Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2-hydroxybutyric acid

Journal Pre-proof Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2hydroxybutyric acid Ningning Zheng, Yu Gu, Ying Hong, Lili Sheng, Linlin Chen, Feng Zhang, Zimiao Weng, Weidong Zhang, Zean Zhang, Wei Jia, Houkai Li PII:

S2095-1779(19)30567-2

DOI:

https://doi.org/10.1016/j.jpha.2019.11.003

Reference:

JPHA 500

To appear in:

Journal of Pharmaceutical Analysis

Received Date: 9 July 2019 Revised Date:

11 October 2019

Accepted Date: 5 November 2019

Please cite this article as: N. Zheng, Y. Gu, Y. Hong, L. Sheng, L. Chen, F. Zhang, Z. Weng, W. Zhang, Z. Zhang, W. Jia, H. Li, Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2-hydroxybutyric acid, Journal of Pharmaceutical Analysis (2019), doi: https://doi.org/10.1016/ j.jpha.2019.11.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Xi'an Jiaotong University. Production and hosting by Elsevier B.V. All rights reserved.

1

Vancomycin pretreatment attenuates acetaminophen-induced liver

2

injury through 2-hydroxybutyric acid

3

Ningning Zheng1, Yu Gu1, Ying Hong1, Lili Sheng1, Linlin Chen1, Feng Zhang1,

4

Zimiao Weng2, Weidong Zhang1,3, Zean Zhang4, Wei Jia5,6*, Houkai Li1*

5

1. Institute of Interdisciplinary Integrative Medicine Research, Shanghai

6

University of Traditional Chinese Medicine, Shanghai 201203, China

7

2. Dalian Medical University, Dalian 116044, China

8

3. School of Pharmacy, Second Military Medical University, Shanghai 200433,

9

China

10

4. Center for Drug Safety Evaluation and Research, Shanghai University of

11

Traditional Chinese Medicine, Shanghai 201203, China

12

5. University of Hawaii Cancer Center, Honolulu, Hawaii 96813, USA

13

6. Shanghai Key Laboratory of Diabetes Mellitus and Center for Translational

14

Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital,

15

Shanghai 200233, China

16

*Correspondence author:

17

Wei Jia, University of Hawaii Cancer Center, Honolulu, Hawaii 96813, USA;

18

[email protected];

19

Houkai Li, Shanghai University of Traditional Chinese Medicine, Shanghai

20

201203, China; [email protected];

21 22 1

23

Abstract

24

Liver injury caused by acetaminophen (AP) overdose is a leading public

25

health problem. Although AP-induced liver injury is well recognized as the

26

formation of N-acetyl-p-benzoquinone (NAPQI), a toxic metabolite of AP,

27

resulting in cell damage, emerging evidence indicates that AP-induced liver

28

injury

29

microbiota-involved mechanism remains largely unknown. In our study, we

30

found that vancomycin (Vac) pretreatment (100 mg/kg, twice a day for 4 days)

31

attenuated AP-induced liver injury, altered the composition of gut microbiota,

32

and changed serum metabolic profile. Moreover, we identified Vac

33

pretreatment elevated cecum and serum 2-hydroxybutyric acid (2-HB), which

34

ameliorated AP-induced cell damage and liver injury in mice by reducing AP

35

bioavailability and elevating GSH levels. Our current results reveal the novel

36

role of 2-HB in protecting AP-induced liver injury and add new evidence for gut

37

microbiota in affecting AP toxicity.

is

also

associated

with

gut

microbiota.

However,

the

gut

38 39

Key words: Liver injury; Gut microbiota; Acetaminophen; Vancomycin;

40

2-Hydroxybutyric acid

41 42

1. Introduction

43

Acetaminophen (AP) is a widely used over-the-counter medication for

44

relief of pain and fever. However, liver damage caused by AP overdose is a 2

45

leading public health problem. AP-induced hepatotoxicity accounts for 46% of

46

all acute liver failure in the United States [1] and 40-70% of all cases in Great

47

Britain and Europe [2]. About 85% of AP undergoes phase Ⅱ conjugation to

48

sulfated and glucuronidated metabolism in liver, and 10% of AP undergoes

49

phaseⅠoxidation to N-acetyl-p-benzoquinone (NAPQI), which is catalyzed by

50

CYP2E1, CYP1A2, CYP3A4 and normally conjugated with glutathione (GSH)

51

to nontoxic metabolites. Overdose of AP increases NAPQI production resulting

52

in depletion of GSH, as well as binding with cellular proteins leading to cell

53

injury [3-5].

54

Gut microbiota usually modulates the oral drug bioavailability or half-life

55

by altering the capacity of drug-metabolizing enzymes or expression of genes

56

involved in drug metabolism in host tissues [6]. Clayton et al. [7] have

57

observed that individuals with a high level of pre-dose urinary p-cresol, a

58

microbial metabolite, show low post-dose urinary ratios of sulfated AP to

59

glucuronic AP, suggesting that gut microbiota might affect the metabolism of

60

AP. Moreover, Lee et al. have evaluated the effects of gut microbiota on AP

61

metabolism in antibiotic-treated rats. They find antibiotic-treated rats have a

62

higher level of AP glutathione conjugates in blood than untreated rats,

63

suggesting the impacts of gut microbiota on AP metabolism [8]. Moreover,

64

Gong et al. [9] have demonstrated that AP-induced rhythmic variation in

65

hepatotoxicity is at least partially due to the production of gut microbial

66

metabolite,1-phenyl-1,2-propanedione, that depletes hepatic GSH levels. 3

67

Such evidence suggest that microbial metabolites also play important role in

68

affecting

69

mechanism remains largely unexplored due to the extremely complex

70

relationship between gut microbiota and host.

AP-induced

hepatotoxicity.

However,

the

microbiota-related

71

In our present study, we observed that vancomycin (Vac) pretreatment

72

attenuated AP-induced hepatotoxicity, as well as the alteration of gut

73

microbiota composition, convincing the microbial impacts on AP-induced

74

hepatotoxicity. Furthermore, untargeted and targeted metabolomics revealed

75

Vac pretreatment increased serum levels of 2-hydroxybutyric acid (2-HB),

76

which attenuated AP-induced hepatotoxicity both in vitro and in vivo.

