Sodium Butyrate Inhibits the Inflammation of ... - ACS Publications

Subscriber access provided by Karolinska Institutet, University Library

Bioactive Constituents, Metabolites, and Functions

Sodium Butyrate Inhibits the Inflammation of LPS-induced Acute Lung Injury in Mice by Regulating the TLR4/NF-#B Signaling Pathway Jing Liu, Guangjun Chang, Jie Huang, Yan Wang, Nana Ma, Animesh Chandra Roy, and Xiangzhen Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06359 • Publication Date (Web): 20 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Journal of Agricultural and Food Chemistry

1

Sodium Butyrate Inhibits the Inflammation of LPS-induced Acute Lung Injury

2

in Mice by Regulating the TLR4/NF-κB Signaling Pathway

3

Jing Liu, Guangjun Chang, Jie Huang, Yan Wang, Nana Ma, Animesh-Chandra Roy, and Xiangzhen Shen*

4 5

College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095,

6

P. R. China

7

*Corresponding author:

8

Xiangzhen Shen,

9

Department of Veterinary Clinical Science, College of Veterinary Medicine, Nanjing Agricultural

10 11

University,

12

Nanjing, 210095, P. R. China;

13

Phone: +86 25 84395505;

14

Fax：+86 25 84398669;

15

Email：[email protected]

16

ACS Paragon Plus 1 Environment


17

Abstract

18

Bacterial pneumonia is a common disease in dairy herds worldwide, which brings

19

great economic losses to farmers. Sodium butyrate (SB), an inhibitor of histone

20

deacetylase, plays an important role in limiting inflammation. The purpose of this study

21

was to investigate the protective effects of SB on LPS-induced acute lung injury (ALI)

22

in mice and explore the potential mechanism of SB protection. Thirty ICR mice were

23

randomly divided into three groups (n=10): a PBS intratracheal instillation group, an

24

LPS intratracheal instillation group, and an SB gavage group (SB was given 1 h before

25

the LPS stimulation). After 12h, samples of the blood and lung tissue were collected

26

from the mice for experimental analysis. The results showed that the concentration of

27

inflammatory cytokines (IL1β and TNF-α); myeloperoxidase (MPO) activity in the

28

lung tissue and blood; the protein abundance of Toll-like receptor 4 (TLR4), nuclear

29

factor kappa B (P65), phosphorylated P65 (p-P65), inhibitor κBα (IκBα) and

30

phosphorylated IκBα (p-IκBα); and the relative mRNA expression of genes associated

31

with inflammation, such as TLR4, NF-κB, IL1β,IL6, and TNF-α, were significantly

32

upregulated in the LPS group compared with the PBS group. However, the SB addition

33

markedly down-regulated the levels of these parameters in the LSB group compared

34

with those in the LPS group. Furthermore, the structure of the lung tissue from the LPS

35

group was severely disrupted compared to that of the PBS group. However, with SB

36

administration, the severe structural disruption was relieved. In addition, an

37

immunohistochemical analysis showed that positive immunoreactions to TLR4, P65,

38

and TNF-α were significant in the LPS group; however, SB addition markedly


Page 2 of 39

Page 3 of 39


39

attenuated this phenomenon. In conclusion, the ALI mouse model was successfully

40

established with an intratracheal instillation of LPS. Furthermore, gavage with sodium

41

butyrate inhibited inflammation in LPS-induced ALI.

42

Keywords: lipopolysaccharide (LPS); acute lung injury (ALI); inflammation; sodium

43

butyrate

44



45

Introduction

46

The bovine respiratory disease complex (BRDC) is generally known as shipping

47

fever, pneumonia, and bronchopneumonia. Investigations have shown that BRDC

48

poses a great challenge to labor expenses, drug costs, and death around the world.1-3

49

BRDC is an infectious respiratory disease characterized by multifactorial causes.4

50

Bacterial pneumonia is a common BRDC respiratory disease in the cattle industry

51

throughout the world. The pathogenesis of bacterial pneumonia can involve

52

predisposing factors, which result in functional deficiencies in the immune system that

53

reduce the resistance to bacterial infection.5 Many reports have shown that the important

54

bacteria involved in bacterial pneumonia are part of the Pasteurellaceae family, which

55

is classified as gram-negative bacteria.6 Antibiotics are always selected to control the

56

pneumonia induced by bacteria. In North America, cattle are administered antibiotics

57

for BRDC control and clinical treatment.7 Although antibiotics can reduce BRDC, they

58

are often less effective and further antibiotic treatments are required for bacterial

59

pneumonia.8 In addition, resistance against antibiotics caused by the long-term

60

application of antibiotics and scientific means of keeping animals healthy and

61

improving animal welfare have attracted people’s attention worldwide. Therefore, there

62

is an urgent need for natural substances that can both reduce the incidence of bacterial

63

pneumonia and solve the problem of susceptibility against infection.in the cattle

64

industry.

