Vitamin D3 Targeted Regulation Of VDR-NLRP6 Signaling Pathway Improves Ulcerative Colitis:Effects And Mechanism Study

Objective:Ulcerative colitis（UC）is a chronic,nonspecific inflammatory bowel disease.Patients with UC often experience vitamin D3（VD3）deficiency due to long-term diarrhea.Supplementation of VD3 helps to improve the imbalance of trace elements in UC patients and plays an adjuvant therapeutic role.However,more research is needed on the mechanism by which VD3 improves UC.Therefore,the main objectives of this study are:1)to clarify the role of VD3 in alleviating UC through clinical samples,colitis mouse models,and in vitro cell models;2)to explore the molecular mechanisms by which VD3 alleviates UC in vivo and in vitro models,and to validate these findings in clinical samples.Methods:1)Fifteen patients with ulcerative colitis（UC group）and fifteen patients undergoing colon polyp resection（control group）who were treated at the First Affiliated Hospital of Xinjiang Medical University from June 2022 to February2023 were randomly enrolled.General information,laboratory examinations,serum samples,and colon tissue samples of the patients were collected.Experimental indicators of the two groups were compared;histopathological changes in intestinal tissues of UC patients were observed by H&E staining and electron microscopy,and the levels of inflammatory factors in colon tissues of UC patients were detected using ELISA.The Vitamin D receptor（VDR）expression levels in patients in the control group and UC patients were determined by immunohistochemistry,WB,and immunofluorescence experiments.The correlation between VDR expression levels and clinical indicators of UC patients was analyzed.2)WB and q RT-PCR were used to detect the expression levels of VDR in colon tissues of colitis mice and control group mice.3)Colitis mouse models were induced by administering 3%dextran sulfate sodium（DSS）,and the treatment group was simultaneously given VD3（100ug/kg）by intraperitoneal injection.The micewereadministered with thedrug consecutivelyforseven days.During theexperiment,the mice’s general condition,food and water intake were observed daily,and changes in body weight,stool characteristics,and presence of bloody stools were monitored;In mice,intestinal permeability measurements were performed using FITC-dextran,and the levels of intestinal tight junction molecules were determined.;H&E staining and electron microscopy were used to observe the pathological changes in the intestines of the mice;q RT-PCR was used to detect the levels of colonic inflammatory factors（IL-1β,TNF-α,IL-18）in mice.4)16s RNA sequencing was used to detect the abundance and diversity of intestinal flora in each group of mice.5)Transcriptomic sequencing was used to detect differentially expressed genes and enriched pathways in the colon of mice from each group,and validation was performed on samples from in vivo and in vitro models using WB and q RT-PCR.6)ATAC-seq was used to map the entire genome chromatin,especially genes related to the vitamin metabolic pathway.7)nlrp6^-/-mice were constructed,and the expression levels of VDR,NLRP6,ASC,and Caspase-1 in wild-type and nlrp6^-/-mice treated with DSS and VD3 were detected by WB and q RT-PCR.8)A lentiviral NLRP6 knockdown cell line was established in MIEC cells,and WB and q RT-PCR detected the expression levels of VDR,NLRP6,ASC,and Caspase-1.9)Protein-protein interaction network analysis of the NLRP6-（Caspase-1）/IL-1b signaling pathway was performed using STRING.Results:Part 1:1)The white blood cell count,neutrophil count,erythrocyte sedimentation rate,C-reactive protein,procalcitonin,and fecal positive rate of UC patients were all higher than those of the control group（P<0.001）,while the serum levels of VD3 and albumin in the UC group were lower than those in the control group（P<0.001）;2)Through HE staining and electron microscopy,atrophy or even disappearance of the glandular mucosal layer in UC patients was observed,and a decrease in the number and blurring of mitochondria,endoplasmic reticulum,lysosomes,and cell nuclei of colon epithelial cells in UC patients was noted.q RT-PCR detected a significant increase in IL-1βand IL-18 levels in colon tissues of UC patients compared to normal individuals（P<0.01,P<0.001）;3)Immunohistochemistry,WB,and