Effect of Vıtamın B12 on Acetic Acıd Induced Colıtıs

Inammatory bowel disease (IBD) is a chronic, relapsing, and remittent inammatory disease of the gastrointestinal tract. Nutritional deciency may be instrumental and contributable in this disease, among which vitamin B12 deciency has been identied in IBD. Since the relationship between vitamin B12 and IBD remains controversial, in this study, we have examined the effect of vitamin B12 supplementation on acetic acid (AA)-induced colitis in rats.


Introduction
In ammatory bowel disease (IBD), including two major typical forms, ulcerative colitis (UC) and Crohn's disease (CD), is a chronic, relapsing and remitting in ammatory disease of the gastrointestinal (GI) tract.
The relationship between individual genetic factors that regulate the innate and adaptive immune system, enteric microbiota, the enteric immune system, and environmental factors, particularly nutritional factors, are key determinants of IBD pathogenesis, and it has been suggested that an excessive enteric immune response to gut ora or nutritional antigensis involved [1]. Since multiple factors can contribute to in ammation of the intestinal mucosa and nutritional de ciencies may occur due to malabsorption, in ammation, and resection, it has been postulated that nutritional de ciencies may actually evoke or in uence colitis.
For instance, as far as nutritional factor is concerned, there is a lower incidence of IBD in the west of Canada, where more fresh fruit and vegetables are consumed than in the east of Canada, where fewer of these foods are consumed [2]. Furthermore, IBD is traditionally higher in developed countries where the consumption of fresh vegetables and fruit is lower than in developing countries. De ciency of folate and vitamin B12 may be involved in the pathogenesis of IBD, and the consequence of such a de ciency is hyperhomocysteinemia [3]. Homocysteine may aggravate in ammation of the intestinal mucosa via an upregulation of proin ammatory genes. Previous studies have shown that homocysteine modulates TNFα mediated cytotoxicity [4], in addition to inducing the production of in ammatory cytokines and chemokines, such as monocyte chemoattractant protein (MCP-1) and IL-8 [5]. Furthermore, oxidative stress, another determinant of IBD pathogenesis, is provoked in hyperhomocysteinemia. Excess homocysteine is reduced by oxygen radicals and by free sulfhydryl groups that can damage DNA, lipids, and proteins, and may cause mutations [6]. SOD activity is increased in hyperhomocysteinemia [7]. Folate produces epigenetic changes and affects the interaction between gut microbiota and the enteric immune system [8].
Although experimental and cohort studies have shown evidence of an association between the pathogenesis of IBD and vitamin B12 and folate de ciency, the evidence regarding the plasma homocysteine levels of IBD patients vs. those of controls is con icting [9], as is that relating to the comparison of vitamin B12 and folate levels in IBD patients vs. controls [10]. In this background, the present study aimed to examine the effect of vitamin B12 administration on acetic acid (AA)-induced colitis in rats.

Rat animals
A total of 28 male Wistar-Albino rats weighing between 250 and 350 g were included, and were kept in separate cages in groups of two or three, at a constant temperature of 23°C and in a 12-hour light/dark cycle. They were fed a standard diet, and food and water were available ad libitum. All animals were maintained under fasting conditions for 24 h before undergoing surgical procedures, and no antibiotics were given before or after the procedures. Approval for the study protocol was granted by the Experimental Animals Ethics Committee of Gaziosmanpasa University Medical Faculty (2014-HAYDEK-29). All experimental, surgical, and laboratory procedures were applied in Gaziosmanpasa University Medical Faculty Experimental Research Centre and Gaziosmanpasa University Medical Faculty Biochemistry and Histology Laboratories.

Induction of acetic acid (AA)-colitis
The colitis model was induced by inserting a soft 6 mm pediatric-feeding catheter into the anus of each rat, and advancing the tip by 8 cm. One mL of 4% AA (pH 2.3) solution was slowly transrectally injected. In order to spread AA into the colon lumen, 2 mL of air was put into the catheter. Physical trauma was reduced by withdrawing the catheter slowly, and the rats were held upside down by the tail for 30 s to prevent any leakage of the administered substance. The experimental procedures were carried out under general anesthesia via an intramuscular administration of 75 mg/kg ketamine hydrochloride (Ketalar 500 mg acon; P zer, Istanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Rompun 2% acon; Bayer, Istanbul, Turkey).

