Impact of Cyanocobalamin and Methylcobalamin on Inflammatory

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Food Safety and Toxicology

Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel Disease and the Intestinal Microbiota Composition Xuan Zhu, Shasha Xiang, Xiao Feng, Huanhuan Wang, Shiyi Tian, Yuanyuan Xu, Lihua Shi, Lu Yang, Mian Li, Yubiao Shen, Jie Chen, Yuewen Chen, and Jianzhong Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05730 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Journal of Agricultural and Food Chemistry

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Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel

2

Disease and the Intestinal Microbiota Composition

3 4

Xuan Zhu1#, Shasha Xiang1#, Xiao Feng1, Huanhuan Wang2, Shiyi Tian1, Yuanyuan

5

Xu1, Lihua Shi1, Lu Yang2, Mian Li3, Yubiao Shen4, Jie Chen1, Yuewen Chen1, and

6

Jianzhong Han1*

7 8

1School

9

Hangzhou 310018, China

of Food Science and Bioengineering, Zhejiang Gongshang University,

10

2School

11

3Zhejiang

12

4Yangtze

13

#These

14

*Corresponding

15

Dr. Jianzhong Han, [email protected], School of Food Science and

16

Bioengineering, Zhejiang Gongshang University, No. 18 Xuezheng Str., Hangzhou,

17

Zhejiang Province, 310018, China

of Medicine, Hangzhou Normal University, Hangzhou, 310018, China Huakang Pharmaceutical Co., Ltd., Kaihua, 324302, China Delta Institute of Tsinghua University, Jiaxing, 314000, China

authors contributed equally to the work author:

18 19

Abstract:

20

Patients with inflammatory bowel disease (IBD) are usually advised to supplement

21

various types of vitamin B12, since vitamin B12 is generally absorbed in colon. Thus,

22

in the current study, the influence of cyanocobalamin (CNCBL) or methylcobalamin

23

(MECBL) ingestion on IBD symptoms will be investigated. Then, whether and how

24

the application of various cobalamins would modify the taxonomic and functional

25

composition of the gut microbiome in mice will be examined carefully. Dextran 1

ACS Paragon Plus Environment


26

sulfate sodium-induced IBD mice were treated with MECBL or CNCBL; disease

27

activity index (DAI) scores and intestinal inflammatory condition of mice were

28

evaluated. Faecal samples were collected; microbiota composition was determined

29

with a 16s rRNA analysis, functional profiles were predicted by PICRUSt and

30

short-chain fatty acids were measured. The consequence of higher relative

31

abundances of Enterobacteriaceae and isomeric short chain fatty acids by cobalamins

32

treatment revealed that a high concentration of CNCBL, but not MECBL,

33

supplementation obviously aggravated IBD. A microbial ecosystem rich in

34

Escherichia/Shigella and low in Lactobacillus, Blautia, and Clostridium XVIII was

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observed in IBD mice after a high concentration of CNCBL supplementation. In

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cobalamin-dependent enzymes, CNCBL was more efficient in adenosyl-cobalamin

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system than MECBL, vice versa in MECBL system. The distinct effects of various

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cobalamins were associated with the distribution and efficiency of vitamin

39

B12-dependent riboswitches. CNCBL had a strong inhibitory effect on all

40

riboswitches, especially on btuB and pocR riboswitches from Enterobacteriaceae.

41

CNCBL aggravated IBD via enhancing the proportion of Enterobacteriaceae

42

organisms through riboswitch and enzyme systems. The present study provides a

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critical reference for offering a suitable amount and type of cobalamin during a

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symbiotic condition.

