Inflammation ( # 2014) DOI: 10.1007/s10753-014-9886-1

Protective Effects of Lactobacillus plantarum NDC 75017 Against Lipopolysaccharide-Induced Liver Injury in Mice Xinyan Peng1,2,3 and Yujun Jiang1,2,4

Abstract—This study investigated the protective effect of Lactobacillus plantarum NDC 75017 (L. plantarum NDC 75017) against acute liver injury induced by lipopolysaccharide (LPS). Thirty male mice were randomly divided into the control, LPS, and LPS + L. plantarum NDC 75017 groups. In the LPS + L. plantarum group, the mice were orally pretreated with L. plantarum NDC 75017 for 15 days. At 16 days, the mice in the LPS and LPS + L. plantarum NDC 75017 groups were intraperitoneally injected with LPS at 4 mg/kg body weight, whereas the control mice were treated with an equal amount of saline. After 8 h, the serum alanine transaminase (ALT), aspartate aminotransferase (AST), and histology changes were examined. The oxidative stress markers and pro-inflammatory cytokines in the liver were also examined. Meanwhile, the expression of nuclear factor κB (NF-κB) mRNA and toll-like receptor 4 (TLR4) in the liver was determined by qRT-PCR. The LPS group showed an increase in ALT and AST, whereas the LPS + L. plantarum NDC 75017 group showed a significant decrease. In addition, pretreatment with L. plantarum NDC 75017 can attenuate LPS-induced oxidative stress and inflammatory response. Furthermore, the increase of hepatic NF-κB and TLR4 mRNA induced by LPS was significantly downregulated by the pretreatment with L. plantarum NDC 75017. These data show that pretreatment with L. plantarum NDC 75017 protects against LPS-induced oxidative stress and inflammatory injury in the liver of mice, which may be attributed to the inhibition of the TLR4-NF-κB pathway. KEY WORDS: Lactobacillus plantarum NDC 75017; liver; toll-like receptor 4; nuclear factor κB; mice.

INTRODUCTION Lipopolysaccharide (LPS) is a major component of the cell wall from Gram-negative bacteria (GNB). It can induce some clinical symptoms, including endotoxemia, sepsis, and septic shock [1–3], which have been attributed to the production of bioactive lipids, reactive oxygen species (ROS), and inflammatory cytokines [4]. Toll-like receptors (TLRs) are inductors of an innate immune response and are involved in many liver injuries 1

Key Laboratory of Dairy Science, Ministry of Education, Department of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China 2 National Research Center of Dairy Engineering and Technology, Northeast Agricultural University, Harbin, Heilongjiang 150086, China 3 College of Food Engineering, Ludong University, Yantai, Shandong 264025, China 4 To whom correspondence should be addressed at Key Laboratory of Dairy Science, Ministry of Education, Department of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China. E-mail: [email protected]

induced by microbial toxins [5]. Of note, the specific receptor of LPS is TLR4 [6], which mediates the activation of nuclear factor κB (NF-κB) during LPS-induced inflammatory response [7–10]. The expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), IL-6, and IL-12, would be subsequently activated. The activation of NF-κB can also induce the expression of inducible nitric oxide synthase (iNOS) and further cause the release of nitric oxide (NO), in turn exacerbating oxidative stress [5, 11]. The use of probiotics is one of the new strategies for preventing and treating liver diseases, including acute hepatic failure [12], nonalcoholic fatty liver disease [13], and alcoholic liver disease [14]. Some studies have reported that probiotics provide protection against liver injury by bacteria or other toxins [9, 15, 16]. One of the potential mechanisms of probiotics is their ability to decrease proinflammatory production, such as that of TNF-α, IL-6, and interferon gamma (IFN-γ) , via downregulation of NF-κB or TLR4 signaling [12, 17] or through their anti-oxidative ability [18]. Lactobacillus plantarum (L. plantarum) NDC

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Peng, and Jiang 75017, a potential probiotic, showed anti-inflammatory and cholesterol-lowering activities in vitro in our previous study [19]. We hypothesize that L. plantarum NDC 75017 can be beneficial for protecting against acute liver injury through its anti-inflammatory and anti-oxidative abilities. In this study, we evaluated the hepatoprotective effects of L. plantarum NDC 75017 on LPS-induced liver injury in mice. To further study its potential mechanism, we examined the expression of TLR4 and NF-κB mRNA in liver tissue.

MATERIALS AND METHODS Chemicals and Reagents Escherichia coli LPS (serotype O55:B5) was purchased from Sigma (St Louis, MO, USA). TNF-α, IL1β, IL-6, IL-10, and IL-12 (p70) enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D (USA). Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), NO, and iNOS assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China). The BCATM protein assay kit was purchased from Wuhan Boster Bio-engineering Limited Co. (Wuhan, China). TRIzol was purchased from TakaRa Biotechnology Co., Ltd (Dalian, China). The other materials were analytical reagents.

approved by the Animal Care and Use Committee of Heilongjiang Province, People’s Republic of China. Experiment Design Animal experiments began after 5 days of adaptation. Thirty male mice were randomly divided into three groups (n=10 in each group): healthy control group (control), LPS-induced liver injury group (LPS), and L. plantarum NDC 75017 protective group (LPS + L. plantarum). The mice in the control and LPS groups received oral gavages with normal saline, whereas the mice in the LPS + L. plantarum group were gavaged with 0.1 ml/10 g body weight of L. plantarum NDC 75017 (1×109 CFU/ml) for 15 days prior to exposure to LPS. At 16 days, the mice in the LPS + L. plantarum and LPS groups were intraperitoneally (i.p.) injected with LPS at 4 mg/kg. The control mice were injected with an equal volume of saline. As in our pilot study, 8 h after LPS or saline injection, blood was collected under anesthesia and the liver tissues were dissected for examination of histological and biochemical changes. The blood was centrifuged at 3,000×g for 15 min at 4 °C; the separated serums were collected and stored at −20 °C for testing. The liver tissues were divided into two parts: one part was fixed in 10 % neutral formalin for histological analysis, and the other was stored in liquid nitrogen for examination. Biochemical Assays