77 78

2. Materials and Methods

79

2.1 Animals, cell lines and reagents

80

Male SD and Wistar rats (160-180 g) and C57 mice (18-22 g) were

81

purchased from Shanghai SLAC laboratory animal company and housed at

82

constant temperature (24 ± 2℃) with a 12 h light-dark cycle. BRL3A (ATCC

83

number: CRL-1442) and AML12 (ATCC number: CRL-2254) cells were

84

purchased from ATCC (Manassas, MA, USA). Vac was purchased from Lilly

85

Company (USA). AP, 2-HB, phenacetin were purchased from Sigma-Aldrich

86

(St. Louis, MO, USA). Sodium (±)-2-hydroxybutyrate-2,3,3-d3 (d3-2-HB) was

87

purchased from C/D/N Isotopes Inc (Canada). ALT, AST kits were purchased

88

from Nanjing Jiancheng Bioengineering Institute (China). GSH & GSSG and 4

89

CCK-8 kits were purchased from Beyotime Biotechnology (China). Gut

90

bacterial DNA extraction kit was purchased from QIAGEN company (Germany).

91

TRIzol reagent, reverse transcript enzyme and SYBR Green master mix were

92

purchased from Invitrogen (Carlsbad, CA). CYP2E1 (Cat: ab28146,

93

Lot:GR324351-11), CYP3A4 (Cat: ab3527, Lot: GR3210692-3), NQO-1 (Cat:

94

ab2346, Lot: GR3550-44) primary antibodies were purchased from Abcam

95

(USA). Collagenase type I (Cat: LS004196 ) was purchased from Washington

96

Biochemical Corporation. Percoll (Cat: P1644) was purchased from

97

Sigma-Aldrich.

98 99

2.2 Animal experiments and sample collection

100

The animal experiments were conducted under the Guidelines for Animal

101

Experiment of Shanghai University of Traditional Chinese Medicine and the

102

protocol was approved by the institutional Animal Ethics Committee. First, to

103

distinguish the gut microbial impacts on AP-induced acute liver injury, male SD

104

rats were divided into three groups as following: Con group (n=6), AP (i.g)

105

group (n=14) and AP (i.p) group (n=8). After overnight fasting, rats were

106

administered with single-dose of AP either orally (2.0 g/kg) or intraperitoneal

107

injection (1.0 g/kg) respectively, and sacrificed 24 h after AP administration. In

108

the second animal experiment, SD rats were divided into four groups as

109

following: Con group (n=6), Vac group (n=6), AP group (n=29) and Vac_AP

110

group (n=21). Vac and Vac_AP groups were orally given Vac (200 mg/kg/d) [10] 5

111

for continued 4 days prior to the administration of a single dose of AP (2.0

112

g/kg). After 24 h, all rats were anesthetized with 2% pentobarbital sodium and

113

samples were collected and stored at −80 °C for subsequent analysis.

114

Meanwhile, a paralleled experiment was conducted on Wistar rats by oral

115

gavage of 1.0 g/kg of AP based on our previous dosage experiment (n=20).

116

For the pharmacokinetic study of AP, serum samples in AP and Vac_AP

117

groups were collected at different timepoints (n=4-8).

118 119

2.3 Biochemical and liver histological analysis

120

Serum ALT, AST, liver and cellular GSSG/GSH were analyzed according

121

to the instructions. Liver tissues were maintained in 10% neutral formalin, then

122

embedded in paraffin and liver tissues were sliced at 5 μm for hematoxylin

123

and eosin (H&E) staining. The degree of liver injury was scored according to

124

reference [11].

125 126

2.4 Untargeted metabolomics analysis on serum sample with GC-MS

127

Serum samples stored at −80 °C were thawed and vortexed for 5 s at

128

room temperature. A total of 30 µL of serum sample was added to tube

129

containing 10 µL of internal standard (0.1 mg/mL dulcitol), and vortexed for 5 s.

130

Then, 120 µL of ice-cold methanol/chloroform (3:1) was added, the resulting

131

mixture was vortexed for 30 s and placed at −20 °C for 20 min before

132

centrifugation at 16 000 g and 4 °C for 15 min. Quality control (QC) sample 6

133

was prepared in parallel by pooling a small part of serum from each sample

134

and analyzed with the same procedure. The supernatant was utilized for

135

untargeted metabolomics analysis with GC-MS. The instrumental analysis and

136

data preprocessing was described as our previous method [12].

137 138

2.5 Quantitation of AP and 2-HB with targeted metabolomics in serum

139

and cecum samples

140

For serum samples, 5 µL of d3-2-HB (20 µg/mL, internal standard for

141

2-HB) and 5 µLof phenacetin (2 µg/mL, internal standard for AP) were added

142

to 25 uLof serum sample, and 165 µL of methanol was added into each

143

sample to precipitate protein. After votex for 1 min, the samples were

144

centrifuged (14000 rpm, 4 °C, 10 min) and the supernatant was transferred

145

into tubes for further analysis. For 2-HB calibration curve, 25 µL of 2-HB

146

standard solutions (125 ng/mL, 250 ng/mL, 500 ng/mL, 1 µg/mL, 2.5 µg/mL, 5

147

µg/mL,10 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL) were mixed with 5 µL of

148

d3-2-HB (20 µg/mL) and 170 µL of methanol and prepared with the same

149

method. For AP calibration curve, 2.5 µL of AP standard solutions (10 ng/mL,

150

20 ng/mL, 100 ng/mL, 500 ng/mL, 1 µg/mL, 2 µg/mL, 5 µg/mL,10 µg/mL, 20

151

µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, 500 µg/mL) were mixed with 22.5 µL

152

of blank serum, 5 µL of phenacetin (2 µg/mL) and 170 µL of methanol and

153

prepared with the same method.

154

A total of 50 mg cecum content of each mouse was weighed and added 7

155

with 400 µL of water. The samples were grinded thoroughly and centrifuged

156

(14000 rpm, 4 °C, 10 min) . 100 µL of supernatant was transferred into the tube

157

and 295 µL of methanol and 5 µL of d3-2-HB (20 µg/mL) were added, after

158

votex for 1 min, the samples were centrifuged (14000 rpm, 4 °C, 10 min).The

159

supernatant was transferred into a new tube and concentrated to dry under

160

nitrogen. The dried sample was dissolved in 100ul of methanol, after votex for

161

2 min and centrifuged at 14000 rpm for 2 min, the supernatant was transferred

162

into tubes for further analysis. For calibration curve of 2-HB, 100 µL of

163

standard solution (31.25, 62.5, 125, 250, 500, 1000 ng/mL) was mixed with 5

164

µL of d3-2-HB (20 µg/mL) and 295 µL methanol and prepared with the same

165

method.