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Keywords: Cyanocobalamin, Methylcobalamin, Gut microbiome, Inflammatory

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bowel disease, Enterobacteriaceae

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2


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Introduction

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Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is

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an uncontrolled chronic and recurring inflammatory response in the gastrointestinal

52

tract that has been linked to mucosal immunity dysregulation and alterations in the

53

intestinal microbiota.1 IBD commonly involves the terminal ileum, which is the major

54

site of vitamin B12 absorption. Vitamin B12 abnormalities are common in IBD patients

55

with a prior ileal or ileocolonic resection.2 Therefore, IBD patients, especially those

56

who are elderly, are usually advised to supplement vitamin B12. Cobalamin (vitamin

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B12), one of the water-soluble vitamins, is the general name for naturally occurring

58

organometallic compounds containing cobalt and works as a co-factor for two

59

important human enzymes. Cobalamin deficiency is believed to be related with

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disturbances in cell division, neuropathy, nervous system disease and pernicious

61

anemia.3 To prevent such fatal diseases caused by cobalamin deficiency, daily intake

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of 2.4 µg cobalamin is recommended.4 Cobalamin includes four bio-active forms. The

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adenosyl group could be replaced by a methyl group, a hydroxyl group, and a cyano

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group

65

Cyanocobalamin (CNCBL) does not exist naturally but nowadays is used as a

66

supplementary nutrient for humans and stocks.

to

form

methyl-,

hydroxo-,

and

cyano-cobalamin,

respectively.

67

Vitamin B12 is the only vitamin produced exclusively by bacteria and archaea,

68

but used by many domains of life. Synthesis of vitamin B12 is energetically costly,

69

requiring nearly 30 different enzymes.5 Only 20% of prokaryotes have the genetic

70

capacity to produce it, such as Aerobacter, Agrobacterium, Alcaligenes, Azotobacter,

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Bacillus,

72

Mycobacterium,

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Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus, and

Clostridium,

Corynebacterium,

Norcardia,

Flavobacterium,

Propionibacterium,

3


Micromonospora,

Protaminobacter,

Proteus,


74

Xanthomonas.5,6 In gut microbial ecology, the limited sources of vitamin B12 are

75

recognized as a precious commodity. As such availability, vitamin B12 has been

76

suggested to impart a fundamental contribution, not only as nutrition but also as signal,

77

to the spatial and functional organization of gut micro-ecology.5,6 However, the IBD

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symptoms of some patients are aggravated after supplementation with different kinds

79

of vitamin B12, as observed in our studies and other investigations.7 Multiple causes

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can be attributed in this deregulation, but many studies have shown that dysbiosis of

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the gut microbiota8 and genes involved in interactions of host-microorganisms are

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linked to IBD9. Arelative abundance of species such E. coli, Ruminococcus gnavus,

83

Alistipes putredinis, and Clostridium difficile were reported to be associated with

84

IBD.10 Meanwhile, both the human and mouse intestinal microbiota are dominated by

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the same members of 15,000 species and only varied between 1-160 species, which

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can be considered to be similar in broad terms (Neill 2010).11 Nevertheless, we lack

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an understanding of how different cobalamins shape the composition of the gut

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microbiome, how cobalamin mediates the growth of Enterobacteriaceae by enzymes

89

or riboswitches, and further, how it influences IBD.

90

Riboswitch is a 5’-untranslated leader sequence of the correspondent mRNA,

91

which regulates translation initiation and gene expression by binding to a specific

92

molecule.12 Vitamin B12 dependent riboswitch maybe act as a regulator during IBD

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development. Expression of cobalamin biosynthetic cob operon and transporter btuB

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gene was repressed by the presence of CNCBL, methylcobalamin (MECBL),

95

andadenosyl-cobalamin (ADCBL).13 It has been reported that ADCBL directly binds

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to riboswitch region leading to a conformational change in the secondary structure of

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mRNA which masks the ribosome-binding site (RBS), and thus inhibiting gene

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expression.14 Degnan et al. demonstrated that 313 genomes of gut microbiota include 4


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809 vitamin B12 riboswitchespredicted to regulate 3,868 genes, most of which are

100

related with vitamin B12 related enzyme, transporter, and isoezyme, however part of

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which have not previously been associated with vitamin B12.6 Consequently, those

102

results suggest that vitamin B12 riboswitches may shape gut microbiota ecology due to

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its wide association with a relative abundance range of vitamin B12-dependent and/or

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-regulated proteins expression.

