Sodium Butyrate Inhibits the Inflammation of Lipopolysaccharide

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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

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Journal of Agricultural and Food Chemistry

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

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

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

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

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P. R. China

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*Corresponding author:

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Xiangzhen Shen,

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

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University,

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Nanjing, 210095, P. R. China;

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Phone: +86 25 84395505;

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Fax：+86 25 84398669;

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Email：[email protected]

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ACS Paragon Plus 1 Environment


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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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immunohistochemical analysis showed that positive immunoreactions to TLR4, P65,

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


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

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

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butyrate inhibited inflammation in LPS-induced ALI.

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

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butyrate

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Introduction

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

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

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

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

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

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throughout the world. The pathogenesis of bacterial pneumonia can involve

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

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

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

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

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

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

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

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

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application of antibiotics and scientific means of keeping animals healthy and

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

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

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

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industry.

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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as neutrophils and macrophages, into inflamed tissues.15

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

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cheese.16 It can also be produced by

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endogenous bacterial anaerobic fermentation of fiberpolysaccharides.17 Previous

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

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

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

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

ruminants and monogastric species through



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

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

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

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

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

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supplementation suppresses the adaptive response to inflammation in LPS-stimulated

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

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

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

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

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regarding respiratory inflammation induced by gram-negative bacteria in ruminants

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

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

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

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

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

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

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

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regulating the TLR4/NF-κB pathway.

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Materials and methods

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Ethics statement

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

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Committee of Nanjing Agricultural University. Sampling procedures adhered to the

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

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Beijing, China).

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Chemical

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

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

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

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sodium butyrate. Analytical grade chloroform and isopropanol were obtained from

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

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were of analytical grade.

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Animals and the experimental design

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

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

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

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

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

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

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

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



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

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

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

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

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Sample collection

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

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

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

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

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

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

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Enzyme-linked immunosorbent assay

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

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

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

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

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

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

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duplicate. Additionally gradient concentration was obtained for establishing standard

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

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

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


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

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

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

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MPO activity assay

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

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

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

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

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

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

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

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

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

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

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

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

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respectively.

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Histological analysis

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

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

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

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



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

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

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

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

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

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

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

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

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

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sections from each animal.

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RNA analysis

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

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

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

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

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

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

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

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

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

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

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obtained from the reverse transcriptional reaction was diluted eightfold and subjected


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

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

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

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

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

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

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

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

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

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Western-Blot analysis

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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incubations with the primary and secondary antibodies, the nitrocellulose membranes

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

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

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

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

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

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for the target protein band.

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Immunohistochemical analysis


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

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

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

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deparaffinization, the sections for immunohistochemical analysis were treated for

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

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

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

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

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

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

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

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

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

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treated by immunohistochemical analysis were captured by using abiological

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

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Statistical analysis

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

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

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

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0.05 was considered statistically significant.

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Results

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Concentration levels of inflammatory cytokines in the plasma from the mice

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

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

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

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

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

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similar to that detected in the PBS group.

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MPO analysis

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

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

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

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

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

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and plasma, P < 0.05).

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Histopathological analysis of the lung tissue

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

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

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

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

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

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


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

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

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

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tissue of the mice

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

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

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

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

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

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

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

Sodium Butyrate Inhibits the Inflammation of Lipopolysaccharide

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