immunofluorescence experiments revealed a significant decrease in VDR expression in colon tissues of UC patients（P<0.01）;4)Correlation analysis found that the expression level of VDR was positively correlated with serum VD3 levels in UC patients,and negatively correlated with white blood cell count,erythrocyte sedimentation rate,and CRP levels;5)WB and q RT-PCR tests found a significant decrease in VDR expression levels in the intestinal tissues of colitis model mice.Part 2:1)Compared to the control group,the rate of change in body weight of UC group mice was significantly lower（P<0.01）,and the rate of change in body weight of the VD3 group also decreased but was higher than that of the UC group（P<0.01）.The disease activity index（DAI）of the UC group was significantly higher（P<0.01）,while the DAI score of the VD3 group decreased compared to the UC group（P<0.01）;2)The colon length of UC group mice was significantly shorter than that of the control group（P<0.001）,while the colon length of VD3 group mice was higher than that of the UC group（P<0.05）;3)Measurement of intestinal permeability in mice using FITC-labeled dextran（FITC-dextran）and determination of the levels of intestinal tight junction molecules revealed that compared to the control group,the intestinal permeability of mice in the UC group was significantly higher than that of the control group（P<0.01）.The VD3 group decreased intestinal permeability compared to the UC group（P<0.05）;4)HE staining and electron microscopy showed disrupted intestinal mucosa in UC group mice,with extensive damage to epithelial cells,while the pathological damage in the intestines of VD3 group mice was significantly reduced.q RT-PCR detected a significant increase in colonic inflammatory factors in UC group mice（P<0.001）,while the inflammatory factors in VD3 group mice decreased compared to the UC group;5)16s RNA sequencing revealed a decrease in the abundance of intestinal flora in DSS group mice,an increase in pathogenic bacteria,and a decrease in beneficial bacteria.Treatment with VD3 alleviated the dysbiosis of intestinal flora;6)Transcriptomic sequencing of mouse colon tissues revealed differences in genes and pathways related to vitamin metabolism.There were 16differentially expressed genes in this pathway,among which VDR was downregulated in UC,while NLRP6,Caspase-1,PYD,and PYCARD were upregulated;7)Immunohistochemistry,WB,and q RT-PCR tests found that after treatment with VD3 in colitis mice,the expression of VDR increased,while the expression of NLRP6,Caspase-1,and ASC decreased.Similar results were obtained in primary mouse intestinal epithelial cells（MIEC cells）in vitro.Part 3:1)ATAC-seq and Ch IP experiments revealed that VDR downregulates the expression of NLRP6 by inhibiting the transcriptional activity of the NLRP6 promoter;2)WB and q RT-PCR tests found that UC induction increased the NLRP6 levels in nlrp6^-/-mice,but VD3 treatment decreased its expression level（P<0.05）.In nlrp6^-/-mice of the UC group and the VD3 treatment group,the expression levels of Caspase-1 and ASC were similar to the expression of NLRP6（P<0.05）;3)WB and q RT-PCR tests found that LPS treatment increased the NLRP6 levels in si RNA-NLRP6 MIECs cells,but VD3 decreased its expression level（P<0.05）.The expression levels in the LPS treatment group and the VD3 treatment group of si RNA-NLRP6 MIECs cells.Discussion:1)In UC patients,serum VD3 levels decrease,inflammatory markers increase,and VDR expression levels decrease.Furthermore,VDR expression is positively correlated with serum VD3 levels and negatively correlated with inflammatory markers.Consistent results were also obtained in DSS-induced colitis mice and IL-1β-induced MIECs cell models;2)It was found in both colitis mice and inflammation cell models that supplementation of VD3 led to a reduction in inflammation-related markers.Additionally,compared to the colitis group of mice,there were significant differences in the expression of VDR,NLRP6,Caspase-1,ACS,PYD,and PYCARD molecules in the VD3 treatment group of mice,indicating that VD3 exerts an anti-inflammatory effect through modulating the VDR-NLRP6 pathway;3)In in vivo and in vitro experiments,it was elucidated that VDR downregulates NLRP6 expression by inhibiting the transcriptional activity of the NLRP6 promoter.