Experimental group
The total 28 rats were randomized into four groups of seven. Group 1 (Control saline) was the control group, in which the rats received transrectal injections of saline; group 2 (AA colitis control) was the control colitis group, in which AA was administered into the colon of the rats; group 3 (VitB12 alone) was the vitamin B treatment group, in which 1 mg/kg of vitamin B12 was intraperitoneally administered 5 minutes after saline injection, with treatment then continuing for 3 consecutive days; group 4 (VitB12 treatment in AA colitis ) was the AA-induced colitis group in which 1 mg/kg vitamin B12 was intraperitoneally administered 5 minutes after colitis induction, with treatment then continuing for 3 consecutive days. All rats were sacri ced on day 4 after the induction of colitis by cervical decapitation. Before the procedure, 30 mg/kg hydrochloride and 5mg/kg xylazine were administered as anesthesia.
Intracardiac blood samples were drawn via making an incision of the abdomen and accessing the heart through the diaphragm. All blood samples were stored at -80°C until the day of analysis.
Clinical changes after AA administration and macroscopic ndings Body weights were obtained before and after the colitis induction procedure. A new parameter, "weight change" (WC) was obtained according to the formula "body weight after the procedure -body weight before the procedure". Macroscopic assessment of the colon was made after the rats had been sacri ced. The longitudinally removed colon was opened and washed with saline. Mucosal lesions were then macroscopically scored, according to the Morris Scoring System [11], as shown in Table I.

Microscopic changes
Colonic samples were chosen according to the Morris Scoring System. The colon of each rat was macroscopically assessed, and samples of the region with the highest macroscopic score were obtained. These samples were then xed in 10% buffer -neutral formaldehyde solution for 36 h. The xed samples were embedded in para n, and 5 µm sections were obtained by cutting the para n blocks. Hematoxylin and eosin were used for staining after melting the para nized samples. Histologic assessment was carried out by a researcher blinded to the group information. A total of 10 sections from the seven rats in each group were considered, and an average of 20 microscopic views was assessed for each group. These views were analyzed via a computer-assisted light microscope (Nikon Eclipse 200,serial no: T1al 944909, Japan) with an integrated camera (Nikon Ds-Fi1, Japan), and transferred to the monitor for analysis using a Nis element program. In ammation score (IS) was reported by the researcher, using a coding system that evaluates the intensity of in ammation in the colon strata. The four-level grading system is shown in Table II [12].

Biochemical measurement
The blood samples were centrifuged at 4,000 rpm for 10 min at 4°C, and the removed plasma was stored at -80°C. Glutathione (

Statistical analysis
All statistical analyses were conducted by using SPSS (Version 22.0, SPSS Inc., Chicago, IL, USA). Descriptive statistics were presented as mean ± standard deviation and median (min-max). Normality distributions of the data were assessed by Shapiro-Wilk test. The signi cance of the difference between two paired groups was evaluated using Wilcoxon signed rank test. The signi cances of the difference between more than two groups were evaluated by using Kruskal-Wallis Test (non-parametric analysis of variance) since data did not meet the assumptions of a parametric Analysis of variance (ANOVA) test. Post-hoc test conducted after Kruskal-Wallis test in order to determine signi cant differences among the multiple groups with pairwise comparison. P value < 0.05 was considered statistically signi cant.

