The Role And Mechanism Of Vitamin D In Preventing The Recurrence Of Ulcerative Colitis

Ulcerative colitis (UC) is a group of chronic intestinal inflammatory disease, the etiology of which is still unknown. The natural history of UC is characterized by chronic relapsing and remitting episodes. Currently, the goal of the available treatments is only to induce and to maintain remission of symptoms and mucosal inflammation. Therefore, it is an urgent task to find an effective approach to maintain remission of ulcerative colitis.Recent understanding of UC is a result of a genetic predisposition to environmental triggers. The onset and reactivation of UC are triggered by environmental factors that transiently break the impaired mucosal barrier, which make pathogenic enteric bacteria can not be eliminated timely, leading to pathogenic chronic intestinal inflammation. The intestines are situated in a complex environment of bacteria, and prior to the onset, where exist an increased mucosal permeability in UC patients. The weakening of intestinal barrier is probably the initiating factor in the relapse of UC. Therefore, to remove the surface bacteria adhesion to the intestinal wall might be the key to the prevention of UC recurrence.Antibiotic peptides, which have been demonstrated with activity against a wide range of microorganisms including bacteria, protozoa, yeast, fungi, viruses and even tumor cells,contribute to the maintenance of intestinal mucosal barrier of bacterial invasion. Human defensins are the most important antibiotic peptide in gut, playing a crucial role in controlling pathogen invasion in IBD. It mainly expresses Humanβ-defensin 1 (HBD-1) and humanβ-defensin 2 (HBD-2) in colonic tissue.It has been proved that active vitamin D, 1,25(OH) 2D3, regulated the expression and activation of antibiotic peptides. Vitamin D deficiency is highly prevalent among patients with IBD, irrespective of established or new-onset IBD, or even IBD is well controlled. Compelling data in mice have shown that vitamin D and signaling through the vitamin D receptor (VDR) dictated the outcome of experimental UC, and that vitamin D prevented and ameliorated symptoms of experimental IBD murine. But the required 1,25-(OH) 2D3 concentration for biological effect is far higher than the plasma concentrations of 0.03～0.06 ng/ml,which suggests there may have a form of local activation of vitamin D. 25-Hydroxyvitamin D3 1-alpha -hydroxylase(CYP27B1), the key rate-limiting enzyme of vitamin activation, has been detected in colonby Zehnder D in 2001. We speculated that CYP27B1 may play a key role in the regulation of the expression of human defensins in colonic tissue. Therefore, we conducted the following study:(1) Investigate the expression of CYP27B1, HBD-1 and HBD-2 on the protein and transcription level and to explore the relationship between CYP27B1 and human defensins in UC patients and normal intestinal biopsy specimens; (2) By over-expression strategy, construct recombinant adenovirus vector containing CYP27B1 gene and analysis of expression of HBD1 and HBD2 in vitro; (3) Investigate the efficacy of vitamin D combined with mesalazine in maintaining remission of UC, evaluation of the feasibility of active vitamin D as a maintenance therapy for UC patient in quiescent period. The project contain three parts as below:Part 1: The expression of CYP27B1 in colonic mucosa of UC and the correlation with HBD1 and HBD2Objective: To investigate the expression of CYP27B1, HBD1 and HBD2 on the protein and transcription level in colonic mucosa of UC, and to explore the correlation between CYP27B1 and human defensins.Methods: The UC patients were final diagnosed according to clinical data, biochemical indicator and endoscopic characteristics. The expressions of CYP27B1, HBD1 and HBD2 in colon mucosa of UC patients and healthy people were determined by immunohistochemical staining. Then, the mRNA and protein expression level of CYP27B1, HBD1 and HBD2 were assessed by real-time fluorescent qunatitative reverse transcription-polymerase chain reaction (real-time Q-PCR) and Western blot, respectively. The groups were as follows: contorl group: normal colon mucosa; uninflammatory group: noninflammatory colon mucosa of UC; infl group: inflammatory colon mucosa of UC.Results: (1) A total of 35 patients (19 men and 16 women)were collected and divided into mild(8), moderate(18) and heavy(9) group; (2) The results of immunohitochemical assay of colonic mucosa showed that CYP27B1 protein located in the membrane of epithelium, near the of cavity surface, and in the nuclears of Inflammatory cells, respectively, HBD-1 was expressed in surface and crypt epithelium in uninflamed and inflamed colon and in crypt. HBD-2 protein expressed abundantly in the cytoplasm of epithelium with inflammation, and nucleus of Inflammatory cells, respectively. CYP27B1 expressed in the three groups, and the expression in inflammatory group was significantly higher than the control group and uninflammatory group (P<0.05). The expression of HBD1 in control group was significantly higher than that of uninflammation group and inflammatory group (P<0.05, P<0.01, respectively). The expression of HBD2 in inflammatory group was conspicuous and higer than that in contorl group and uninflammatory group in which there were almost no expression. Pearson's correlation analysis showed that the expression of CYP27B1 in inflammatory mucosa was positively correlated with HBD2; (3) The data measured by real time PCR showed the mRNA expressions of HBD1 in uninflammatory group and inflammatory group were significantly lower than that in Con group (P<0.01). Meanwhile, mRNA expressions of HBD2 and CYP27B1 in inflammatory group were significantly higher than that in control group (P<0.01); (4) The data measured by western blot showed the protein expressions of HBD1 in uninflammatory group and inflammatory group were significantly lower than that in control group (P<0.05, P<0.01, respectively). Meanwhile, protein expressions of HBD2 and CYP27B1 in inflammatory group were significantly higher than that in