166

The quantitation of AP and 2-HB was analyzed with LC20AD (Shimadzu)

167

coupled with AB Sciex Triple Quadrupole 4500. AP and 2-HB was separated

168

through a C18 column (5 µm, 2 × 25 mm). The mobile phase was water with

169

0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). At a flow

170

rate of 0.25 mL/min, a gradient was used as follows： 0-1.0 min：3% B, 1.0-1.1

171

min: 3% B to 50% B , 1.1-2 min: 50% B , 2.0-2.1 min: 50% B to 3% B, 2.1-2.9

172

min: 3% B. The column was maintained at 40 ℃, and the samples were kept

173

at 4 °C in an auto sampler during the entire analysis. The response of AP and

174

phenacetin were measured in positive mode ([M+H]+). The Q1 Mass (Da) / Q3

175

Mass (Da) for AP and phenacetin was 152.0 / 110.1 and 180.1 / 110.0,

176

respectively. The response of 2-HB and d3-2-HB was measured in negative 8

177

mode ([M-H]-). The Q1 Mass (Da) / Q3 Mass (Da) for 2-HB and d3-2-HB was

178

103.0 / 57.1 and 106.0 / 59.1, respectively. Data were acquired and processed

179

with Analyst 1.5.2 system software (AB SCIEX™, Foster City, CA, USA).

180 181

2.6 Gut microbiota analysis with 16S rRNA gene sequencing

182

Gut bacterial DNA extraction and 16S rRNA gene sequencing were

183

conducted according to the method previously published [12]. Briefly, bacterial

184

DNA of cecum contents was extracted with bacteria DNA stool mini kit

185

following the manufacturer’s instructions. The extracted DNA samples were

186

used as template for amplification of the V3 region of 16S rRNA gene. The

187

PCR amplification, pyrosequencing of PCR amplicons and quality control were

188

performed on Ion PGM™ System according to reference [13]. Raw fastq data

189

was quality-filtered for further analysis. Operational taxonomic units (OTUs)

190

were delineated at 97% similarity level with Mothur software. Raw fastq files

191

were subsequently deposited to the Sequence Read Archive database under

192

the accession number PRJNA546452.

193 194

2.7 Preparation of primary hepatocytes from mice

195

Mouse primary hepatocytes were isolated by two-step collagenase

196

perfusion protocol [14]. Briefly, mice were anaesthetized with 1% pentobarbital

197

sodium solution by intraperitoneal injection. Then, the mouse liver was

198

perfused with perfusion medium I via portal vein for 10 min. Next, the liver was 9

199

perfused with medium containing collagenase type I for 10 min. After adequate

200

digestion, the liver was dissected in sterile dish and passed through a 100 µm

201

filter followed by centrifugation for 2 min at 600 rpm. The supernatant was

202

discarded and hepatocytes pellet was resuspended in DMEM medium. The

203

above washing step was repeated for three times. Then the hepatocytes were

204

purified with percoll regent and then seeded on plates with 10% FBS DMEM

205

for further experiments.

206 207

2.8 Cell viability test under the treatment of 2-HB

208

1 x 104 cells per well were seeded on a 96 well plate. After 24 h, cells were

209

incubated with or without 2-HB (5~40 mM) for 24 h or 48 h. Cell viability was

210

measured with CCK-8 kit, with the addition of CCK-8 regent (10%) into the

211

medium following the incubation at 37 ℃ for 1 h. The absorbance was

212

measured at 450 nm (the main wavelength) and 600 nm (the secondary

213

wavelength) and the viability was calculated as the percentage of control.

214 215

2.9 The protective effects of 2-HB on AP-induced cell injury

216

First, the IC50 of AP in BRL3A, AML12 and mouse primary hepatocytes

217

were evaluated. Different concentrations of AP (3.125, 6.25, 12.5, 25, 50 mM)

218

was incubated with cells for 24 h or 48 h. The cell viability was analyzed with

219

CCK-8 kit. And these cell lines were separately treated with AP at its IC50

220

dosage and different concentrations of 2-HB. BRL3A cells were treated with 9 10

221

mM of AP and 2-HB (5, 10,20,40 mM) for 48 h. AML12 cells were treated with

222

14 mM of AP and 2-HB (5, 10, 20, 40 mM) for 48 h. Mice primary liver cells

223

were treated with 17 mM of AP and 2-HB (5, 10,20,40 mM) for 24 h. Cell

224

viability was measured with CCK-8 kit and calculated as the percentage of

225

control group.

226 227

2.10 Functional investigation of 2-HB in AP-induced acute liver injury in

228

mice

229

To study the impact of 2-HB on AP-induced liver injury, male C57 mice

230

were divided into four groups as following: Con group (n=5), 2-HB group (n=6),

231

AP group (n=7) and 2-HB+AP group (n=7). 2-HB (250 mg/kg) were

232

intraperitoneally injected into C57 mice in 2-HB and 2-HB+AP groups for 4

233

days, and 30 min after the last injection, 400 mg/kg of AP were orally

234

administrated to mice in AP and 2-HB+AP groups. After 24 h, all mice were

235

anesthetized with 2% pentobarbital sodium and samples were collected and

236

stored at −80 °C for subsequent analysis. For the pharmacokinetic study of AP,

237

serum samples in AP and 2-HB+AP groups were collected at the time points of

238

0, 10 min, 0.5 h, 1 h, 3 h, 8 h, and 24 h. Three mice in AP or 2-HB+AP group at

239

every time point were used in this study and each serum sample was analyzed

240

twice with instrument (n=6).

241 242

2.11

β-GD activity assay in cecum content 11

243

A total of 100 mg of cecum contents from each rat of Con, Vac, AP and

244

Vac_AP was homogenized with 100 µL of lysis buffer and grinded with

245

magnetic beads for 1 min. After centrifuged at 8000 g for 10 min (4 °C), the

246

supernatant was transferred to another clean tube for further analysis. Total

247

protein concentration of each sample was assayed and the samples were

248

adjusted to the same protein concentration before β-glucuronidase (β-GD)

249

assay. The assay was performed based on articles previously published [15].