65

Lipopolysaccharide (LPS), the principal virulence element of the cytoderm of

66

gram-negative bacteria, has been thought to be the critical risk factor for ALI.9,10 As the


Page 4 of 39

Page 5 of 39


67

specific receptor of LPS, TLR4 can recognize gram-negative bacteria. Research has

68

demonstrated that as soon as LPS is recognized by TLR4, the NF-κB signaling pathway

69

is activated in the lung.11 Inhibitors of the nuclear factor kappaB kinase (IKK) complex,

70

consisting of IKKα and IKKβ, are the primary regulators involved in the activation of

71

the NF-κB signaling pathway.12 According to our knowledge, under normal conditions,

72

NF-κB exists in the cytoplasm in a non-activated state due to its inhibition by IκB,

73

which can combine with NF-κB. Phosphorylation occurs when IκB is activated by the

74

activated IKK complex. As a result, NF-κB is separated from phosphorylated IκB,

75

leading to the activation of NF-κB. Phosphorylated NF-κB, the active form of NF-κB,

76

is transferred into the nucleus and then initiates the expression of pro-inflammatory

77

genes, such as TNF-α, IL6,and IL1β.13,14 Generally, we can observe cytokines,

78

including TNF-α and IL1β, at inflammatory sites and in the peripheral blood after they

79

are released by macrophages and neutrophils. Moreover, the inflammatory response

80

can be promoted by cytokines by initiating the infiltration of innate immune cells, such

81

as neutrophils and macrophages, into inflamed tissues.15

82

Butyric acid, a short-chain fatty acid (SCFA),is naturally found in butter and

83

cheese.16 It can also be produced by

84

endogenous bacterial anaerobic fermentation of fiberpolysaccharides.17 Previous

85

studies have indicated that by reducing the activation of NF-κB and abolishing the

86

expression of LPS-induced pro-inflammatory cytokines, butyrate can suppress the

87

inflammatory response induced by LPS.18,19 Sodium butyrate(SB), one of the butyrate

88

salts, is often used in place of butyric acid in animal studies and practical industry

ruminants and monogastric species through



89

applications due to its physical properties, including that it is solid, stable, and much

90

less odorous.20 It has been reported that SB can ameliorate the inflammatory response

91

and attenuate organ dysfunction in different disease models.21 According to Dai et al.,20

92

SB plays an important role in attenuating high-concentrate diet-induced inflammation

93

in the rumen epithelium of dairy goats. More recently, Xu et al. proposed that SB

94

supplementation suppresses the adaptive response to inflammation in LPS-stimulated

95

bovine hepatocytes.22 Moreover, in recent years, investigations on the ability of SB to

96

diminish respiratory inflammation in mice have also been conducted. Previous study

97

showed that,23 the intraperitoneal injection of SB alleviates LPS-induced ALI in mice

98

via inhibiting HMGB1 release. However, according to our knowledge, few studies

99

regarding respiratory inflammation induced by gram-negative bacteria in ruminants

100

have been conducted to investigate the anti-inflammatory mechanism of SB.

101

By analyzing the primary literature, we can conclude that SB has great potential in

102

modulating the LPS-induced inflammatory response. Therefore, we hypothesized that

103

SB supplementation can attenuate gram-negative bacterial pneumonia in ruminants. In

104

this study, we established an ALI mouse model by intratracheal instillation of LPS and

105

oral treatment with SB. The objective of this study was to investigate the potential

106

mechanism by which SB inhibits inflammation in LPS-induced ALI in mice by

107

regulating the TLR4/NF-κB pathway.

108


Page 6 of 39

Page 7 of 39


109

Materials and methods

110

Ethics statement

111

All of the experiments were conducted with the approval of the Animal Care and Use

112

Committee of Nanjing Agricultural University. Sampling procedures adhered to the

113

Guidelines for Experimental Animals of the Ministry of Science and Technology (2006,

114

Beijing, China).

115

Chemical

116

Sodium butyrate (C4H7NaO2, CAS number 156-54-7) was purchased from YuanYe

117

Biotechnology, Shanghai, China. A Milli-Q system (Bedford, MA, United States) was

118

used to produce ultrapure water, which was used as the solvent for the dilution of

119

sodium butyrate. Analytical grade chloroform and isopropanol were obtained from

120

Shanghai Lingfeng Chemical, Shanghai, China. All other reagents used in the study

121

were of analytical grade.

