| Objective1.To observe the distribution of T lymphocytes in peripheral blood of patients with psoriasis vulgaris.2.To observe the effects of different concentrations of YinXieYiHao(YXYH)serum on the levels of cytokine IL-4,IL-6,IL-17,IL-23,INF-??TNF-??TGF-??25HVD3(VD)and the protein and gene expression level of VDR?STAT4in psoriatic cell model,to elucidate the therapeutic effects of YXIH prescription on psoriasis at molecular level,to explore the mechanism of drug metabolism and provide more basic scientific research support for the modernization and popularization of traditional Chinese medicine compound.3.To explore the mechanism of VDR signaling network and STAT signaling pathway,clarify the mechanism of YXIH in the treatment of psoriasis vulgaris by interfering with vitamin D receptor and regulating STAT signaling pathway,to clarify the basis for clinical promotion of YXIH.Method:Diagnose 80 psoriasis vulgaris patients from the outpatient department of dermatology of the first affiliated Hospital of Guangzhou University of traditional Chinese Medicine,meeting the criteria of inclusion and exclusion,and Score according to PASI after signing an informed consent.To extract 10 ml peripheral venous blood in each patients,then T lymphocytes were isolated and cultured in low,medium and high concentrations of YXYH serum,normal control group(blank)and positive control group(triptolide intervention)to be control groups,to observe the distribution of T lymphocyte subtype CD3+CD4+and CD3+CD8 in culture supernatant.At the same time,as mature molecular biological techniques,ELISA,RT-PCR and WB were used to detect the concentration of IL-4?IL-6?IL-17?IL-23?INF-??TNF-??TGF-??25HVD3(VD)in psoriatic cell model,and to compare the expression levels of VDR and STAT4 protein and gene,detect the fluorescence intensity of VDR and STAT4 in T lymphocytes of each group by immunofluorescence double labeling,comparative statistical analysis of data.Results:1.Effect on the distribution of T lymphocytes in peripheral blood The expression of T lymphocytes in human peripheral blood was different in different concentrations of serum containing drugs,compared with the normal control group,the distribution ratio of CD3+CD4+ increased significantly in Low,medium,high dosage groups of traditional Chinese medicine and positive control group(P<0.01),The proportion of CD3 CD4 in each group showed a significant upward trend with the increase of drug concentration(P<0.01),the proportion of CD3 CD8 increased,but the fluctuation was obviously lower than that of CD3 CD4.The ratio of CD4/CD8 in each group was less than 1,The ratio of CD4/CD8 in the low,middle and high dose groups of traditional Chinese medicine was gradient increased(P<0.01).2.Effect on the concentration of cytokine in psoriatic cell model.Compared with the normal control group,the concentration of cytokine IL-6?IL-17?IL-23?INF-??TNF-??VD in the low?middle?high dose group and the positive control group were significantly lower(P<0.01),the decrease was most obvious in the high dose group and the positive control group;Compared with the positive control group,the concentration of IL-6?IL-17?IL-23?INF-??TNF-??VD in the low? middle dose group was significantly higher(P<0.01),no change in high dose group(P>0.05);the concentration of cytokine were obvious gradient differences in each groups of traditional Chinese Medicine,low dose group > middle dose group > high dose group(P<0.01).Compared with the normal control group,the concentration of IL-4?TGF-?in the low?middle?high dose group and the positive control group were significantly increased(P<0.01),especially in the high dose group and the positive control group;compared with positive control group,the concentration of IL-4?TGF-?in the low and middle dose group was posignificantly recuded(P<0.01),but the concentration of IL-4?TGF-?in the high dose group was not significantly decreased(P>0.05).There was a significant gradient difference between the three groups(P<0.01),and the effect of high dose group was the best.3.Effects on the protein concentration and gene expression of VDR and STAT4.Comparison of expression of VDR protein:compared with the normal control group,the expression of VDR protein in the low,medium,high dose and positive control groups of TCM was significantly increased(P<0.01));compared with the positive control group,the expression of VDR in the low and middle dose group decreased significantly(P<0.01),but there was no significant difference between the high dose group and the positive control group(P>0.05).Comparison of expression of STAT4 protein: compared with the normal control group,the expression of VDR protein in the low,medium,high dose and positive control groups of TCM was significantly decreased(P<0.01);compared with the positive control group,the expression of VDR in the low and middle dose group increased significantly(P<0.01),and the STAT4 expression in the high dose group was higher(P<0.05).Comparison of VDRmRNA expression levels:compared with the normal control group,the expression of VDRmRNA protein in the low,medium,high dose and positive control groups of TCM was significantly increased(P<0.01);Compared with the positive control group,the expression of VDRmRNA in the low and middle dose group decreased significantly(P<0.01),but there was no significant difference between the high dose group and the positive control group(P>0.05).Comparison of expression of STAT4 mRNA protein: compared with the normal control group,the expression of VDRmRNA protein in the low,medium,high dose and positive control groups of TCM was significantly decreased(P<0.01);compared with the positive control group,the expression of VDR in the low and middle dose group increased significantly(P<0.01),and the STAT4 expression in the high dose group was the same(P>0.05).Conclusion YXIH can reduce the concentration of cytokine IL-6?IL-17?IL-23?INF-??TNF-??VD in psoriatic cell model,can promote the expression of IL-4?TGF-?,YXIH could up-regulate the expression of VDR protein and gene,and inhibit the expression of STAT4 protein and gene.The results showed that By comparing different dose YXIH serum groups,we can show a certain dose dependence.The higher the drug concentration,the more obvious the inhibitory effect on inflammatory factor,the more obvious the promoting effect on VDR signal expression,but the negative correlation with STAT4 signal pathway.The results suggest that YXIH has obvious inhibitory effect on inflammation,and its mechanism may be related to the regulation of VDR signal network and theexpression of STAT4 signal. |
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