250

Briefly. P-nitrophenyl-b-D-glucuronide acid (PNPG) was used as the probe

251

substrate of β-GD. The incubation mixture with a total volume of 0.1 mL was

252

consisted of 48 µL of 0.2 M phosphate buffer (pH 6.8), 50 µL of sample and 2

253

µL of PNPG (200 mM, final concentration). PNPG hydrolysis was performed at

254

37 °C for 30 min. The signal of the hydrolytic metabolite of PNPG (PNP) was

255

detected at 405 nm every 1 min, by a Synergy H1 Hybrid Multi-Mode

256

Microplate Reader (BioTek, USA). The reaction rate was calculated based on

257

the OD405 and used for the comparison of β-GD activity between groups.

258 259

2.12 Real-time RT-PCR analysis on gene expression

260

Total RNA of liver tissue was extracted using TRIzol reagent (Invitrogen,

261

Carlsbad, CA) and quantified by UV absorption (Colibri, Titertek Berthold).

262

Following quantification, reverse transcript reaction was performed with a

263

reverse transcript enzyme (Life technologies, Invitrogen) according to the

264

manufacturer’s instructions. Quantitative amplification by PCR was carried out 12

265

using SYBR Green Master Mix regent by Biorad CFX Connect system. Cycle

266

numbers of both 18s (as an internal control) and cDNAs of interest were used

267

to calculated relative expression levels of the genes of interest.

268 269

2.13 Western blot

270

Liver protein was extracted with a commercial lysis buffer (Shanghai

271

beyotime Biotechnology Company) added with 1% of phosphatase inhibitor

272

cocktail 2 and phosphatase inhibitor cocktail 3 (Sigma) according to

273

well-established protocols. The concentration of total protein was determined

274

with Pierce™ BCA protein assay kit (Thermo Scientific). Then the samples

275

were adjusted to the same concentration and mixed with loading buffer, and

276

heated at 100℃ for 10 min. A total of 40 µg protein was loaded into each lane

277

and separated by 10% SDS-PAGE gel, and then transferred to a PVDF

278

membrane (Millipore). The membranes were blocked with 10% milk at room

279

temperature for 90 min and then washed with TBST (20 mM Tris-HCl, 137mM

280

NaCl, and 0.1% Tween20, pH7.5) for three times at 10 min interval, following

281

the incubation with primary antibodies for NQO-1 (Abcam), CYP2E1 (Abcam)

282

and CYP3A4 (Abcam) overnight at 4 ℃. The membranes were washed with

283

TBST for three times, and incubated with the HRP-conjugated secondary

284

antibodies for 2 h at room temperature. The membranes were exposed with

285

SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific,

286

USA) and the bands were quantified with Amersham Imager 600 system 13

287

(General Electric Company, USA).

288 289

2.14 Liver CYP450 enzymatic activity assay

290

S9 fractions were prepared from livers to determine the activities of

291

CYP2E1 and CYP1A2 according to previous method with some modifications

292

[16]. Briefly, 250 mg of liver tissue in each group (n=5-10) was mixed with 1 mL

293

of 100 mM potassium phosphate containing 0.15M potassium chloride

294

(pH=7.4) and grinded with magnetic beads for 1 min. After centrifuged at 9000

295

g for 20 min, the supernatant was transferred to another clean tube and tested

296

for the total protein concentration with BCA method. Each assay contained 99

297

µL of 0.1 M potassium phosphate buffer (pH 7.4), a NADPH-generating system

298

(containing 0.1 M glucose 6-phosphate, 10 mM NADP+, and 10 IU/mL

299

glucose-6-phosphate dehydrogenase, 40 mM MgCl2), 20 µl of S9 fraction and

300

1 µL of probe substrate. Following a 3 min preincubation period in the absence

301

of probe substrate, the reaction mixture was incubated for 30 min at 37 °C, and

302

the reaction was stopped by adding 100 µl acetonitrile. LC20AD (Shimadzu)

303

coupled with AB Sciex Triple Quadrupole 4500 system was utilized to measure

304

the

305

(acetaminophen).

metabolites

of

CYP2E1

(6-hydroxy-chlorzoxazone)

and

CYP1A2

306 307 308

2.15 PICRUSt analysis with 16S rRNA gene sequencing data Phylogenetic

Investigation

of

Communities 14

by

Reconstruction

of

309

Unobserved States (PICRUSt) analysis was performed with 16S rRNA gene

310

sequencing data[17]. This method predicts the gene family abundance from

311

the phylogenetic information with an estimated accuracy of 0.8. The OTU table

312

was used as the input for metagenome imputation and was first rarefied to an

313

even sequencing depth prior to the PICRUSt analysis. Next, the resulting OTU

314

table was normalized by 16S rRNA gene copy number. The gene content was

315

predicted for each individual. Then the predicted functional composition

316

profiles were collapsed into level 3 of KEGG (Kyoto Encyclopedia of Genes

317

and Genomes) database pathways.

318 319

2.16 Statistical analysis

320

Data are shown as means ± SEM unless otherwise noted. Statistical

321

significance was determined with the unpaired two-tailed Student’s t test. The

322

statistical significance of hepatic steatosis scores was evaluated with

323

non-parametric Kruskal-Wallis test followed by the MannWhitney U test. p<

324

0.05 was considered statistically significant.

325 326

3. Results

327

3.1 Vac pretreatment attenuates AP-induced acute liver injury in rats

328

First of all, to confirm the impact of gut microbiota on AP-induced liver

329

injury, male SD rats were administered with single-dose of AP either orally (2.0

330

g/kg) or through intraperitoneal injection (1.0 g/kg). We found that serum ALT 15

331

and AST levels were significantly elevated in both oral administration and

332

intraperitoneal injection groups compared to control group. However, dramatic

333

inter-individual variation in serum ALT and AST was only present in orally

334

administrated rats (Figure 1A), but not those under intraperitoneal injection

335

(Figure 1B). Given the fact that the absorption process of intraperitoneal

336

injection is absent of impact from gut microbiota, high inter-individual variation

337

of AP-induced liver injury in orally administrated rats was probably due to gut

338

microbial influence on AP pharmacokinetics. Then, the microbial impact on

339

AP-induced liver injury was investigated in male SD rats pretreated with or

340

without Vac for 4 consecutive days prior to a single-dose AP (2.0 g/kg)

341

administration. After 24 h of AP treatment, we found that pretreatment of Vac

342

significantly attenuated serum ALT and AST elevation compared to that of AP

343

alone (Figure 1C), which was convinced by liver histological analysis (Figure

344

1D). Meanwhile, a paralleled experiment was conducted in Wistar rats, which

345

showed consistent results with that observed in SD rats (Supplementary

346

Figure 1A and B). These results indicated that Vac pretreatment could

347

attenuate AP-induced acute liver injury in rats.