122

Animals and the experimental design

123

Thirty ICR mice (female, 8-10 weeks old, and 20-25 g each) were obtained from

124

Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China). They were housed

125

in independent ventilation cages (IVCs) at 22±2℃ with sufficient water and food on a

126

12h light/dark cycle. The mice were housed in these controlled conditions for at least 7

127

days to acclimatize them to the laboratory. The mice were randomly separated into three

128

groups, which were the PBS (Solarbio, Beijing, China) group, LPS (Sigma-Aldrich,

129

L2880, LPS from E. coli 055:B5) group and SB (YuanYe Biotechnology, Shanghai,

130

China) + LPS (LSB) group. The mice were anesthetized by inhaling isoflurane (100



131

mg/kg) and immediately administered LPS by tracheal instillation (7.5 mg/kg in PBS).

132

The mice in the PBS group received the same volume of PBS, but without LPS, as the

133

LPS group. To investigate the protective effects of SB, mice were treated by intragastric

134

administration of SB (25 mg/kg) 1 h before the LPS treatment.

135

Sample collection

136

Twelve hours after the LPS administration, Eight hundred microlitre mouse blood was

137

collected into an anticoagulant tube containing EDTA-Na2 and centrifuged at 3,000×g

138

and 4°C for 15 minutes to isolate the plasma, which was then stored at -20°C for future

139

analysis. When sampling the lung tissue, the mice were anesthetized with inhaled

140

isoflurane and then sacrificed by cervical dislocation. The lung tissue samples were

141

flash frozen in liquid nitrogen and then stored at -70℃ for future analysis.24,25

142

Enzyme-linked immunosorbent assay

143

The concentration of inflammatory cytokines in the plasma of the mice was detected

144

by using ELISA kits (IL1β, YFXEP00028, TNF-α, YFXEM00031, Yi Fei Xue

145

Biotechnology, Nanjing, China) according to the manufacturer’s instructions. The

146

range of the ELISA kits were 5 ng/L-100 ng/L and 25 ng/L-500 ng/L for IL1β and TNF-

147

α, respectively. Briefly, plasma from mice was incubated in coated 96-wells plates for

148

2h at 37℃. The plasma was diluted to a certain concentration, followed by applying in

149

duplicate. Additionally gradient concentration was obtained for establishing standard

150

curves. Then washing the plates followed by incubation with detection antibody. The

151

conjugate was added into the plates for 30 min after washing it again, followed by

152

adding substrate solution. Stopping buffer was used to end the chromogenic reaction.


Page 8 of 39

Page 9 of 39


153

The absorbance values were detected at 460 nm and according to standard curves,

154

concentration of inflammatory cytokines could be acquired. Intra and inter assay

155

variations of ELISA kits (IL1β and TNF-α) (%CV) were less than 9 and 11 respectively.

156

MPO activity assay

157

Myeloperoxidase (MPO) activity in the lung tissue and blood of the mice was detected

158

by using an MPO kit (A044, Nanjing Jiancheng Bioengineering Institute, Nanjing,

159

China). According to the manufacturer’s instructions, initially, the lung tissue samples

160

were homogenized with 5% extraction buffer, and the plasma was diluted with an equal

161

amount of extraction buffer. Reaction buffer was added into the mixture mentioned

162

above at a ration 9：1, followed by water bath, 37℃ for 15 min. after that chromogenic

163

agent was mixed into the mixture fully, followed by 37℃ water bath for 30 min. then

164

terminating agent was used to end the chromogenic reaction. Enzymatic activity was

165

determined by measuring the absorbance of the colorimetric reaction at 460 nm using

166

a spectrophotometer (UV-5600, Metash Instruments Co., Ltd., Shanghai, China) and

167

expressed as units per gram of total protein (u/g). The detected range of MPO kit was

168

2.9-393.3 U/L, and its intra and inter assay variations (%CV) were 3.9 and 6.66

169

respectively.

170

Histological analysis

171

The lung tissue samples for histological analysis were immersed in a 4%

172

paraformaldehyde solution for fixation, and then the fixed lung tissue samples were

173

embedded in paraffin after being dehydrated. Five-micron-thick sections were cut,

174

stained with hematoxylin and eosin (H&E) and visualized. Light microscopy was used



175

to observe these slides, and the images were captured by using a high-resolution digital

176

camera and NIS Elements F 3.0 image acquisition software (Nikon Corporation,

177

Minato-ku, Tokyo, Japan). As previously described, the semi-quantitative scoring

178

method was used to assess the histopathological changes in the lung tissue.9 Briefly,

179

according to the severity of each indication observed in the lung tissue, including

180

interstitial inflammation, inflammatory cell infiltration, congestion and edema. A

181

numerical score was assigned from 0 (normal) to 4 (severe) for each indication. Finally,

182

the individual scores for the four indications can be added to obtain the total lung injury

183

score. In consideration of observer bias, two blinded observers assessed two tissue

184

sections from each animal.