Results
The effect of vitamin B12 on AA-induced colitis The body weights of the rats in all four groups were measured pre-experiment and post-experiment. The Control saline group rats gained weight after saline injection, while the AA colitis control group rats lost weight following AA injection; the difference was signi cant in both groups (p = 0.028; p = 0.018, respectively) (Table III, Fig. 1). There was no signi cant weight change in the other two groups. The new WC parameter was obtained by subtracting the post-experiment weight from the pre-experiment weight. A post hoc analysis showed that the WC differences between the AA colitis control and Control saline, AA colitis control and VitB12 alone, Control saline and VitB12 treatment in AA colitis groups were signi cant (p = 0.002, p = 0.027, and p = 0.03, respectively). There was no difference between the AA colitis control and VitB12 treatment in AA colitis groups (Table III, Fig. 1). Post hoc analyses revealed signi cantly different mean macroscopic scores between the control saline group vs. the VitB12 treatment in AA colitis group and the control saline groups vs. the AA colitis control and VitB12 alone and AA colitis control groups (p = 0.032, p < 0.001, and p = 0.001, respectively) (  Fig. 2). Post hoc analysis showed mean differences in IS between the VitB12 treatment in AA colitis and VitB12 alone groups, the AA colitis control and VitB12 alone groups, and the AA colitis control and control saline groups (p = 0.016, p < 0.001, and p = 0.005, respectively), as shown in Table V (Fig. 3). Hematoxylin and eosin stained para n sections of four groups can be seen Fig. 4. AA colitis control group has the greatest intensity of in ammatory cells. Control saline group appears to be normal colon tissue. The intensity of in ammation treatment decreased with VitB12 treatment in VitB12 alone and VitB12 treatment in AA colitis group but the only difference between VitB12 alone and AA colitis control is signi cant. There is no signi cant difference between AA and VitB12 treatment in AA colitis groups.
Changes of biochemical measurement after vitamin B12 administration in AA-induced colitis All biochemical parameters and mean values are shown in Table VI. There was no difference between groups in the mean of MPO (p = 0.095) (Table VI, Fig. 5). Different mean IL-1β values were observed between the VitB12 treatment in AA colitis and control saline groups, and the VitB12 treatment in AA colitis and VitB12 alone groups (p = 0.006, and p = 0.001, respectively), as shown in Table VI. Signi cantly different mean plasma IL-6 values were shown between the VitB12 treatment in AA colitis and control saline groups, the VitB12 treatment in AA colitis and the AA colitis control groups, and the VitB12 treatment in AA colitis and VitB12 alone groups (p = 0.048, p = 0.023, and p = 0.009, respectively), as shown in Table VI. Signi cant differences in the mean serum TNF-α values between the VitB12 treatment in AA colitis and VitB12 alone groups and the control saline and VitB12 treatment in AA colitis groups (p = 0.025 and p = 0.021, respectively) were observed, as shown in Table VI. There was a signi cant difference between the VitB12 treatment in AA colitis and VitB12 alone groups (p = 0.001) in mean SOD values (Table VI). Post hoc analysis revealed a signi cant difference in the mean GSH between the VitB12 treatment in AA colitis and VitB12 alone groups, and the VitB12 treatment in AA colitis and AA colitis control groups (p = 0.028 and p = 0.003, respectively, as shown in Table VI. Signi cantly different mean serum MDA levels between the VitB12 treatment in AA colitis and VitB12 alone groups and the AA colitis control and VitB12 alone groups were observed (p = 0.002 and p = 0.010, respectively), as shown in Table VI.