348 349

3.2 Vac pretreatment decreases the bioavailability of AP and ratio of

350

GSSG/GSH

351 352

To investigate whether the attenuation of AP-induced liver injury by Vac pretreatment

was

associated

with 16

changes

of

AP

bioavailability,

353

pharmacokinetics of AP were compared between AP and Vac_AP groups

354

either in SD or Wistar rats. The results showed that serum concentrations of

355

AP at the 2nd ,4th, 8th, 24th h and AUC0-t were significantly lower in Vac_AP

356

group than AP group in either SD or Wistar rats (Figure 2A, Supplementary

357

Figure 1C). Since the serum concentration of AP is partially dependent on the

358

extent of AP reabsorption in intestinal tract through deglucuronidation of

359

conjugated AP by bacteria-derived β-GD [18, 19], we therefore expected

360

whether Vac pretreatment altered the activity of β-GD or not. The activity of

361

β-GD was measured in cecum contents. The results showed that the activity of

362

β-GD was significantly inhibited by Vac pretreatment (Figure 2B), suggesting

363

that the reduced serum concentration of AP in Vac-pretreated rats was

364

probably associated with the inhibition of bacterial β-GD activity.

365

Since the AP-induced liver injury is partly due to depletion of GSH [5], the

366

ratio of GSSG/GSH was compared between AP and Vac_AP groups. We

367

found that the ratio of GSSG/GSH was dramatically reduced in Vac_AP rats

368

compared to AP rats (Figure 2C), suggesting that Vac pretreatment increased

369

levels of GSH, rather than its oxidative product GSSG. In line with the

370

increased levels of GSH, Vac pretreatment resulted in up-regulated mRNA

371

expression of Nqo-1 and Gclc gene, and down-regulated Tnf-α and Il-1β

372

(Figure 2D).

373

In addition, the protein expression of CYP2E1 and CYP3A4, and enzyme

374

activity of CYP2E1 and CYP1A2 was measured in liver tissue, which regulate 17

375

the formation of toxic NAPQI from AP [20, 21]. There were no significant

376

differences in CYP3A4 protein expression and CYP1A2 activity between the

377

two groups (Figure 2E and F, Supplementary Figure 2), except for the

378

up-regulated activity of CYP2E1 in Vac_AP group (Figure 2G and H,

379

Supplementary Figure 2). Collectively, these results suggested that the

380

protection of Vac pretreatment against AP-induced liver injury may not due to

381

the reduction of NAPQI product, but associated with decreased bioavailability

382

of AP and increased ratio of GSH/GSSG.

383 384

3.3 Vac pretreatment elevates 2-HB in both cecum and serum

385

In addition to the pharmacokinetics change, previous study indicates that

386

endogenous metabolite derived from gut microbiota may also contribute to

387

AP-induced hepatotoxicity[9] . We therefore investigated the mechanism

388

underlying the protective effect of Vac pretreatment against AP-induced liver

389

injury using GC-MS-based untargeted metabolomics on serum samples. First

390

of all, we observed that Vac treatment not only significantly altered the

391

metabolic profile at baseline, but also in AP treated-rats (Figure 3A),

392

suggesting that Vac treatment could on one hand alter the metabolism of host,

393

on the other hand, reverse the endogenous metabolism in AP-treated rats.

394

Then, differential metabolites among groups were determined with the double

395

criteria of VIP>1.0 in OPLS-DA model and p<0.05 in Student’s t test, in which a

396

total of 13 differential metabolites were identified and visualized with a 18

397

heatmap (Figure 3B). These 13 differential metabolites were further uploaded

398

to MetaboAnalyst (https://www.metaboanalyst.ca/) for pathway enrichment

399

analysis, and 9 pathways were enriched with criteria of p< 0.05 including

400

pathways involved in propanoate metabolism, amino acids metabolism (i.e.

401

phenylalanine and tyrosine, beta-alanine, asparate, alanine, cysteine,

402

malate-asparate shuttle and glucose-alanine cycle) and urea cycle (Figure

403

3C). Propanoate metabolism pathway was the most significant pathway with

404

the lowest p value, which included 2-HB, beta-alanine, 2-ketoglutaric acid and

405

glutamic acid. All these four metabolites were increased by AP treatment

406

compared to Con group, whereas 2-HB was one of the most significantly

407

altered metabolites (Supplementary Table 1). Furthermore, we quantified the

408

concentrations of 2-HB in both cecum content and serum with or without AP

409

treatment by using targeted metabolomics based on LC-MS/MS. The results

410

showed that Vac pretreatment significantly elevated 2-HB levels in cecum

411

content with or without AP treatment (Figure 3D), suggesting gut-derived 2-HB

412

was stimulated in Vac. Consistent with this finding, Vac pretreatment also

413

increased levels of 2-HB in serum before AP treatment (Figure 3E). However,

414

serum 2-HB level was lower in Vac_AP than AP group 24 h after AP treatment,

415

which was in line with the untargeted metabolomics result (Figure 3E).

416

To test whether the increased concentration of 2-HB by Vac treatment was

417

due to altered gut microbiome, we compared the gut microbiota profiles by

418

using 16S rRNA sequencing between AP and Vac_AP groups. The PCoA 19

419

showed that AP and Vac_AP group were clearly separated (Figure 3F). At the

420

phylum level, the relative abundance of Firmicutes and Bacteroides were

421

dramatically decreased, while the abundance of Proteobacteria and

422

Fusobacteria were greatly enriched in Vac_AP group (Figure 3G). Then, a

423

total of 50 genera with relatively higher abundance was chosen and compared

424

between AP and Vac_AP groups, in which the relative abundance of 9

425

gram-negative and 16 gram-positive bacteria genera were either significantly

426

increased or decreased by Vac pretreatment (Supplementary Figure 3).

