| Objective:Based on Wnt/β-catenin signaling pathway,the effect of Zuogui pill(ZGP)on immune function of offspring mice and its mechanism were studied from the perspective of thymus.Methods:1.Theoretical research:To clarify the relationship between congenital kidney deficiency(CKD)and immunity,to explore the theoretical mechanism of CKD affecting immune function,and to explore the treatment of CKD.2.In vivo research:60 3-month-old SPF grade SD rats in estrus.Male and female SD rats were divided into two groups according to the ratio of 2:1 to prepare 30 pregnant rats every night.Pregnant rats were randomly divided into normal group,model group,thymosin group(0.003g/L),high-dose ZGP group(800g/L)and low-dose ZGP group(200g/L),with 6 rats in each group.Normal group was given normal saline by gavage;model group was given normal saline by gavage;thymosin group was given thymosin capsule solution by gavage;high-dose ZGP group and low-dose ZGP group were given ZGP suspension by gavage.The pregnant rats were stimulated by compound stress until their offspring were born.The thymus and spleen of offspring rats was extracted on the day of birth,and the thymus index of offspring rats was calculated.The T cell types in thymus and spleen of offspring rats were detected by flow cytometry.The expression of Wnt4,β-Catenin,Foxn1 gene and protein in thymus of offspring rats were detected by RT-PCR and Western Blot.SPSS 22.0 was used to analyze the data.The results were expressed as mean plus minus standard deviation(x±s).One way ANOVA was used for comparison between groups.3.In vitro research:Thymic epithelial cells(TECs)of SD rats were cultured and identified by broad-spectrum cytokeratin(PCK)immunofluorescence staining.CCK-8 method was used to determine the effect of serum containing ZGP and Wnt4 inhibitor(ICG-001)on cell proliferation rate.Western Blot was used to detect the inhibitory effect of different concentrations of ICG-001.The best experimental concentration of serum containing ZGP and ICG-001 was determined by combining the two methods.TECs were divided into control group,kidney tonifying group,Wnt inhibitor control group and Wnt inhibition kidney tonifying group.RT-PCR and Western Blot were used to detect the expression of Wnt4,β-Catenin and Foxn1.ELISA was used to detect the content of thymosinβ4(TMSβ4)and thymosinα1(Tα1).Result:1.CKD can affect the related functions of Yin and Yang,Qi,blood,body fluid and five zang organs,while Yin and Yang,Qi,blood,body fluid and five zang organs are related to immunity.2.Compared with the normal group,the thymus index of the model group decreased(P<0.05);Compared with the model group,the thymus index and spleen index of the thymosin group and the high-dose ZGP group increased(P<0.05).Flow cytometry showed that:compared with the normal group,the number of SP cells in the thymus of the model group increased(P<0.05),and the number of CD4~+/CD8~+in the thymus of the model group increased(P<0.05),CD4~+/CD8~+in the spleen of the model group decreased(P<0.05);compared with the model group,the number of SP cells in the thymus of thymosin group,high-dose ZGP group and low-dose ZGP group decreased(P<0.05),and the number of SP cells in the thymus of thymosin group,high-dose ZGP group and low-dose ZGP group decreased(P<0.05),The CD4~+/CD8~+in thymus decreased(P<0.05),and the CD4~+/CD8~+in spleen increased(P<0.05).RT-PCR and Western Blot showed that:compared with the normal group,the expression of Wnt4,β-Catenin,Foxn1 gene and protein in the thymus of the model group was decreased(P<0.05);compared with the model group,the expression of Wnt4,β-Catenin,Foxn1 gene and protein in the thymus of thymosin group,high-dose ZGP group and low-dose ZGP group was increased(P<0.05).3.The purified cells were identified as TECs by PCK immunofluorescence staining.CCK-8 test showed that ICG-001and 10%of the serum containing ZGP had no effect on the cell proliferation rate.Western Blot analysis showed that ICG-001could inhibit the expression of Wnt4 in TECs.25μm ICG-001 and20%serum containing ZGP were selected as experimental drug concentration.ELISA results showed that:compared with the control group,the content of TMSβ4 and Tα1 in Wnt inhibitor control group decreased(P<0.05),the content of TMSβ4 and Tα1 in kidney tonifying group increased(P<0.05);compared with Wnt inhibition control group,the content of TMSβ4 and Tα1 in Wnt inhibitor kidney tonifying group increased(P<0.05).The results of RT-PCR and Western Blot showed that compared with the control group,Wnt4,β-Catenin and Foxn1 in the Wnt inhibitor control group were significantly higher The gene and protein contents of Wnt4,β-Catenin and Foxn1 in the kidney tonifying group were increased(P<0.05);compared with the inhibitor control group,the gene and protein contents of Wnt4,β-Catenin and Foxn1 in the kidney tonifying group were increased(P<0.05).Conclusion:1.CKD can affect the immune state of the body by affecting the functions of yin and Yang,Qi,blood,body fluid and five zang organs.It is one of the new methods to prevent and treat congenital kidney deficiency by tonifying kidney and filling essence of mother.2.Compound stress method can successfully reproduce the model of CKD.3.The abnormal expression of Wnt4/β-catenin signaling pathway in thymus affects the differentiation,development and migration of T cells,which may be one of the mechanisms of immune disorder in offspring rats with CKD.4.ZGP can activate Wnt4/β-catenin signaling pathway in thymus,increase the expression of Foxn1,promote the secretion of thymosin by TECs,and regulate the differentiation,development and migration of T cells,which may be one of the mechanisms of tonifying kidney and filling essence to improve the immune disorder of CKD. |
PDF Full Text Request