105

Thus, in the current study we utilized 60 mice to dissect the reasons for IBD

106

aggravation after supplementation with various cobalamins. We experimentally

107

examined whether the application of cobalamin would aggravate IBD and modify the

108

taxonomic and functional composition of the gut microbiome in mice.

109

Materials and Methods

110

Animals

111

Male C57BL/6 mice (10 weeks old) were provided by the Centre of Laboratory

112

Animals, Hangzhou Normal University. Mice were housed under a 12 h dark/light

113

cycle at a controlled room temperature (22 °C) and a relative humidity of 50% and

114

were provided standard laboratory chow. Before the experimental procedures, animals

115

were acclimatized for five days. The Animal Care and Use Committee of the School

116

of Medicine, Hangzhou Normal University, approved the experimental protocols in

117

this study.

118

Induction of colitis and treatment

119

Sixty mice were randomly divided into 10 groups: normal control (NC) group (group

120

1); dextran sulphate sodium (DSS)+saline treated group (group 2); DSS+CNCBL

121

treated group at low (0.0125 mg/L) (group 3), medium (0.125 mg/L) (group 4), and

122

high (1.25 mg/L) (group 5) concentrations; DSS+methylcobalamin (MECBL) treated

123

group at low (0.0125 mg/L) (group 6), medium (0.125 mg/L) (group 7), and high 5



124

(1.25 mg/L) (group 8) concentrations; CNCBL (1.25 mg/L) (Sigma-Aldrich, St.

125

Louis, Missouri, US) treated group (group 9); and MECBL (1.25 mg/L)

126

(Sigma-Aldrich, St. Louis, Missouri, US) treated group (group 10). For induction of

127

acute colitis, 5% w/v DSS (MP Biochemicals, USA) was dissolved in drinking water

128

and administered to mice for 5 days. For cobalamin treatment, certain concentrations

129

of CNCBL or MECBL were prepared in drinking water and administered to mice in

130

the last 3 days together with DSS treatment. Cobalamin is stable under pH value

131

between 5.0 and 8.0. DSS consists of dextran and sulfate units, both of which do not react

132

with cobalamin in those pH conditions. The high concentration of cobalamins was

133

calculated from recommend intake of human being (1.5 mg/day) to dosage for mouse

134

according to body surface. See the details of IBD induction and treatment in S Fig. 1.

135

Mice were sacrificed on day 7, and colon tissues were rapidly removed and measured

136

for length and weight. After washing out the faeces in the colonic lumen with saline,

137

the colons were fixed in 4% neutral paraformaldehyde solution for morphologic

138

evaluation. The remaining portions were stored in liquid nitrogen for further

139

investigation.

140

Evaluation of the disease activity index (DAI)

141

Throughout the experiment, we recorded the following parameters of each mouse

142

daily to determine the DAI according to a previously reported protocol15,16: loss of

143

body weight, stool consistency, and presence of occult or gross bleeding in faeces.

144

The detailed information is listed in S Table 1.

145

Evaluation of the histological score

146

The colon samples were collected and histological scores were evaluated following by

147

previous protocol.17 Briefly, colons were fixed in a 4% phosphate-buffered

148

paraformaldehyde solution for 24 h, dehydrated via a graduated ethanol series and 6


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embedded in paraffin blocks. Colon sections (5 mm) were cut and stained with H&E.

150

Morphological changes in the colons were evaluated by two pathologists who did not

151

know the experimental design.

152

Quantitative polymerase chain reaction (Q-PCR)

153

Colons were ground rapidly in liquid nitrogen. Total RNA was isolated using TRIzol

154

reagent (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol.