control group (P<0.01, P<0.05, respectively).Conclusions: (1) There was significant difference in the expression of CYP27B1, HBD-1 and HBD-2 between normal colon mucosa and colon mucosa of UC. The level of HBD-1 was lower in noninflammatory and inflammatory colon mucosa of UC than in normal colon mucosa. On the contrary, the level of CYP27B1 and HBD-2 were higher in noninflammatory and inflammatory colon mucosa of UC. The results suggested that CYP27B1, HBD1 and HBD2 may be involved in the pathogenetic procedure of UC; (2) The expression of CYP27B1 in inflammatory colon mucosa was positively correlated with HBD2. The correlation between HBD-1and CYP27B1 need to be further proved.Part 2: Construction of CYP27B1 recombinant adenovirus vector and its effexts on expression of defensins in Caco-2 cellsObjective: To investigate regulatory effects of over-expression of CYP27B1 on expression of HBD-1and HBD-2 in Caco-2 cells.Methods: (1) To construct the CYP27B1 adenovirus vector, genomic fragment containing CYP27B1 was generated by RT-PCR using total RNA from Caco-2 cells. The cDNA was first cloned into pTA2 vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-CYP27B1. Subsequently, CYP27B1 was constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. The positive recombinant pAdEasy-1 containing CYP27B1 gene was linearized by PacⅠand transfected into 293 cells to package recombinant adenovirus. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein(Ad-GFP)was used as control. (2) To confirm the recombinant adenovirus particles containing CYP27B1 gene, The expression of CYP27B1 in infected Caco-2 cells was measured by western blot and real-time PCR.③To gain the over-expression of CYP27B1 in Caco-2 cells, the cells were infected by the recombinant adenovirus particles containing CYP27B1. HBD1 and HBD2 expression in Caco-2 was measured by western blot and real-time PCR. Cells were grouped as follows: (1)control group, cells were cultured in DMEM medium containing 10% FBS; (2)Ad-GFP group, Caco-2 cells were infected with adenovirus expressing green fluorescent protein alone; and (3)Ad- CYP27B1 group, Caco-2 cells were infected with adenovirus harboring genes CYP27B1 and GFP.Results: (1) Construction of recombinant adenovirus vector containing CYP27B1 gene: The size of the amplified PCR product was 1527bp. The results of sequencing were consistent with those from GenBank. CYP27B1 gene was subcloned into adenoviral shuttle vector pAdTrack-CMV. Then, CYP27B1 was successfully constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. Recombinant adenovirus containing CYP27B1 were obtained by transfecting into 293 cells. The adenovirus virus titer was about 1011U/mL; (2) The expression of CYP27B1 in Caco-2 cells was detected after recombinant adenovirus particles infected cells for 48 hours. The mRNA expression of CYP27B1 in Caco-2 cells was measured by real time Q-PCR. The relative values of CYP27B1 mRNA were calculated and compared with untreated control group(The relative value in con group was assigned to 1 ). Obviously, the mRNA expression of CYP27B1 in the Ad-CYP27B1 group was significantly higher than that in the control group (P < 0.01). The result of western blot analysis showed that protein expression levels of CYP27B1 in Ad-CYP27B1 group were significantly higher than that in control group (P<0.01). No significant differences were observed in the mRNA and protein expressions of CYP27B1 between control group and Ad-GFP group (P > 0.05). Overall, the cells were successfully infected by the recombinant adenovirus particles containing CYP27B1; (3) Infected Caco-2 cells were observed by fluorescent microscope after the cells were infected by recombinant adenovirus. The transfection efficiency was about 48% at 24h post-transfection and 80% at 48h post-transfection; (4) To evaluate the effect of CYP27B1 on induction expression of HBD-1 and HBD-2 in Caco-2 cells, the expressions of HBD-1 and HBD-2 in Caco-2 were detected by real-time PCR and western blot after Ad-CYP27B1 infected the cells post 48 h. The results showed that the mRNA and protein expressions of HBD-1 in Ad-CYP27B1 group were significantly higher than that in control group (P < 0.05). The mRNA and protein expressions of HBD-1 in Ad-CYP27B1 group were also significantly higher than that in control group (P<0.01, P<0.05). Additionally, there were no significant differences in the mRNA and protein expression of HBD1 and HBD2 between Ad-GFP group and control group (P>0.05).Conclusions: CYP27B1 can improve the expression of HBD-1 and HBD-2 in the Caco-2 cells, and promote the antibacterial activity of colonic mucosa, and further protect the colon mucosa barrier. Part 3: Efficacy of Vitamin D in Maintaining Remission of Ulcerative ColitisObjective: At present there is no satisfactory remission maintenance therapy for Ulcerative colitis (UC). Evidence indicated that there existed a correlation between incidence and vitamin D deficiency. The goal of this work was to evaluate the efficacy of vitamin D combined with mesalazine in maintaining remission of UC.Methods: A total of 52 eligible patients were enrolled in a single-blind prospective randomized controlled observation. Patients were randomized to receive either mesalazine 1500 mg/d alone (MSL group, n=26) or combination with Rocaltrol 0.25μg/d (V+M group, n=26). The endpoint of the study was defined as the incidence of relapse.Results: For frequent relapse patients, although relapse rates were similar between the V+M group (69.2%) and the MSL group (83.3%; P=0.409), patients in the V+M group were found to relapse later than patients in the MSL group (Log-rank P=0.039) by comparing the duration of remission. The differences in maintaining remission time for these two groups are statistically significant (224±39 vs 134±27, respectively; P=0.026)Conclusions: The efficacy of combined treatment with vitamin D and mesalazine in maintaining remission of UC is superior to treatment with mesalazine alone, and also can prolong the remission time and improve the cumulative remission rates for frequent relapse patients.