Mechanisms Of Vitamin D Regulation Of Treg Cell Immune Response Through β-catenin Pathway

Objective:This research examined the role and significance of vitamin D and Treg cells in the development of asthma.Furthermore,it elucidated the probable molecular processes by which vitamin D affected the inflammatory,immunological response of Treg cells at the level of cells via theβ-catenin pathway.Methods:1.From January 2021 to April 2022,58 patients with acute exacerbations of asthma at Shanxi Medical University’s Second Hospital were collected as the experimental group,all of whom met the inclusion criteria,and 33 patients with health check-ups at our hospital during the same period were collected as the control group.Each research subject’s fasting blood was taken and processed to extract serum and peripheral blood mononuclear cells.ELISA was used to determine serum 25（OH）D₃levels.Flow cytometry was used to determine the proportion of Treg cells.The expression level of Foxp3 m RNA,a particular marker on the surface of Treg cells,was determined by RT-q PCR.The serum inflammatory factors IL-10 and IL-17 were evaluated using a flow-through multi-factor assay technique.2.In vitro experiments:mouse Treg cells were cultured in vitro.There were four groups set up:a blank control group,a TGF-β1 induction group,a 1,25（OH）₂D₃intervention group,and an ICG-001 intervention group.Except for the blank control group,each well received TGF-β1 at a dose of 2ng/ml,1,25（OH）₂D₃at an intervention dose of 1μmol/l,and ICG-001 at an intervention dose of 5μmol/l.The dose was chosen and applied based on the findings of the subject’s prior research.Western blot analysis was performed on the expression levels of Treg cell-specific markers and the positive regulatory protein Foxp3.RT-q PCR was used to assess the amounts of Foxp3m RNA,IL-10m RNA,and IL-35m RNA.Co IP was used to examine the alterations inβ-catenin/TCF andβ-catenin/Foxo1 transcriptional complexes in each group.A dual luciferase reporter gene assay was used to evaluate the transcriptional activity of the Foxo1 transcription factor on the downstream Foxp3 promoter.Results:1.There were no statistically significant variations in gender or age between the groups of all the research participants（P>0.05）.ELISA findings revealed that serum25（OH）D₃values were significantly lower in individuals with acute asthma exacerbations than in healthy controls（P<0.05）.Flow cytometry results showed that the proportion of Treg cells in patients with acute asthma exacerbation was lower than in healthy participants（P<0.001）.RT-q PCR findings revealed that the Treg cell-specific marker Foxp3 was expressed at lower levels in asthma patients（P<0.05）.According to the findings of the flow-based multifactor assay technique,serum expression levels of the anti-inflammatory factor IL-10 were significantly lower and pro-inflammatory factor IL-17 was significantly higher in asthma patients with acute exacerbations,and the IL-17/IL-10 ratio was significantly abnormal in asthma patients with acute exacerbations compared to healthy subjects（P<0.05）.Pearson correlation analysis found a significant positive association between the amount of anti-inflammatory factor IL-10 expression and the fraction of Treg cells（r=0.5388,P<0.01）.There was no significant relationship between the pro-inflammatory factor IL-17 and the Treg cells（r=-0.1264,P>0.05）.In individuals with acute asthma exacerbation,there was a negative link between serum25（OH）D₃levels and the IL-17/IL-10 ratio（r=-0.3987,P<0.01）and a positive correlation with Treg cells（r=0.4110,P<0.01）.2.Results of cell biology experiments:When compared to the control group,Foxp3protein expression was raised in the TGF-β1-induced group,1,25（OH）₂D_3,and ICG-001intervention treatments（P<0.05）.Foxp3 protein expression was higher in the1,25（OH）₂D₃and ICG-001 intervention-treated groups than in the TGF-β1-induced group（P<0.01）.There was no significant difference between the 1,25（OH）₂D₃and ICG-001intervention groups（P>0.05）.The expression of Foxp3,IL-10,and IL-35 was significantly elevated after TGF-β1 treatment of the cells,and the manifestation of Foxp3,IL-10,and IL-35 was significantly enhanced following 1,25（OH）₂D₃or ICG-001intervention treatment（P<0.05）.The difference in intervention therapy groups between1,25（OH）₂D₃and ICG-001 was not statistically significant（P>0.05）.The TGF-1-induced group had moreβ-catenin/Foxo1 binding than the control group（P<0.05）,while the expression ofβ-catenin/TCF binding was virtually unaffected due to TGF-β1’s strong pro-fibrotic impact.Theβ-catenin/TCF binding level was significantly lower in the 1,25（OH）₂D₃and ICG-001 intervention-treated groups compared to the TGF-β1-induced group（P<0.05）,indicating that TCF and Foxo1 compete for binding toβ-catenin,leading to different transcriptional complexes to regulate downstream target genes.The difference in intervention treatment groups between the 1,25（OH）₂D₃and ICG-001 was not statistically significant（P>0.05）.Furthermore,the TF ratio indicator（β-catenin/TCF transcriptional complex versusβ-catenin/Foxo1 transcriptional complex）was used to compare the levels of the transcriptional complexes in each group,and the results demonstrated that the TF ratio indicator trend was opposite to the expression level of the Treg cell-specific marker Foxp3,indicating that Treg cell proliferation activation was closely related to the reduction of the TF ratio level.The results of the dual luciferase reporter gene tests revealed that TGF-β1 induction increased transcription factor Foxo1 activation of the downstream Foxp3 promoter（P<0.05）.The transcription factor Foxo1 activated the downstream Foxp3 promoter more significantly in the1,25（OH）₂D₃and ICG-001 intervention-treated groups than in the TGF-β1-induced group（P<0.05）.The outcomes of the 1,25（OH）₂D₃and ICG-001 intervention groups were comparable,with no statistically significant changes（P>0.05）.Conclusion:1.Vitamin D deficiency and abnormal Treg cell response are involved in the development of asthma,and vitamin D may exert anti-inflammatory effects by promoting Treg cell immunosuppressive function,which in turn may have therapeutic effects on asthma.2.Vitamin D regulated theβ-catenin pathway,which shifted theβ-catenin key transcription factor from TCF-mediated transcription to Foxo1-mediated transcription,blockedβ-catenin/TCF binding,increasedβ-catenin/Foxo1 binding,promoted Foxo1transcriptional activation of the downstream Foxp3 promoter,and increased the expression of the Treg cell-specific marker Foxp3 and anti-inflammatory factors,thereby enhanced the function and activity of Treg cells against inflammation and attenuated the inflammatory immune response.