155

Total RNA were then converted into cDNA using a High-Capacity cDNA Reverse

156

Transcription (RT) Kit (Takara, Shiga, Japan). The quantification of gene expression

157

was determined by real-time Q-PCR followed by the SYBR Green PCR Master Mix

158

(Roche) standard protocol. The relative expression levels were determined via the

159

ΔΔCt method, using β-actin as an endogenous control. The primers used in the assays

160

are listed in S Table 2.

161

DNA isolation, PCR, and 16S rRNA gene analysis

162

DNA from different samples was extracted using a MicroElute Genomic DNA Kit

163

(D3096-01, Omega, Inc., USA) according to a previously described method with a

164

small modification.18 The total DNA was eluted in 50 µL of elution buffer by a

165

modified version of the procedure described by the manufacturer (QIAGEN), and the

166

samples were stored at -80 °C until PCR amplification (LC-Bio Technology Co., Ltd.,

167

Hang Zhou, Zhejiang Province, China).

168

Bacteria 16S rRNA sequencing genes (V3-V4 regions) were amplified from the

169

whole

genome

of

furu

samples

via

the

primer

170

5'-ACTCCTACGGGAGGCAGCAG-3'

171

5'-GGACTACHVGGGTWTCTAAT-3') according to a previous method with a small

172

modification.18 All reactions were conducted in 25 µL (total volume) mixtures

173

including approximately 25 ng of a genomic DNA extract, 12.5 µL PCR Premix, 2.5

and

7


pair

(319F 806R


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174

µL of each primer, and PCR-grade water to adjust the volume. PCR reactions were

175

performed in a Mastercycler Gradient Thermocycler (Eppendorf, Hamburg, Germany)

176

set to the following conditions: initial denaturation at 98 °C for 30 s; 35 cycles of

177

denaturation at 98 °C for 10 s, annealing at 54/52 °C for 30 s, and extension at 72 °C

178

for 45 s; and the final extension at 72 °C for 10 min.

179

The PCR products were normalized by an AxyPrepTM Mag PCR Normalizer

180

(Axygen Biosciences, Union City, CA, USA), which allowed skipping the

181

quantification step regardless of the PCR volume submitted for sequencing. The

182

amplicon pools were prepared for sequencing with AMPure XT beads (Beckman

183

Coulter Genomics, Danvers, MA, USA), and the size and quantity of the amplicon

184

library were assessed on the LabChip GX (Perkin Elmer, Waltham, MA, USA) and

185

with the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA,

186

USA), respectively. The PhiX Control library (V3) (Illumina) was combined with the

187

amplicon library (expected at 30%). The library was clustered to a density of

188

approximately 570 K/mm2. The libraries were sequenced on the 300PE MiSeq runs,

189

and one library was sequenced with both protocols using the standard Illumina

190

sequencing primers, eliminating the need for a third (or fourth) index read.

191

16S rRNA gene sequences were processed and modified as in previously

192

described methods.19 The reads were controlled and confirmed by QIIME

193

(quantitative

194

http://qiime.org/tutorials/processing_illumina_data.html),

195

(https://github.com/torognes/vsearch, v2.3.4), FASTQC, and FLASH (Fast Length

196

Adjustment of Short reads, v1.2.8, http://ccb.jhu.edu/software/FLASH) quality filters.

197

A Cd-hit method was introduced to select operational taxonomic units (OTUs) by

198

generation of an OUT table.20 When the similarity of OTUs was over 97%, sequences

insights

into

8


microbial

ecology, Vsearch.

Page 9 of 33


199

were assigned to one unit. RDP (Ribosomal Database Project) classifiers were applied

200

to distribute the 16S rRNA gene into distinct taxonomic categories by aligning

201

representative sequences to taxonomically annotated sequences.21 To calculate the

202

alpha diversity, we rarefied the OTU table and calculated two metrics: the Chao1

203

metric, which estimates the richness, and the Shannon index. Principal component

204

analysis (PCA) was carried out by all taxa relative abundances. The heatmap of an

205

important family of the gut microbiome was created by Mev. 4-9-0. A Spearman

206

correlation based on the relative abundances of various genera in bacterial

207

communities of this study was applied. Co-occurrence networks were plotted via

208

Cytoscape 3.4.0 software. When the p value was < 0.1, a correlative connection was

209

plotted between two families. PICRUSt software was used to predict the gene

210

functions

211

(http://huttenhower.sph.harvard.edu/galaxy/).

