| Aim: To verify the clinical efficacy,safety and effect on Wnt signaline pathway of blood-heat syndrome of psoriasis in the treatment of cooling blood and restrain Yang therapy by clinical research.To discuss the role of epigenetic modified Wnt pathway in blood-heat syndrome of psoriasis from the perspective of DNA methylation modification of WIF-1 gene to regulate Wnt signaling pathway,and further explain the mechanism of cooling blood and restrain Yang therapy through the basic research.Methods: A randomized controlled trial was utilized in the first part.80 subjects were randomized divided into syndrome differentiation with cooling blood and restrain Yang treatment group and the fixed treatment group.Each group was given oral medication for 8 weeks.Psoriasis Area Severity Index(PASI)was used to evaluate the clinical efficacy and safety.Meanwhile,the normal tissue was used as the control,and patients with the blood-heat syndrome of psoriasis,patients with effective and ineffective treatment of cooling blood and restrain Yang therapy were selected.The expression and distribution of WIF-1,STAT3,Wnt-5a and β-Catenin were detected by immunohistochemistry.The second and third parts were the combination of in vivo and ex vivo experiments.In vivo,skin lesions of psoriasis patients and normal subjects were taken as the research object.Detection of WIF-1 methylation status,related genes and protein expression by sulfite amplicon sequencing,q PCR,Western blot and immunohistochemistry.In vitro,Ha Ca T cells were used as the research object.WIF-1si RNA transfection and demethylation were used to evaluate the effect of WIF-1expression silencing on Wnt signaling pathway regulation of cell proliferation and apoptosis.Results : In the first part,the total effective rate of syndrome differentiation with cooling blood and restrain Yang treatment group(87.5%)was higher than that of the fixed treatment group(84.21%),but there was no significant difference in the curative effect between the two groups(p>0.05).The PASI scores of the two groups were significantly decreased after 8 weeks of treatment(P<0.001).No prominent adverse drug reactions occurred during the study period.Compared with normal tissues,the WIF-1/STAT3 ratio was significantly decreased(P<0.05);the Wnt-5a/STAT3 ratio was significantly increased(P<0.05).In the second part,the methylation rate of WIF-1 gene promoter was 86.296% in the skin lesion of blood-heat syndrome of psoriasis patients,which was significantly higher than that of normal tissues(77.778%,P<0.05),among which the methylation levels of Cp G sites in promoters 45,86,100,197,221,240,247,266,276,285,353 and 366 were significantly increased(P<0.05),and WIF-1 m RNA and protein expression were significantly down-regulated(P<0.05).The methylation degree,apoptosis rate and Ki67 expression of WIF-1 gene in IL-17-stimulated Haca T cells were significantly increased(P<0.05).The 5-Aza-d C treatment significantly decreased the methylation degree,apoptosis rate and Ki67 expression of WIF-1 gene(P<0.05).In the third part,the methylation rate of CpG locus in the WIF-1 promoter region of the patients with effective treatment of cooling blood and restrain Yang therapy was79.259%,which was significantly lower than that of ineffective treatment of cooling blood and restrain Yang therapy and patients with blood-heat syndrome of psoriasis(86.296%)(P<0.05),among which the methylation levels of Cp G sites in promoters45、100、122、177、197、221、247 and 366 were significantly decreased(P<0.05),WIF-1m RNA and protein expression were significantly up-regulated(P<0.05).The methylation degree,apoptosis rate and Ki67 expression of WIF-1 gene in Haca T cells cultured in serum of Haca T cells cultured in serum of cooling blood and restrain Yang therapy were significantly descended(P<0.05).Conclusions: To summary,the hypermethylation status of the WIF-1 gene promoter results in abnormal activation of the Wnt pathway in blood-heat syndrome of patients with psoriasis vulgaris.Therapeutic effect of cooling blood and restrain Yang therapy treated blood-heat syndrome of psoriasis is achieved by reducing the methylation level of WIF-1 promoter region and inhibiting Wnt signaling pathway. |
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