Discussion
In the present study, we evaluated the effect of vitamin B12 administration on a rat model of AA-induced colitis under the hypothesis that vitamin B12 supplementation is essential in the prevention of IBD. As results, VitB12 treatment in AA colitis group's values of IL-6, TNF-α, MDA, IL-1β, SOD, GSH were lower than AA colitis control group, but only IL-6 and GSH parameters reached the signi cant level. Furthermore,in ammation score and a macroscopic score of VitB12 treatment in AA colitis group were also lower than AA colitis control group, but the difference was not statistically signi cant. Few studies have investigated the association between vitamin B12 and colitis pathogenesis, and, to the best of our knowledge, the present study is the rst and only experimental model that examined the effect of vitamin B12 supplementation modulates in ammation in a colitis rat model. Our results signi ed that although vitamin B12 can in uence colitis, their in uences seemed to marginal and supplementary.
Clinical trials that aimed to nd an association between the pathogenesis of IBD and vitamin B status have also been published, as has an observational prospective trial that investigated the effect of Vitamin B on the course of IBD. The prevalence of hyperhomocysteinemia and the effect of hyperhomocysteinemia on disease activity were assessed via longitudinal follow-up of patients with UC and CD, there was no association between homocysteine levels and disease activity. Instead, the prevalence of hyperhomocysteinemia was higher than in a normal population, although there was no control arm [13]. Retrospective studies have reported a higher prevalence of hyperhomocysteinemia. Erzin et al. [14] reported that 56% of IBD patients, and Peyrin-Biroulet et al. [15] stated that 26% of CD patients have elevated homocysteine levels. Hyperhomocysteinemia can be a result of impaired vitamin absorption due to in ammation, de ciency resulting from a poor diet, and due to a resected or diseased ileum in the CD. Another retrospective study [16] revealed that people with CD had a higher prevalence of vitamin B12 de ciency than did those with UC and a control group. People with prior ileal or ileocolic resection have a particularly higher prevalence. There was no difference between the UC and control groups in this study, a nding that may be interpreted as vitamin B12 de ciency being a result of resection rather than intestinal in ammation. Another study reiterated these results. In a Swiss cohort, only CD patients with stenosis or intestinal surgery had vitamin B12 de ciency [16].
As the studies that assessed the serum folate and vitamin B12 levels of people with IBD patients have produced inconsistent results, meta-analyses have been conducted. The rst meta-analysis, determined that people with CD have signi cantly higher levels of plasma homocysteine than do controls. There was no difference between UC and CD patients [17]. A recently published meta-analysis compared serum folate and vitamin B12 levels in people with IBD and healthy individuals. Interestingly, it found no difference in the mean of vitamin B12, but the folate levels did differ; people with UC patients had signi cantly lower serum folate levels than controls, but people with CD did not have different levels of folate from the control group [10]. The con icting results may be due to the methodology used; one of these meta-analyses measured plasma homocysteine levels, while the other considered vitamin B12 and folate levels.
Although resection due to CD or intestinal in ammation can alter the absorption of vitamin B12 or folate, IBD can be a consequence of nutritional de ciency. Homocysteine is a sulfur-containing amino acid that plays a role in two major pathways: remethylation to methionine, which requires folate and vitamin B12, and transsulfuration to cystathionine, which requires vitamin B6. Irrespective of whether it is due to a methyl-de cient diet vitamin B de cient diet, or to genetic defects of enzymes that are involved in homocysteine metabolism (e.g., methyl tetra hydro folatereductase), hyperhomocysteinemia is known as one of the etiologic factors of thrombotic events [18]. It causes vascular in ammation via several mechanisms, one of which is increased production of reactive oxygen species. Similar activation takes place in the intestinal mucosa. Hyperhomocysteinemia promotes microvascular in ammation with aggravating endothelial in ammation, resulting in vascular cell adhesion molecule − 1 upregulation, MCP-1 production, and p38 phosphorylation [19]. This is thought to promote mucosal in ammation and activate several in ammatory and oxidative stress pathways [20].
Antioxidant mechanisms are essential for protecting the colonic mucosa from the harmful effect of in ammation [21]. Increasing antioxidant defense mechanisms in the colon mucosa, via pharmacologic therapies, may be bene cial in IBD treatment. The present study is the rst to investigate vitamin B12 as an antioxidant therapy for IBD, although many antioxidant compounds have yielded promising results as such treatment [22]. We expected that vitamin B12 supplementation will decrease the level of homocysteine that modulates in ammation on intestinal mucosa. Our study demonstrated that vitamin B12 supplementation decrease level of IL-6 and GSH in an AA-induced colitis rat model. However, the same results are not observed with all in ammation and oxidative stress biomarkers. Furthermore IS and macroscopic score difference between AA colitis control and VitB12 treatment in AA colitis group were not signi cant. There are not experimental colitis models that used vitamin B12 supplementation as an antioxidant therapy which we can compare with the present study. One