427

Functional annotation of gut bacteria showed that propanoate metabolism

428

pathway was significantly enriched in Vac_AP group than AP group (Figure

429

3H), which is consistent with the observation of serum metabolomics (Figure

430

3C). In addition, since 2-HB is generated from 2-oxobutanoate by lactate

431

dehydrogenase (LDH) (from Kyoto Encyclopedia of Genes and Genomes

432

database, https://www.kegg.jp/), which is also present in some kinds of gut

433

microbiota [22-25], we specially analyzed the abundance of LDH-expressing

434

bacteria. Interestingly, we observed that the relative abundance of

435

LDH-expressing bacteria was also increased in Vac_AP group (Figure 3I).

436

Alltogether, our results suggest that increased 2-HB by Vac treatment may due

437

to altered gut microbiota.

438 439 440

3.4 2-HB attenuates AP-induced cellular toxicity To investigate the biological function of 2-HB, different hepatocytes were 20

441

treated with 2-HB at a series of concentrations including rat-derived liver cell

442

line BRL3A, mice-derived liver cell line AML12 and mice primary hepatocytes.

443

Interestingly, 2-HB treatment enhanced cell viability to different extent in

444

AML-12 and mice primary hepatocytes, but not BRL3A (Supplementary

445

Figure 4A-C). We then asked whether 2-HB could influence AP-induced cell

446

toxicity. The IC50 of AP on these three cell lines were determined first

447

(Supplementary Figure 4D-F), which were used for subsequent experiments

448

with or without addition of 2-HB for 24 or 48 h. We found that addition of 2-HB

449

significantly attenuated the AP-induced cell toxicity regardless of species

450

difference in cell origin (Figure 4A-C). Representative photographs of BRL3A

451

cells treated with AP for 48 h in the presence or absence of 2-HB co-culture

452

were presented in Figure 4D. In line with the cell viability results, AP treatment

453

resulted in a large portion of cells dead and floating, which was obviously

454

improved by 2-HB (Figure 4D). Furthermore, we observed that 2-HB addition

455

decreased the ratio of GSSG/GSH in mice primary hepatocytes compared to

456

AP alone (Figure 4E). Our current results indicated that 2-HB protected

457

AP-induced cell toxicity in either rat- or mouse-derived cell lines.

458 459

3.5 2-HB treatment protects AP-induced acute liver injury in mice

460

Given the observed evidence, we hypothesized that the attenuation on

461

AP-induced liver injury by Vac pretreatment was probably due to 2-HB

462

production. We observed the impact of 2-HB on AP-induced liver injury in a 21

463

group of male C57 mice, which were orally given with single-dose of AP (400

464

mg/kg) following 3 days of 2-HB (250 mg/kg per day, i.p) treatment. Results

465

showed that AP treatment induced significant liver injury in mice, whereas

466

2-HB pretreatment effectively protected mice from severe liver injury (Figure

467

5A and B). Moreover, we found that 2-HB decreased the peak concentration of

468

serum AP and reduced AUC0-t (Figure 5C). In addition, the serum

469

concentration of 2-HB was also detected before and after AP administration.

470

We observed that intraperitoneally injection of 2-HB definitely increased serum

471

levels of 2-HB, especially within the 1st h after AP treatment (Figure 5D).

472

Moreover, 2-HB pretreatment also inhibited mRNA expression of genes

473

involved in mediating inflammation (Tnf-α, Il-1β) (Figure 6A), cell proliferation

474

process (Btg2, Ccng1) and DNA damage induced genes (Ddit4l, Gadd45α)

475

(Figure 6C) that were stimulated by AP treatment, as well as the regulation of

476

genes in anti-oxidative pathways such as Nqo-1, Txnrd1, Car3 (Figure 6B)

477

and down-regulation of genes related with hepatotoxicity (Krt8, Krt18) (Figure

478

6C). In addition, 2-HB did not alter the protein expression of hepatic CYP2E1

479

or NQO-1 in AP-treated mice (Figure 6D, Supplementary Figure 5). Overall,

480

these data highlighted that 2-HB was protective against AP-induced liver injury

481

in mice.

482 483 484

4. Discussion In our current study, we demonstrated that Vac pretreatment attenuated 22

485

AP-induced liver injury in rats, and altered the composition of gut microbiota.

486

Then, our untargeted metabolomics revealed the significant metabolic change

487

by Vac pretreatment. Moreover, we found 2-HB was elevated in serum by Vac

488

pretreatment, which could reduce AP-induced toxicity in either rat- or

489

mouse-derived cell lines or primary hepatocytes, as well as acute liver injury in

490

mice.

491

AP-induced liver injury has been extensively investigated so far; however,

492

the underlying mechanism is not fully elucidated. In addition to the

493

conventional theory of NAPQI production [3, 5], emerging evidence has

494

indicated that the extent of AP-induced liver injury is dependent on microbial

495

composition because of the extensive co-metabolism on xenobiotic between

496

gut microbiota and host [7, 8]. Previously, Jeremy et al. demonstrate that the

497

character of baseline metabolic profile is predictive for the extent of

498

AP-induced liver injury [11], in which microbial metabolites such as taurine are

499

involved. Moreover, the variation of urine p-cresol in human subject influences

500

the metabolism of AP [7]. Recently, the impact of gut microbiota on AP-induced

501

liver injury is associated with changes of GSH levels [9] or alteration of AP

502

metabolism [8, 11]. In our current study, we first observed that obvious

503

inter-individual variation in levels of serum ALT and AST existed in AP orally

504

treated rats, but not in those with intraperitoneal injection. It is reasonable that

505

the bioavailability of orally administrated xenobiotics is apt to be impacted by

506

more factors than intraperitoneal injection, in particular gut microbiota [6]. We 23

507

further found that Vac pretreatment decreased the levels of serum ALT, AST

508

and the extent of liver necrosis caused by AP in either SD or Wistar rats. Vac is

509

a potent antibiotic against gram-positive bacteria and with limited systemic

510

impacts due to non-absorptive character in intestine [10, 26, 27]. As a result,

511

Vac is frequently used for investigating the role of gut microbiota in various

512

studies [28-30]. Our results convinced that alteration of gut microbiota was a

513

critical factor in determining the extent of AP-induced liver injury. Moreover, we

514

found that Vac pretreatment significantly decreased the bioavailability of AP.

515

Since the bioavailability of AP is influenced by intestinal absorption of AP,

516

which is deglucuronidated by microbiota produced β-GD [18, 19], we showed

517

that Vac pretreatment greatly depleted the activity of β-GD in cecum contents.