212

Bacterial culture and manipulation

213

Escherichia coli E4 with a capacity of shiga toxin synthesis was kept at -20 °C at

214

Zhejiang Gongshang University and was grown in LB medium at 37 °C aerobically.

215

Lactobacillus reuteri DSM20058 was kept at -20 °C at Zhejiang Gongshang

216

University and was cultured anaerobically at 37 °C in liquid MRS media. Cells were

217

grown for 48 h in corresponding media, washed, and re-suspended in triplicate at

218

OD600 in vitamin B12 analysis media supplemented with 0.02 mg/L MECBL or 0.02

219

mg/L CNCBL. Triple parallel tests of each strain under various cobalamin

220

supplementations were conducted. OD600 measurements of both strains were

221

internally collected every 2 h for 48 h. The supernatants of E. coli E4 were collected

222

every 6 h from 30 h to 48 h. The shiga toxins were analysed via colorimetric ELISA

223

immunoassay previously described by Gehring et al. with tiny modification.22 All

for

the

OTU

tables

via

9


KEGG

database


224

wells were filled with 200 µL of PBST (PBS containing 0.05% Tween 20),

225

immediately emptied by rapidly inverting the plate. 100 µL supernatants of E. coli E4

226

were added into wells for 30 min. The wells were washed twice with PBST. Then,

227

100 μL of HRP-Antibody conjugate cocktail was added to each well followed by

228

static incubation for 20 min. Wells were washed twice with PBST and then once with

229

PBS. To each well, 50 µL of tetramethylbenzidine and 50 µL of H2O2 were separately

230

added and reacted for 10 min. 50 µL of H2SO4 were added to stop the reaction. The

231

contents of shiga toxin were determined by OD450.

232

Riboswitch sensor comparison and construction

233

Six genera of gut microbiota (S Table 3), including three Gram-negative genera and

234

three Gram-positive genera, were chosen to scan for vitamin B12-dependent

235

riboswitches. We subsampled 6 publicly available human gut microbial genomes23

236

into a custom database that includes a single representative of each species. The

237

methods of searching for riboswitches were reported by Degnan et al.24

238

To construct cobalamin ribosensor switches, the above determined fragments

239

containing an inferred riboswitch and RBS from gut microbiota (S Table 3) were

240

amplified. The fragments containing a riboswitch and RBS were PCR amplified. The

241

amplicon was cloned in p519 ngfp between the pnpt2 promoter and GFP (S Fig. 2)

242

and sequenced as described in a previous work25. For heterologous expression and

243

riboswitch examination, the recombinant plasmids were transformed into an E. coli

244

BL21 (DE3) strain, as described ina previous work25. The decrease in normalized

245

GFP intensity by active cobalamin suggested a positive relationship between vitamin

246

B12 and GFP expression, and vice versa, cobalamin had no ability to turn riboswitches

247

off.

248

Effect of various cobalamins on the vitamin B12-dependent enzyme activity system 10


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249

The method of glycerol dehydrase (GDH) activity determination was as described by

250

Yamada et al., with a small modification (S methods 1).26 The method for

251

determination of cobalamin effects on methionine synthase activities was described

252

by Banerjee et al. (S methods 2).27

253

Statistics

254

All parameters were recorded for individuals within all groups. All data are shown as

255

the mean±SD. The data were analyzed by variance and Duncan’s test for multiple

256

comparisons with SPSS ver. 17.0. A value of p< 0.05 was considered significant.