Lactobacillus Strain and Growth Conditions L. plantarum NDC 75017 was isolated from a Chinese traditional fermented yogurt (from the Tongliao range of Inner Mongolia, China). The bacterium was anaerobically grown at 30 °C in de Man-Rogosa-Sharpe (MRS) broth (Difco, USA) overnight, as in our previous study [19]. The bacterial cells were collected by centrifugation at 3,000×g for 10 min, washed three times with PBS, and adjusted to 1×109 colony-forming units (CFU)/ml for oral administration to mice. Animals Male Kunming mice (weighing 18–22 g) were obtained from the experimental animal center at the Harbin Veterinary Research Institute (HVRI). Every five animals were kept in a cage. Room temperature was kept at 24±1 °C and relative humidity at 55±10 %. The mice were placed under a 12-h light/dark cycle (light from 7 a.m. to 7 p.m.). All animals were fed with a standard laboratory diet and given water ad libitum. All of the animal experiments were

The levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) in the mice were determined using the 756P UV-visible spectrophotometer according to the manufacturer’s instructions (Jiancheng Institute of Biological Engineering, Nanjing, China). Measurement of MDA, SOD, GSH, NO, and iNOS Levels Parts of the liver tissue were homogenized by a weight (g)/volume (ml) proportion of 1:9 in cold physiological saline to prepare 10 % tissue homogenates and then centrifuged at 3,000g (4 °C) for 15 min. The supernatants were collected to examine the changes of MDA, SOD, GSH, NO, and iNOS levels in the liver tissues of the mice. The supernatants were isolated, and their protein concentrations were measured using the BCA protein assay, according to manufacturer’s instructions (Wuhan Boster Bioengineering Limited Co., Wuhan, China). The contents of MDA, NO, and GSH and the activities of iNOS and SOD were examined according to the manufacturer ’s

L. plantarum NDC 75017 Protects Against LPS-Induced Liver Injury instructions (Jiancheng Institute of Biological Engineering, Nanjing, China).

Histopathological Examination After 48 h, the liver tissues were fixed in 10 % formalin. They were then dehydrated through graded alcohol and embedded in paraffin. They were washed with running water overnight, and then sequentially treated with graded alcohol and xylene, and then embedded in paraffin. Finally, they were sectioned with a microtome at 5-μm thickness and stained with hematoxylin and eosin (H.E) for histopathological assessment.

Statistical Analysis All data were expressed as mean ± SD. All statistical analyses were carried out using the SPSS 16 software (Chicago, USA). A one-way analysis of variance (ANOVA), followed by a least significant difference (LSD) test, was used to compare between any two means when the variance was homogeneous, or Dunnett’s T3 test was used. Values of p < 0.05 represented significant differences.

RESULTS Changes of ALTand AST Levels in Serum

Measurements of IL-1β, TNF-α, IL-6, IL-10, and IL-12 Levels in Liver Tissue The concentrations of cytokines, including IL-1β, TNF-α, IL-6, IL-10, and IL-12, in the supernatants of the liver homogenates were assayed by mouse ELISA kits according to the manufacturer’s protocol (R&D Systems, Minneapolis, USA). The optical densities (OD) were determined by the microplate reader at 450 nm.

Quantitative Reverse Transcription-PCR Examination Total RNA was isolated by using TRIzol extraction according to the manufacturer’s instructions (Invitrogen Inc, Carlsbad, CA). The quality of RNA was verified by evaluating OD 260 nm/280 nm. One microgram total RNA was reverse-transcribed to cDNA using the Prime Script RT-PCR kit (Takara, Dalian, China). RT-PCR analysis for NF-κB was performed using a Light-Cycler 480 System (Roche Applied). The primer sequences and the conditions in which PCR was performed are as follows: NF-κB: forward, 5′-CAC TGT CTG CCT CTC TCG TCT-3′, reverse, 5′ -AAG GAT GTC TCC ACA CCA CTG-3′; TLR4: forward, 5′-TCT GCC TTC ACT ACA GAG ACT-3′, reverse, 5′-AGT CTT CTC CAG AAG ATG TGC-3′; β-actin: forward, 5′-GAG ACC TTC AAC ACC CCA GC-3′, reverse, 5′-ATG TCA CGC ACG ATT TCC C-3′. Standard cycling conditions were used, including a preamplification step of 95 °C for 10 min, followed by amplification for 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 20 s. The mean threshold cycle (Ct) was calculated to determine the amount of target, normalized to an endogenous reference (β-actin) and relative to a calibrator (2−ΔΔCt) [20].

The levels of serum ALT and AST were determined 8 h after the challenge with LPS. Figure 1 shows the results. Compared with the control group, the levels of serum ALT and AST increased significantly to 106.4± 12.4 and 170.4±19.8 U/l (both p

Protective effects of Lactobacillus plantarum NDC 75017 against lipopolysaccharide-induced liver injury in mice.

This study investigated the protective effect of Lactobacillus plantarum NDC 75017 (L. plantarum NDC 75017) against acute liver injury induced by lipo...
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