基于Wnt/β-catenin信号通路探讨Tregs介导的苏木提取物抗ApoE-/-小鼠动脉粥样硬化机制OA

Objective:To investigate the effects of Sappanwood Extract(SEAE)and/or T regulatory cells(Tregs)on the Wnt/β-catenin pathway in ApoE-/-mice with atherosclerosis(AS),and to elucidate their anti-atherosclerosis mechanisms.Methods:A total of 10 C57BL/6J mice served as the control group,and 40 ApoE-/-mice models were successfully established and randomly divided into the model group,the Sappanwood Extract group,the T regulatory cells group and the combined group,with 10 mice in each group.Following intervention,tissue samples were collected and examined for pathological morphological changes in the aortic sinus using haematoxylin and eosin(HE)staining.Enzyme linked immunosorbent assay(ELISA)was used to measure serum levels of interleukin-6(IL-6),interleukin-17(IL-17),interleukin-10(IL-10)and transforming growth factor-β(TGF-β).Immunohistochemistry was used to examine the expression of the aortic fork head box protein P3(FOXP3).Flow cytometry was used to determine the number of Tregs in spleen.Western blotting was used to detect Wnt1,β-catenin,c-Myc,histone deacetylase 9(HDAC9)and TIP60 proteins in aorta,and FOXP3 protein in spleen.Co-immunoprecipitation was used to to determine the acetylation level of FOXP3 protein in spleen.Results:The aortas of the model group exhibited typical pathological morphological changes characteristic of atherosclerosis.The pathological morphology of the aortic sinuses in the Sappanwood Extract group,T regulatory cells group and combined group showed improvement compared with the model group,with the combined group demonstrating greater improvement than the T regulatory cells group and the Sappanwood Extract group.Serum IL-6 and IL-17 levels in the model group were higher than those in the control group(P＜0.01),while IL-10 and TGF-β levels were lower(P＜0.01).The relative expression of HDAC9,Wnt1,β-catenin and c-Myc protein in the aorta of the model group were higher than that in the control group,while the relative expression of TIP60 protein was lower(P＜0.01).In the model group,the relative expression of FOXP3 protein in the spleen was lower,the acetylation level of FOXP3 protein was lower,and the proportion of CD4+CD25+FOXP3+T cells among CD4+CD25+T cells was lower than that in the control group(P＜0.01).In mice from the Sappanwood extract group,T regulatory cells group and combined group,serum IL-6 and IL-17 levels were lower than those in the model group(P＜0.05 or P＜0.01),while serum IL-10 and TGF-βlevels were higher than those in the model group(P＜0.05 or P＜0.01).FOXP3-positive staining in the aortic sinus was more abundant than that in the model group.In the Sappanwood extract group,T regulatory cells group and combined group,the relative expression levels of HDCA9,Wnt1,β-catenin and c-Myc proteins in the aorta were lower than those in the model group(P＜0.05 or P＜0.01),while the relative expression level of TIP60 protein in the aorta was higher than that in the model group(P＜0.05 or P＜0.01).In the Sappanwood extract group,T regulatory cells group and combined group,the relative expression levels of FOXP3 protein,the acetylation levels of FOXP3 protein,and the proportion of CD4+CD25+FOXP3+T cells among CD4+CD25+T cells were higher than that in the model group(P＜0.05 or P＜0.01).Serum IL-6 and IL-17 levels in the combined group were lower than those in the Sappanwood extract group and T regulatory cells group(P＜0.01),while serum IL-10 and TGF-β levels were higher than those in the Sappanwood extract group and T regulatory cells group(P＜0.01).The relative expression levels of HDCA9,Wnt1,β-catenin and c-Myc proteins in the aortas of mice in the combined group were lower than those in the Sappanwood extract group and T regulatory cells group(P＜0.01),while the relative expression level of TIP60 protein was higher than that in the Sappanwood extract group and T regulatory cells group(P＜0.01).In the combined group,the relative expression levels of FOXP3 protein,the acetylation levels of FOXP3 protein,and the proportion of CD4+CD25+FOXP3+T cells among CD4+CD25+T cells were higher than those in the Sappanwood extract group and T regulatory cells group(P＜0.01).Tregs and SEAE act synergistically to reduce serum IL-6 and IL-17 levels in mice and increase IL-10 and TGF-β levels.They also increased TIP60 protein expression in the aorta,reduced the expression levels of HDCA9,Wnt1,β-catenin and c-Myc protein expression levels in the aorta,and increased FOXP3 protein expression,FOXP3 protein acetylation levels,and the proportion of CD4+CD25+FOXP3+T cells among CD4+CD25+T cells in the spleen(P＜0.05 or P＜0.01).The combined treatment yielded the best results.Conclusion:SEAE and/or Tregs can improve pathological morphological changes in the aortic sinus of AS model mice by suppressing or alleviating inflammatory responses,promoting FOXP3 expression,inhibiting the activation of the Wnt/β-catenin signalling pathway,and increasing the number of Tregs in the spleen.Furthermore,Part of the mechanism of action of SEAE may be mediated by Tregs,namely by affecting the number and function of Tregs and synergizing with exogenous Treg infusion to prevent and treat AS,with the combined use of both yielding the best effect.