185

RNA analysis

186

Fifty milligrams of lung tissue was removed from -70℃ for total RNA extraction by

187

using RNAiso Plus(Takara Co., Otsu, Japan). The extraction protocols were conducted

188

according to the manufacturer’s instructions. Using NanoDrop One spectrophotometer

189

(Thermo Fisher Scientific Inc., Waltham, MA, United States) and agarose gel (1%)

190

electrophoresis, the concentration and quality of RNA can be assessed accurately. Only

191

samples with A260 ： A280 rations between 1.8 and 2.0 were used for later cDNA

192

analysis. Then, 500 ng of total RNA was used to synthesize the first-standard cDNA by

193

using a PrimeScript RT MasterMix Perfect Real Time Kit (Takara Co., Otsu, Japan)

194

according to the manufacturer’s instructions. The relative gene expression levels of

195

TLR4, NF-κB, IL1β, IL6 and TNF-α were evaluated by using real-time PCR. cDNA

196

obtained from the reverse transcriptional reaction was diluted eightfold and subjected


Page 10 of 39

Page 11 of 39


197

to real-time PCR, which was conducted according to the manufacturer’s instructions

198

for ChamQTM SYBR qPCR Master Mix (Vazyem, Nanjing, China). The real-time PCR

199

conditions were denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s

200

and 60°C for 30 s and then running a melting curve. GAPDH was selected as an internal

201

reference gene. Real-time PCR was performed for all the genes on an ABI 7300 real-

202

time PCR system (Applied Biosystems, Foster City, CA). The specific primers listed in

203

Table 1 were commercially synthesized by Shanghai Generay Biotech Co., Ltd.

204

(Shanghai, China). The real-time PCR results are represented as the fold change, which

205

was analyzed using the 2-ΔΔCTmethod according to a previous study.26

206

Western-Blot analysis

207

Approximately Fifty milligrams of lung tissue sample was weighed and transferred into

208

a 2 mL tube with 0.5 mL of ice-cold RIPA protein isolation buffer (Beyotime, Shanghai,

209

China). The mixture of lung and RIPA was treated by POLYTRON PT

210

(KINEMATICA, Switzerland), a mechanism used for homogenizing tissue in RIPA,

211

for 15 s, incubated on ice for 10 minutes and then centrifuged at 4℃ and12, 000 rpm

212

for 20 minutes. Then, a new 1.5 mL tube was used to collect the supernatant containing

213

the total protein. The transfer of the supernatant was performed carefully to avoid

214

touching the pellet at the bottom. Then, it was necessary to determine the concentration

215

of the total protein using a BCA Protein Assay kit (Thermo Fisher Scientific Inc.). Forty

216

micrograms of total protein mixed with protein loading buffer was loaded into 12%

217

sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels to

218

separate the proteins. After separation, SDS-PAGE with blots was stripped and then



219

each blot on the SDS-PAGE was transferred onto nitrocellulose membranes (Pall

220

Gelman Laboratory, Ann Arbor, MI, United States).Immediately, each membrane was

221

blocked at room temperature (25°C) with Tris-buffered saline with Tween 20 (TBST)

222

containing 7% skim milk powder, or 5% bovine serum albumin (BSA) (Solarbio,

223

Beijing, China) when blocking for phosphorylated protein detection, for 2 h, and then

224

incubated with primary antibodies, including anti-TLR4 (1:250, ab13556， Rabbit,

225

Abcam, USA), anti-P65 (1:1000, AF1234, Rabbit, Beyotime, China), anti-p-P65

226

(1:1000, AN371, Rabbit, Santa Cruz, USA), anti-IκBα (1:1000, #2859, Rabbit, Cell

227

Signaling Technology Danvers, MA), anti-p-IκBα (1:1000, #4812, Rabbit, Cell

228

Signaling Technology, Danvers, MA), and anti-GAPDH (1:1000， AG019, Mouse,

229

Beyotime, China). The incubation condition was 4°C overnight. Horseradish

230

peroxidase (HRP)-conjugated secondary antibodies, goat-anti-rabbit (1:1000, A0208,

231

Goat, Beyotime, China) and goat-anti-mouse (1:1000, A0216, Goat, Beyotime, China),

232

were incubated at room temperature for 2 h to detect the primary antibodies. After the

233

incubations with the primary and secondary antibodies, the nitrocellulose membranes

234

were washed 6 times with TBST and each wash was10 minutes long. The results were

235

visualized by using High-sig ECL Western Blotting Substrate (Tanon Science ＆

236

Technology Co., Ltd, Shanghai, China). The signals were recorded as optical results by

237

using a ChemiDoc imaging system (Bio-Rad Laboratories, Inc., USA). Imaging Lab

238

(Bio-Rad Laboratories, Inc., USA), a software package, was used to analyze the results

239

for the target protein band.