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Results and Discussions

258

Cobalamin regulated pathogenesis from DSS-induced IBD mice

259

IBD mouse model was established by 5% DSS treatment as shown in S Fig. 1. Over

260

98% mice survived in all 60 experimental mice. During the period of DSS and

261

cobalamin feeding, the parameters of body weight, stool consistency and occult

262

bleeding were observed and recorded every day, based on which DAI scores were

263

calculated. Data from Fig. 1A showed that DSS treatment for 5 days obviously raised

264

the DAI scores in the mice. On the last day (Day 6) of DSS treatment, the mice lost

265

approximately 15-20% of their body weight, excreted loose stools and showed

266

positive occult blood tests. CNCBL treatment for 3 days seemed to have little effect

267

on the colitis symptoms and even aggravated the colitis symptoms of the mice.

268

Surprisingly, MECBL treatment at medium and high levels for 3 days notably

269

reduced DAI scores compared to DSS group and especially improved the stool

270

consistency.

271

The mice were then sacrificed, and colon samples were collected, weighed, and

272

subjected to histopathological examination. We found that DSS treatment could

273

significantly reduce colon length. Although neither CNCBL nor MECBL regulated 11



274

DSS-induced colon length reduction, a high concentration of MECBL may impair

275

colon weight loss against DSS-induced IBD (Fig. 1B). Histological findings were also

276

evaluated at the end of the experiment (Fig. 1C). H&E staining analysis data indicated

277

that the DSS group showed severe mucosal ulceration, leukocyte infiltration, and

278

massive depletion of crypts, goblet cells, and epithelial cells. Interestingly, the high

279

concentration of CNCBL treatment may even worsen the damnification of colon

280

mucosal tissue and lead to even more severe neutrophile infiltration. Moreover, the

281

DSS-induced mucosal lesions were partially attenuated by MECBL as assessed by

282

microscopy.

283

Cobalamin treatment regulated the local inflammatory reaction from

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DSS-induced IBD mice. We then further evaluated the local inflammatory reactions

285

from DSS- and cobalamin-treated mouse colons. Colon tissues were collected and

286

digested to extract total RNA samples. Then, QPCR experiments were performed to

287

measure the transcription level of some inflammatory cytokines, such as IL-1β, IL-6

288

and TNFα (Fig. 1D). The results revealed that the high-concentration CNCBL

289

treatment could obviously aggravate the DSS-treatment-induced local inflammatory

290

reaction, such as transcription of IL-1β and IL-6, while MECBL treatment effectively

291

released the inflammatory reaction and cytokine transcription against DSS-induced

292

colitis.

293

From our results, CNCBL treatment did not seem to ameliorate the colitis

294

symptoms of mice or even aggravate the symptoms, while MECBL treatment notably

295

reduced the DAI scores, especially improving the stool consistency. H&E staining

296

analysis data indicated that CNCBL treatment worsened the damnification of colon

297

mucosal tissue, which were partially attenuated by MECBL treatment. Furthermore,

298

CNCBL could obviously aggravate the DSS treatment-induced local inflammatory 12


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reaction, whereas MECBL treatment effectively relieved the inflammatory reaction

300

and cytokine transcription against DSS-induced colitis. Inconsistent with our study,

301

previous work demonstrated that CNCBL has an anti-inflammatory effect, as they

302

reduced bacteria by antibiotics.28 Thus, we speculate that CNCBL may not directly

303

deteriorate IBD but may act through certain bacteria.

304

Effects of CNCBL and MECBL on gut microbiome in DSS-induced IBD mice

305

We had interest in the changes of gut microbiomes. Comparison of microorganism

306

profiles in DSS-induced IBD with cobalamin supplementation. A total of 60 samples

307

from stools of mice with different concentrations and types of cobalamin were chosen

308

for microorganism profile analyses. After quality-filtering, the 16S rRNA sequencing

309

results produced 426,316 reads, giving a mean sample depth of 14,210.6 reads with a

310

standard deviation of 6897.6 reads. A total of 3614 operational taxonomic units

311

(OTUs) were obtained, and the relative abundance of OTUs showed the genera

312

relative abundances inthe stools of the mice. The raw sequence reads files of 16S

313

rRNA gene amplicon sequencing in FASTAQ format were deposited in the NCBI

314

Sequence Read Archive (SRA) under the Bioproject number PRJNA451085.

