Hydroxylation Of Vitamin D In Ulcerative Colitis Model Cells And Its Effect On Tight Junctions

Objective:1.To investigate the differences in disease activity,pro-inflammatory cytokines and oxidative stress parameters of patients with ulcerative colitis with different serum vitamin D levels.2.To explore the expression of vitamin D hydroxylase,the hydroxylation of vitamin D,and the influence of vitamin D on tight junctions in inflammatory colorectal cancer cells.Methods:Part Ⅰ:According to the inclusion and exclusion criteria,fifty patients with UC were recruited from the First Affiliated Hospital of Shanxi Medical University and Coal Group General Hospital between December 2016 and January 2019.Fasting venous blood samples was collected.Some of them were sent to community hospitals for blood routine examination,albumin,total protein and erythrocytic sedimentation rate to calculate UCAI score.Other blood was centrifuged and stored at-80℃for detection of serological indexes.25（OH）D₃and inflammatory cytokines were detected by ELISA.Oxidative stress parameters were detected by biochemical kits.According to serum 25（OH）D₃,patients with ulcerative colitis were divided into non-VD deficiency group（≥20 ng/ml）and VD deficiency group（<20 ng/ml）.Then we compared the differences of disease activity,inflammatory cytokines and oxidative stress parameters between the two groups.Part Ⅱ:Human colon cancer cells（SW480,Caco2）were divided into normal group（Control）,LPS group,25（OH）D₃group,25（OH）D₃+LPS group,1,25（OH）₂D₃group,1,25（OH）₂D₃+LPS group.According to the set intervention conditions,SW480 and Caco2 in each group were cultured for 24 hours.The expressions of hydroxylase（CYP27B1 and CYP24A1）,pro-inflammatory cytokines（TNF-αand IL-6）,tight junction proteins（Occludin and ZO-1）were detected by Western blot.The expression of vitamin D hydroxylase（CYP27B1 and CYP24A1）was detected by immunofluorescence.1,25（OH）₂D₃was detected by ELISA.Cell apoptosis was detected by flow cytometry.Results:Part Ⅰ:There were no significant difference in baseline characteristics between non-VD deficiency group and VD deficiency group.ELISA result showed that VD deficiency was common in patients with ulcerative colitis,and only 22.0%of the 50patients with ulcerative colitis had sufficient 25（OH）D3（30-100 ng/ml）.Compared with the non-VD deficiency group,the disease activity index was higher in the VD deficiency group（P>0.05）.Levels of pro-inflammatory cytokines TNF-αand IL-1βin non-VD deficiency group were lower than those in VD-deficient group（P<0.05）,there were no significant difference in CRP,IL-6,IL-17 and IL-23 between the two groups（P>0.05）.Levels of oxidative stress parameters MPO and SOD in the non-VD deficiency group were higher than those in the VD deficiency group（P<0.05）,while there were no significant difference in MDA and GSH-Px between the two groups（P>0.05）.Part Ⅱ:SW480 and Ca CO2 cells were used as subjects,and LPS was used to establish an in vitro model of inflammatory bowel disease.This study showed that vitamin D hydroxylase CYP27B1 and CYP24A1 were expressed in two types of colon cancer cells,and there were no significant difference in expression of CYP27B1 and CYP24A1 protein between LPS group and control group;compared with LPS group,the expression level of CYP27B1 protein was decreased in 25（OH）D₃and 25（OH）D₃+LPS groups;there were also no significant difference in 1,25（OH）₂D₃between 25（OH）D₃and25（OH）D₃+LPS group.The expression level of inflammatory cytokines（TNF-αand IL-1β）protein and cell apoptosis in LPS group were higher than in other groups,while the level of tight junction proteins（Occludin and ZO-1）in LPS group were lower than in other groups.The level of inflammatory cytokines（TNF-αand IL-1β）and cell apoptosis in 1,25（OH）₂D₃+LPS group were lower than in LPS group,while the expression level of tight junction proteins（Occludin and ZO-1）in 1,25（OH）₂D₃+LPS group were higher than in LPS group.Conclusion:Vitamin D hydroxylases CYP27B1 and CYP24A1 were expressed in colon.Inflammatory cytokines may not affect 25（OH）D hydroxylase,but sufficient VD may decrease the expression of CYP27B1.1,25（OH）₂D₃could reduce disease activity,promote the expression of closed tight junction proteins,and inhibit the pro-inflammatory cytokines and cell apoptosis that could destroy tight junction.