518

Therefore, it is possible that the depletion of intestinal β-GD reduces the

519

absorption of AP. Nevertheless, the change of AP bioavailability cannot fully

520

explain the reduced hepatotoxicity by Vac pretreatment. Moreover, the

521

expression of CYP2E1 protein was even up-regulated in Vac pretreated group,

522

which implied that the protective effect of Vac on AP-induced liver injury was

523

not due to reduced NAPQI production.

524

Metabolomics is increasingly applied to investigate the underlying

525

mechanisms of drug activity/toxicity because metabolomics is a unique

526

approach for quantitatively measurement of the time-related metabolic

527

response of living systems to pathophysiological stimuli or genetic modification

528

simultaneously [31, 32]. Gong et al. [9] reveals that gut microbiota determine 24

529

the diurnal variation of AP-induced liver injury through microbial metabolite,

530

1-phenyl-1,2-propanedione by using untargeted metabolomics. In our current

531

study, an untargeted GC-MS-based metabolomics was adopted to test

532

whether there are metabolites that are involved in attenuation of AP-induced

533

liver injury by Vac pretreatment. Our results showed that the metabolic profile

534

of AP was very different from others. Then, a total of 13 differential metabolites

535

were identified between Con and AP group that were restored by Vac

536

pretreatment,

537

AP-induced hepatotoxicity. Moreover, propanoate metabolism was one of the

538

most significantly altered metabolic pathways enriched with these differential

539

metabolites, in which metabolite 2-HB attracted our attention because of its

540

involvement in propanoate metabolism and dramatic fold change between Con

541

and AP groups. Consistently, our targeted metabolomics analysis convinced

542

the elevation of 2-HB by Vac pretreatment in cecum content with or without AP

543

treatment, suggesting that 2-HB production was associated with gut microbiota.

544

Our results showed serum 2-HB level was lower in Vac_AP than AP group

545

after 24 h of AP treatment. Oxidative stress or detoxification of xenobiotics in

546

the liver can dramatically increase the rate of hepatic glutathione synthesis.

547

Under such metabolic stress conditions, homocysteine is diverted from the

548

transmethylation pathway (which forms methionine) into the transsulfuration

549

pathway (which forms cystathionine). alpha-ketobutyrate is produced when

550

cystathionine is cleaved into cysteine that is incorporated into glutathione, and

suggesting

that

these

25

metabolites

were

relevant

with

551

2-HB is formed via a reaction catalyzed by LDH from alpha-ketobutyrate

552

(http://www.hmdb.ca/metabolites/HMDB0000008). So the elevation of serum

553

2-HB in AP group after 24 h of AP treatment may be a stress response

554

because of the upregulation of hepatic glutathione synthesis.

555

Meanwhile, 16S rRNA gene sequencing data indicated that Vac treatment

556

obviously altered the composition of gut microbiota. PICRUSt is a

557

computational

558

reconstruction algorithm to predict which gene families are present and then

559

combines gene families to estimate the composite metagenome, thus can

560

predict the functional composition of a metagenome using a database of

561

reference genomes [17]. Based on the PICRUSt analysis of 16S rRNA gene

562

sequencing data, the abundance of propanoate metabolism pathway involved

563

in 2-HB producing was upregulated in Vac_AP group. At genus level, Vac

564

treatment also upregulated the relative abundance of some LDH expressing

565

bacteria such as Lactobacillus, Clostridium, Fusobacterium, Desulfovibrio

566

[22-25]. As a result, we speculated that the attenuation on AP-induced liver

567

injury by Vac pretreatment might be associated with the increase of 2-HB.

approach

which

uses

an

extended

ancestral-state

568

In line with our hypothesis, our results showed that 2-HB significantly

569

improved cell viability under AP treatment in cell lines from either rats or mouse

570

such as rat-derived BRL3A cells, mouse-derived AML12 cells and mouse

571

primary hepatocytes, suggesting that the hepatoprotective effect of 2-HB is

572

trans-species. Cellular GSH is critical for the detoxification of AP through 26

573

conjugation with the toxic product NAPQI of AP. The accumulated NAPQI will

574

deplete cellular GSH resulting to oxidative stress-induced liver injury [33, 34].

575

We found 2-HB reduced the AP-induced upregulation of liver GSSG/GSH in

576

mouse primary hepatocytes that is consistent with its protective effect against

577

AP-induced cell toxicity. Given the higher inter-individual variation of

578

AP-induced liver injury among rats and observed trans-species protection of

579

2-HB against AP-induced toxicity in cell lines, the biological function of 2-HB

580

was further validated in AP-treated mice, instead of rats. We observed that

581

2-HB prevented AP-induced liver injury in mice. Interestingly, 2-HB also

582

decreased the Cmax and AUC0-t of AP, as well as regulated the expression of

583

genes involved in inflammation, oxidative stress, cell proliferation and DNA

584

damage process. Nevertheless, we are not quite clear about the link between

585

2-HB and AP bioavailability based on our current results. Further investigation

586

will be conducted for explaining their potential relationship.

587

In summary, our current study demonstrates that Vac pretreatment

588

attenuates AP-induced liver injury, alters serum metabolic and gut microbiota

589

profiles in rats. The identified metabolite 2-HB has trans-species protective

590

effect against AP-induced cell toxicity in either rat- or mouse-derived cells,

591

which is further validated in mice. The protective effect of 2-HB is associated

592

with the reduction of AP bioavailability and increase of GSH levels. This study

593

convinces the impacts of gut microbiota on AP-induced hepatotoxicity, and

594

sheds light on the potential to prevent AP-induced hepatotoxicity by 27

595

modulating gut microbiota with antibiotic or other approaches such as prebiotic

596

or probiotic if specific functional bacteria was determined in the future.

597 598

Author contributions

599

Houkai Li and Wei Jia supervised the whole study and revised the

600

manuscript; Ningning Zheng conducted all of the in vivo and in vitro

601

experiments and data analysis; Yu Gu helped in targeted metabolomics of

602

serum and cecum samples with LC-MS/MS; Ying Hong, Lili Sheng and

603

Weidong Zhang helped in study design and data analysis; Linlin Chen helped

604

in the animal experiments; Feng Zhang helped in analysis of hepatic CYP450

605

enzymatic activity; Zimiao Weng helped in the cecum β-GD activity assay;

606

Zean Zhang helped in histological analysis of liver tissue. All authors have read

607

and approved the final version of the manuscript.