315

The alpha diversity index (Chao and Shannon) corresponded to the richness and

316

evenness of the bacterial community in the stools of mice with various treatments.

317

The Chao index showed that DSS supplementation had obviously repressed the

318

bacterial richness in contrast to control and cobalamin supplementation without DSS.

319

MECBL and CNCBL supplementation under DSS treatment did not have a significant

320

effect on the Chao1 index. In Fig. 2 A and B, the Shannon indexes of mice treated

321

with DSS were lower than those in the other mice. Both indices demonstrated that the

322

bacterial diversity and richness were decreased with the supplementation of DSS. The

323

supplementation of cobalamin did not recover the bacterial diversity and richness of 13



324 325

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IBD mice. Bacterial communities from the stools of mice under various treatments were

326

investigated

based

on

OTUs

classified

at

the

phylum,

genus

and

327

Gram-positive/negative levels. The changes in various bacterial phyla are shown in

328

Fig. 2 C. The results showed that the DSS treatment group enhanced the proportion of

329

phylum Firmitus from 45.0% to 77.2%, phylum Proteobacteriafrom 0.6% to 14.7%,

330

and phylum Deferribacteres to 3.0% in the bacterial community and decreased the

331

proportion of phylum Bacteroidetes from 53.6% to 5.0%. The high concentration of

332

CNCBL supplementation under the DSS treatment group enhanced phylum

333

Proteobacteria (10.0%) and Bacteroidetes (57.6%) compared with the low

334

concentration of CNCBL (0.3% and 13.75%). The MECBL treatment group

335

obviously raised the proportion of phylum Firmitus (from 17.64% to 54.0%), phylum

336

Proteobacteria (from 0.3% and 13.8%) and reduced the proportion of phylum

337

Bacteroidetes (from 82.2% back to 45.3%) in contrast to the CNCBL treatment group.

338

In the view of the Gram-positive/negative field, the increased supplementation of

339

CNCBL under treatment with DSS enhanced the ratio of Gram-negative bacteria by

340

up to 68.2%, and vice versa, increasing MECBL repressed the ratio of Gam-negative

341

bacteria.

342

The changes in the bacteria community at the genus level and hierarchical

343

clustering (Fig. 3 A) of the microbiota profiles by a heatmap represented the effects of

344

CNCBL and MECBL on the gut microbiota patterns under IBD situations. The results

345

of hierarchical clustering had the same pattern as DAI scores. The samples taken from

346

the DSS treatment group and the high concentrations of CNBCL under IBD were

347

distinguished from others. The samples taken from mice treated with low

348

concentration of CNCBL or various concentration of MECBL under IBD symptoms 14


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349

showed a similar pattern of microbiome. The control, MECBL, and CNBCL

350

supplementation groups were distributed together. DSS treatment and the high

351

concentrations of CNCBL under IBD stimulated growth of some genera of

352

Ruminococcus (Lachnospiraceae), Clostridium XlVb, Flavonifractor, Clostridium

353

XlVa,

354

Escherichia/Shigella (Fig. 3 B). In contrast, bacteria related to Lactobacillus and

355

genera from Porphyromonadaceae, and Clostridiales were starkly reduced (Fig. 3 B).

356

The group without IBD showed stimulation of the following genera: Lactobacillus,

357

Coprococcus, Ruminococcaceae, Ruminococcus, Lachnospiraceae, Clostridiales,

358

Erysipelotrichaceae, Barnesiella, and Porphyromonadaceae. CNCBL and MECBL

359

with IBD increased the proportion of genus Clostridium with DSS treatment.