experimental colitis model demonstrated that folate supplementation downregulated homocysteine-induced IL-17 and ROR-γt expression in dextran sulfate sodium (DSS) induced colitis rats that were fed a methionine-de cient diet [23]. Homocysteine activated the p38/cPLA2/COX2/PGE2 pathway and also increased the expression of IL-17 and retinoid-related orphan nuclear receptor-γt (ROR-γt), which is the key transcription factor of IL-17. T helper 17 (Th17) cells, which derive from CD4 + T-cells, play an important role, and an imbalance between T regulatory cells and Th17 appears to be critical for the development of IBD [24,25]. Although many cytokines play a role in IBD pathogenesis, some studies have suggested that IL-17, which is the cytokine of Th17, contributes to pathogenesis [26]. The present study evaluated the values of nonspeci c in ammatory markers such as IL-6, IL-1β etc. Maybe evaluating speci c markers of intestinal mucosa such as IL-17 on an experimental colitis model that use antioxidant supplementation will be more bene cial. Furthermore, we used standard diet,not a methyl-de cient diet.
One experimental study on colitis evaluated the role of vitamin B on colitis sequela. In this study, we used dietary vitamin B de ciency and then created an experimental colitis model with DSS infusion. The disease progression and severity were determined by macroscopic changes and in ammation markers. Furthermore, this study determined the in vivo kinetics of the methionine pathway by measuring vitamin B6, vitamin B12, homocysteine and other metabolite levels in colon, plasma, and liver of mice. Interestingly, mortality rates, disease activity index and in ammation markers of mice group that were fed on a control diet were higher when compared with the mice that were fed on a vitamin B de cient diet. This study showed that homocysteine levels were higher in the plasma and colon of the de cient diet mice groups regardless of DSS status. Furthermore, vitamin B6 levels were determined indirectly by calculating its active form of pyridoxalphosphate (PLP). PLP levels were higher in the de cient diet mice groups. This study measured B12 associated metabolites to assess Vitamin B12 de ciency.
Methylmalonic acid (MMA) is converted by an enzymatic reaction and requires VitB12 as a cofactor. MMA levels were higher in plasma when compared between the de cient diet mice and the control diet mice groups. Further, this study indicated that remethylation and glycine levels were increased in the de cient mice group. These results indicated that the diet can overcome VitB6 de ciency but not that of VitB12. Folate was included in the diet so homocysteine level was increased due to the de ciency of vitamin B6 and not vitamin B12. Furthermore, this increase did not contribute to the disease progression or in ammation in DSS induced colitis model [27].
Experimental rodent IBD model (IL-10 knockout mice) was used and the effects of vitamin B6 inadequacy and supplementation were monitored on the severity of intestinal in ammation. Mice were randomly assigned to vitamin B6 de cient, replete, and supplementation group. The de cient and supplementation groups both had lower concentrations of molecular and histological markers of in ammation of colon than the replete group. The lower concentrations of histologic and molecular de ciency markers were in accordance with the study conducted by Benignt et al.; however, this study showed that vitamin B6 supplementation ameliorates intestinal in ammation [28]. The results reported by Benignt et al. and us are against the hypothesis of homocysteine contribution due to the nutritional de ciency of colitis pathogenesis and a decrease in its level with nutritional supplementation. Gut microbiota is associated with the pathogenesis of IBD. VitB12 producing bacteria may increase in case of acute intestinal in ammation and intestinal mucosa may absorb more VitB12 but supplementation of VitB12 may not ameliorate intestinal in ammation. We did not measure the level of VitB12 or its metabolites in colon and plasma. Furthermore, acetic acid-induced colitis model represents acute colitis rather than a chronic relapsing and remittent condition so we did not know the effect of vitamin B12 on a chronic colitis model. The effect of vitamin B6 de ciency and supplementation is associated with colitis rather than VitB12.

Conclusion
We assessed the effect of vitamin B12 on AA-induced colitis and expected to see an anti-in ammatory bene t of giving vitamin B12 as a drug. Our study demonstrated the bene t of vitamin B12 via the mean of in ammatory markers, such as IL-6, and the indirect oxidative stress marker GSH, but other biomarkers did not change. This may be explained by the fact that a methyl or vitamin B12 or/and B6 de ciency in the diet can exert a greater in uence on in ammation than taking vitamin B12. This nutritional de ciency can in uence intestinal in ammation more than taking extra antioxidants. Moreover, trials that use more speci c markers for intestinal in ammation, such as IL-17, may be more informative in this research eld.
In conclusion, the use of vitamin B compounds to provide an anti-in ammatory effect in colitis requires further research in animal models and in clinical trials.

Declarations
Authorship Policy: Seyma ve Zeki Ozsoy contributed to the study concept and design and the data acquisition. Abdullah Özgur Yeniova contributed to the statistical analysis and data analysis and interpretation. Fikret Gevrek contributed to the technical or material support.

Figure 2
Photomicrographs of H&E stained para n sections of the colonic tissues of rats.

Figure 3
Boxplot for macroscopic nding according to rat groups.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Tables.pdf