608 609

Acknowledgments

610

This work was funded by National Natural Science Foundation of China

611

(No. 81873059 & 81673662), & National Key Research and Development

612

Program of China (No. 2017YFC1700200), & Shuguang Scholar (16SG36) at

613

Shanghai Institutions of Higher Learning from Shanghai Municipal Education

614

Commission.

615 616

Conflicts of interest 28

The authors declare that there are no conflicts of interest.

617 618 619

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723 724

Figure legends

725

Figure 1 Vac pretreatment attenuates AP-induced acute liver injury in rats.

726

(A and B) Serum ALT and AST levels. i.g and i.p represent oral gavage or 33

727

intraperitoneal injection with a single dose of 2.0 g/kg or 1.0 g/kg of AP,

728

respectively (n = 6-14). (C and D) Serum ALT, AST, hepatic H&E staining, and

729

histology score in SD rats treated with Vac (100 mg/kg, twice a day) and/or with

730

AP (i.g, 2.0 g/kg). n = 5-6 in Con or Vac group. n = 29 in AP and 20 in Vac_AP

731

groups. Data were mean ± S.E.M. The magnification of H&E staining

732

photograph is 200х. * p < 0.05; ** p < 0.01.

733

Figure 2. Vac pretreatment decreases the bioavailability of AP and ratio

734

of GSSG/GSH. (A) The serum concentrations of AP in SD rats at different time

735

points (n = 4). (B) The activity of β-glucuronidase (β-GD) in cecum. n = 5 in

736

Con or Vac group, and 10 in AP or Vac_AP group. (C and D) Hepatic

737

GSSG/GSH and mRNA expression of genes (n = 6-8). (E and G) The hepatic

738

protein levels of CYP3A4 and CYP2E1 (n = 6) as well as (F and H) enzymic

739

activity of CYP1A2 and CYP2E1 measured with LC-MS/MS (n = 10 in Con or

740

Vac group and 20 in AP or Vac_AP group). Data were mean ± S.E.M. * p <

741

0.05, ** p < 0.01.

742

Figure 3. Vac pretreatment elevates 2-HB in both cecum and serum. (A)

743

PLS-DA plot of serum metabolites (n = 5). (B) Differential metabolites among

744

groups (n = 5). (C) Pathway analysis based on the 13 differential metabolites

745

in B. The quantitation of 2-HB in cecum contents (D) and serum (E) of rats 24 h

746

after AP gavage (n = 5-8). (F-G) PCoA analysis at OTU level and column

747

diagram at phylum level based on 16S rRNA gene sequencing (n = 5). (H) The

748

abundance of propanoate metabolism pathway based on PICRUSt analysis of 34

749

16S rRNA gene sequencing (n = 5). (I) Heatmap of the relative abundance of

750

LDH-producing bacteria at genus level (n = 5). Data were mean ± S.E.M. * p <

751

0.05; ** p < 0.01.

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Figure 4. 2-HB attenuates AP-induced cellular toxicity. (A) BRL3A cells, (B)

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AML12 cells, and (C) mouse primary hepatocytes were treated with AP alone

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or in combination with 5-40 mM 2-HB for indicated periods, and cell viability

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was measured with CCK-8 kit (n=6). (D) Representative photographs of

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BRL3A cells treated with AP alone or in combination with 5-40 mM 2-HB for 48

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h (The magnification of photographs is 100х). (E) Cellular GSSG/GSH was

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analyzed in primary hepatocytes treated with AP alone or in combination of 5

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mM 2-HB for 24 h (n = 3). Data were mean ± S.E.M. * p < 0.05, ** p < 0.01.

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Figure 5. 2-HB treatment protects AP-induced acute liver injury in mice.

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Mice were intraperitoneally injected with 2-HB (250 mg/kg, once a day) for 4

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days and administrated with a single dose of AP (400mg/kg,) 30 min after the

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last injection of 2-HB. (A-B) Serum ALT, AST, and liver histology in mice 24 h

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after AP treatment (n = 5-7). * p< 0.05, ** p < 0.01. (C-D) Serum concentrations

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of AP and 2-HB quantified with LC-MS/MS in mice treated with AP and/or 2-HB

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(n = 6). * p< 0.05, ** p < 0.01 compared to the same time point between the

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two groups. Data were mean ± S.E.M.

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Figure 6. 2-HB normalizes gene expression. (A) Inflammation related genes.

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(B) Oxidative stress related genes. (C) Cell proliferation and DNA damage

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induced genes. (D) Hepatic CYP2E1 and NQO-1 protein levels. n = 5-7 in A-D. 35

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Data were mean ± S.E.M. * p < 0.05, ** p < 0.01.

772 773

Supplementary Figure 1. Vac pretreatment attenuates AP-induced acute

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liver injury and the bioavailability of AP in Wistar rats. (A-B) Serum ALT,

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AST, hepatic H&E staining, and histology score in rats orally gavaged with Vac

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(100 mg/kg, twice a day) and/or AP (1.0g/kg). n = 5 in Con or Vac group, n = 20

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in AP or Vac_AP group. Data were mean ± S.E.M. The magnification of H&E

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staining photograph is 200х. * p < 0.05; ** p < 0.01. (C) The concentration of

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serum AP in rats at different time points, n = 4.

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Supplementary Figure 2. Raw data. Original uncropped scan images of

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Figures 2E and 2G.

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Supplementary Figure 3. Vac alters gut microbiota composition in rats.

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Heatmap analysis of the top 50 genera in AP and Vac_AP group. n = 5. * p <

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0.05, ** p < 0.01 with Wilcoxon rank-sum test.

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Supplementary Figure 4. (A-C) Cell viability of BRL3A cells, AML12 cells, and

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C57 mice primary hepatocytes treated with 5-40 mM 2-HB for indicated time.

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(D-F) The IC50 values of AP in BRL3A, AML12 and C57 mice primary

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hepatocytes determined by non-linear regression. n = 6 in A-F.

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Supplementary Figure 5. Raw data. Original uncropped scan images of

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Figure 6D.

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Supplementary Table 1 Common differential metabolites among groups.

792 36

Highlights 1. Vac pretreatment attenuated AP-induced liver injury in rats. 2. Vac pretreatment elevated metabolite 2-HB both in cecum and serum. 3. 2-HB attenuated the AP-induced hepatotoxicity both in vitro and in vivo.