360

Increasing CNCBL with IBD and DSS treatment stimulated the proportion of genus

361

Oscillospira and genus Mucispirillum. Moreover, an increase in MECBL with IBD

362

decreased the proportion of these genera. On the other hand, increasing MECBL with

363

IBD stimulated the proportion of genus Blautia, which was opposite to an increase in

364

CNCBL with IBD. Both types of cobalamin with IBD increased the proportion of

365

genus Bacteroides, compared with DSS treatment.

Oscillibacter,

Clostridium

IV,

Oscillospira,

Clostridium

and

366

Varieties in bacterial composition were further calculated using principal

367

component analysis (Fig. 3 C and D). The first axis (PC1), the second axis (PC2), and

368

the third axis (PC3) accounted for 24.04%, 18.15%, and 13.12% of the variability,

369

respectively. Samples from the control, CNCBL and MECBL groups exhibited

370

similar bacterial compositions; however, it can be seen that the bacterial compositions

371

among the DSS treatment and CNCBL treatment under IBD conditions were

372

attributed together and were distinct from the others. The rest of the MECBL with

373

IBD samples consisted of one group. 15



374

Results of PCA and heatmap clustering representing the effects of CNCBL and

375

MECBL on the gut microbiota patterns under IBD situations exhibited the same

376

pattern as the DAI scores (Fig. 3A, C and D). In this study, we found that the

377

proportion of phylum Proteobacteriaand Gram-negative bacteria were obviously

378

higher in IBD as well as in IBD with CNCBL, but not MECBL, supplementation (Fig.

379

2 C). Other studies had confirmed that the shift in Proteobacteria was the main reason

380

for gut microbiota dysbiosis and IBD29,30 because those strains secreted

381

lipopolysaccharide endotoxin, which could cause intestinal mucosal injury and

382

inflammation. MECBL was found to improve the proportion of Bacteroides in our

383

study (Fig. 3 B). Cordonnier et al. stated that Bacteroides thetaiotaomicron can

384

inhibit Shiga toxin synthesis from Enterohemorrhagic Escherichia coli through

385

competitive absorption of cobalamin.31 When supplementations of CNCBL exceeded

386

a threshold, cells of Bacteroides spp. are not enough to absorb and storage redundant

387

cobalamin. Strains such of Mucispirillum spp., Ruminococcus spp., Clostridium IV,

388

Lachonspiraceae, Escherichia were fast growing to occupy a suitable position in the

389

competition of gut micriobiome. Therefore, the statistical analysis performed in the

390

present study confirmed that the genus Escherichia/Shigella played an important role

391

during IBD development.

392

The microbiota profiles were used for functional prediction using PICRUSt.

393

Among many of the functions that were significant differentially represented in the

394

different groups (Table 1), a significantly higher production capacity of pathogenic E.

395

coli infection, lipopolysaccharide biosynthesis, lysine degradation, siderophore group

396

non ribosomal peptides, fatty acid metabolism, and amino acid metabolism related

397

genes was observed in DSS treatment and DSS and the high concentration of CNCBL

398

treatment as compared with others (Table 1), indicating CNCBL had an ability to 16


Page 16 of 33

Page 17 of 33


399

develop inflammation. However, MECBL did not show the similar affects, indicating

400

that CNCBL can aggravate IBD via enhancing infection and the relative

401

concentrations of E. coli and other IBD related pathogens. Further, we also observed

402

no big differences of vitamin B12 related genes such as ABC transporters, cysteine

403

and methionine metabolism, and Porphyrin metabolism between different groups.

404

Relationship between DAI, microbiomes, and SCFA in DSS-induced IBD mice with

405

various cobalamin supplementations

406

In order to understand functions of microbiomes during IBD developing, we applied

407

spearman and Canonical correspondence analysis to reveal the relationship between

408

metabolism and gut microbiomes. The Spearman analysis was employed for

409

clustering and explaining the relationship between different genera (family), between

410

DAI scores and genera, and between SCFA and genera. The co-occurrence network

411

was plotted by a correlation efficient higher than 0.8 (p

Impact of Cyanocobalamin and Methylcobalamin on Inflammatory

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