240

Immunohistochemical analysis


Page 12 of 39

Page 13 of 39


241

The immunohistochemical analysis was begun using a process that was consistent with

242

other processes for preparing histological sections. The tissue was fixed with

243

paraformaldehyde, dehydrated, embedded in paraffin, and deparaffinized. After

244

deparaffinization, the sections for immunohistochemical analysis were treated for

245

antigen retrieval and blocked with 3% H2O2 for 25 minutes at room temperature to

246

eliminate endogenous peroxidase activity. The sections were washed with PBS after

247

each step. Then, the sections were blocked with 3% BSA for 30 minutes at room

248

temperature and incubated with a mouse polyclonal antibody (anti-TLR4, anti-p65, or

249

anti-TNF-α) overnight at 4 ℃. After washing with PBS, the sections were incubated

250

with a HRP-conjugated secondary antibody and then washed with PBS. The visualized

251

results of the products were obtained by using a freshly prepared diaminobenzidine

252

(DAB) reaction mixture. Each section was counterstained with hematoxylin for 3

253

minutes, dehydrated and covered with a coverslip. Images of the sections of lung tissue

254

treated by immunohistochemical analysis were captured by using abiological

255

microscope system (Chongqing MIC Technology Co., Ltd., China).

256

Statistical analysis

257

All of the data generated in the present study are expressed as the mean ± standard error

258

of the mean (SEM) and were analyzed by SPSS 20.0 software (IBM Inc., New York,

259

NY, United States) using one-way ANOVA with Dennett’s post-test. A value of P ˂

260

0.05 was considered statistically significant.

261



262

Results

263

Concentration levels of inflammatory cytokines in the plasma from the mice

264

We found that compared with PBS group, the LPS group showed a significant

265

increase in the concentrations of IL1β and TNF-α (P < 0.01, Figure 1A). However, with

266

the intragastric administration of SB 1 h before the LPS treatment, the concentrations

267

of IL1β and TNF-α were significantly decreased compared with those of the LPS group

268

(P < 0.01, Figure 1A). In addition, with the SB treatment, TNF-α remained at a level

269

similar to that detected in the PBS group.

270

MPO analysis

271

The detection of MPO activity in both the lung tissue and blood samples was

272

performed by MPO kit. The results in Figure 1B show that MPO activity was

273

significantly elevated in the LPS-induced ALI in mice compared with the PBS-treated

274

mice (tissue and plasma, P < 0.01). However, the addition of SB generated a marked

275

decline in MPO activity in the LSB group compared with that in the LPS group (tissue

276

and plasma, P < 0.05).

277

Histopathological analysis of the lung tissue

278

As shown in Figure 1C, compared with the lung tissue of the PBS group, that of

279

the LPS group showed an obvious accumulation of a large number of inflammatory

280

cells in the alveolar wall and bronchial wall, a thickened alveoli septum caused by

281

edema leading to a decrease in the alveolar space and interstitial congestion, which was

282

confirmed by the lung injury scores (Table 2). As expected, the addition of SB markedly

283

mitigated the effects of LPS-induced ALI, including edema, congestion, and


Page 14 of 39

Page 15 of 39


284

inflammation in the peribronchovascular interstitium (Figure 1C), and decreased the

285

component scores and total values of the lung injury scores (Table 2).

286

Relative gene expression of TLR4, NF-κB, and inflammatory cytokines in the lung

287

tissue of the mice

288

As shown in Figure 2, the relative gene expression of TLR4 and NF-κB was

289

significantly upregulated in the LPS group compared with the PBS group. However,

290

with the administration of SB, there was a significant decrease in the gene expression

291

of TLR4 and NF-κB in the LSB group compared with the LPS group.

292

The relative gene expression of pro-inflammatory cytokines, including IL1β, IL6,

293

and TNF-α, which are commonly synthesized at inflammatory sites,25 showed an

294

extremely significant increase in the LPS group compared with the PBS group (P

Sodium Butyrate Inhibits the Inflammation of ... - ACS Publications

Recommend Documents