P<0.01,P<0.05,P<0.01)while gene and protein expression of VEGF were decreased(P<0.01)in YXYH-H group.16.Compared with control group,CD3+CD4+ and CD3+CD8+ expressions in peripheral blood of model group were increased(P<0.01)while the ratio of CD4+/CD8+was inverted significantly(P<0.01);Compared with model group,the ratio of CD4+/CD8+in YXYH-M and YXYH-H groups were increased(P<0.01)gradually.Conclusion1.Natural flavonoids and phytosterols are the main material basis for anti-psoriatic effect for YXYH.The targets of YXYH involve inflammation,angiogenesis and abnormal differentiation of keratinocytes.The therapeutic effects are also multi-channels involves proliferation,differentiation,metabolism,inflammation,immunity,tumors,etc.Among them,VEGF pathway and the corticosteroid reflection signal play cross-talk roles.2.VDR is a key factor in the VDR/VEGF signaling pathway.Over-expression of VDR can effectively inhibit the proliferation of Ha Ca T cells mediated by TNF-?.YXYH can up-regulate expression of RAR and RXR while down-regulate expression of VEGF by up-regulating expression of VDR,thereby exerting anti-inflammatory,anti-keratinocyte abnormal proliferation and abnormal neovascularization effects.Over-expression of VDR can effectively increase the inhibitory effect of YXYH on psoriatic cell model,which can provide new ideas for increasing the efficacy of YXYH.3.YXYH can effectively inhibit the cell viability,promote cell apoptosis and inhibit the abnormal ratio of Th1/Th2,alleviate inflammatory micro-environment in psoriatic cell model;it can also be efficient to regulate the immune disorders in psoriatic animal model,but depends on dose. |
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