Protective Effects of Selenium-Enriched Probiotics on Carbon

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The Protective Effects Of Selenium-enriched Probiotics On Carbon Tetrachloride-induced Liver Fibrosis In Rats Yunhuan Liu, Qing Liu, Gengping Ye, Alamzeb Khan, Jin Liu, Fang Gan, Xian Zhang, Shahnawaz Kumbhar, and Kehe Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5039184 • Publication Date (Web): 16 Dec 2014 Downloaded from http://pubs.acs.org on December 18, 2014

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Journal of Agricultural and Food Chemistry

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The Protective Effects Of Selenium-enriched Probiotics On Carbon

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Tetrachloride-induced Liver Fibrosis In Rats

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Short title: Effects Of Selenium-Enriched Probiotics On Rat Liver Fibrosis

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Yunhuan Liu, Qing Liu, Gengping Ye, Alamzeb Khan, Jin Liu, Fang Gan, Xian

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Zhang, Shahnawaz Kumbhar, Kehe Huang*

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Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls,

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Nanjing Agricultural University, Nanjing 210095, China

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∗Correspondence to: Dr. Kehe Huang, College of Veterinary Medicine, Nanjing

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Agricultural University, Nanjing 210095, Jiangsu Province, China

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Tel: +86-25-84395507

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Fax: +86-25-84398669

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: This study aimed to investigate the effects of Se-enriched probiotics

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(SP) on the liver fibrosis induced by CCl4 in rats. The results showed that SP

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significantly decreased serum alanine aminotransferase (87.0±1.96 U/L), aspartate

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aminotransferase (101±3.13 U/L), hepatic hydroxyproline (898±72.5μg/g) and

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malondialdehyde (2.39±0.34 nmol/mg) levels, but increased glutathione peroxidase

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(37.2±3.19 U/mg), superoxide dismutase (201±19.2 U/mg) and glutathione levels

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(3.32±0.25 mg/g) (P < 0.05) in rats treated by CCl4. SP suppressed hepatic

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inflammation and necrosis induced by CCl4. Moreover, SP significantly reduced the

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expression

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inflammation-related gene and induced apoptosis of activated hepatic stellate cells (P

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< 0.05) in rats treated by CCl4. Our results suggest that SP could protect the liver

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fibrosis by attenuating hepatic oxidative stress, suppressing hepatic inflammation

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and inducing apoptosis of hepatic stellate cells.

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KEYWORDS: Liver fibrosis, Selenium-enriched probiotics (SP), Oxidative stress,

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inflammation, Apoptosis.

of

α-smooth

muscle

actin,

collagen,

TGF-β1,

TIMP-1

and

38 39 40 41 42 43 44

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INTRODUCTION

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Hepatic fibrosis is a general pathological basis of hepatic cirrhosis and liver

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cancer which is induced by a variety of etiological factors1. So, prevention and

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treatment of fibrosis is one of the crucial therapeutic goals in hepatology. Some

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studies showed that reduction of oxidative stress is beneficial to ameliorate liver

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fibrosis2. Therefore, radical scavengers and antioxidants are emerged as effective

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antifibrotic agents to prevent liver fibrosis. It was generally believed that

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inflammation could initiate a series of events which leads to liver fibrosis3. The

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activated hepatic stellate cells (HSCs) are major targets for antifibrotic therapy. It has

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been shown that inducing the apoptosis of activated HSCs was an effective method

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to treat established experimental fibrosis4.

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Selenium (Se) is an essential element for human and animals5. It is an essential

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component of some antioxidant enzymes and plays a pivotal role in antioxidant

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defense system6. There are evidences which indicate that Se was decreased in

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chronic liver diseases7. Probiotics have also been considered as possible additive

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therapy in treating liver diseases caused by viral infection, alcoholism and metabolic

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disorders8-10. In addition, probiotics such as Lactobacillus and S. cerevisiae could

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prevent oxidative stress induced liver injury11, 12.

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In our labratory, a newly developed Se-enriched probiotics (SP) product was

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produced

by

culturing

Lactobacillus

acidophilus

(L.

acidophilus)

and

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Saccharomyces cerevisiae (S. cerevisiae) with sodium selenite being added into the

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culture medium in a suitable condition of microenvironment. The strains both have a

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strong ability to convert sodium selenite into organic Se. Previous studies of dietary

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SP supplementation for livestock, poultry and mice showed that the SP has the

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combination effects of selenium and probiotics 13-15.

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Up until now, there have not been any reports concerning the effects of SP on

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liver injury in rats. CCl4 is one of classical hepatotoxicant used to induce liver injury.

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Long-term treatment of CCl4 can cause chronic liver damage and is also a common

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recognized model for inducing hepatic fibrosis

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investigate the effect of SP on CCl4-induced liver fibrosis and further explore the

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underlying mechanisms.

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MATERIALS AND METHODS

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. The aim of current study was to

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Chemicals. GSH-Px, SOD, GSH, MDA and hydroxyproline assay kit was

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obtained from the institute of Jiancheng Biotechnology (Nanjing, China). The kit of

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assay total protein was purchased from the Biyuntian company (Nanjing, China).

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α-SMA monoclonal antibody were purchased from Abcam (Cambridge, UK). CCl4

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was purchased from the institute of Shoude (Nanjing, China).

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Selenium-enriched probiotics and probiotics. Both the Se-enriched probiotics

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(SP) and probiotics (P) products consist of two probiotic strains, L. acidophilus and

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S. cerevisiae. The colony-forming units (CFU) of L. acidophilus and S. cerevisiae in

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both products was about 1011/mL and 109/mL, respectively. The content of total Se in

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the SP is 10.0 mg/L, with >90% being organic Se and >75% being selenomethionine,

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which were detected by AF-610A atomic fluorescence spectrometer17.

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Animals and feeding. The study was carried out according to protocols

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approved by the Animal Care and Use Committee of Nanjing Agricultural University,

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(Certification No.: SYXK (Su) 2011-0036).

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Forty male Wistar rats weighing (200±20g) were purchased from the Center of

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Laboratory Animals, Yangzhou University (Yangzhou, China). The rats were housed

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in a controlled condition at 25±2°C and a 12 h light/dark cycle. The rats were

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acclimatized for more than one week to use. The content of selenium in the basal

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diet was 0.05 mg/kg. The sodium selenite (SS), probiotics (P) and Se-enriched

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probiotics (SP) diets were made by grinding the basal diet into powder which was

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then mixed with SS, P and SP. The doses of SS, P and SP were calculated and mixed

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with the basal diet to obtain three different kinds of new diets and then supplied to

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the rats.

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Experimental procedure. Forty male Wistar rats were randomly divided into

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five groups of 8 rats in each. Each group had 4 replicates, with 2 rats per replicate.

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They are: (A) Control group, (B) CCl4 group, (C) CCl4 + P group, (D) CCl4 + SS

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group, (E) CCl4 + SP group. Group (A) received an intraperitoneal injection of olive

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oil (2 ml/kg body weight) twice weekly for 7 weeks. Groups (B-E) received

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intraperitoneal injection with CCl4 (2 ml/kg body weight, 1:1 in olive oil) twice

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weekly for 7 weeks. Groups (A-B) were fed the basal diet containing 0.05µg/g of Se

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per day. Groups (C-E) were fed the P diet, SS diet, SP diet per day, respectively. The

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number of viable microbial cells was approximately 109 CFU/g in the SP and P diets.

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The final total Se concentration was 0.3µg/g in the SS and SP diets. During the

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experimental period, there were no rats died. Blood and liver samples were collected

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at the end of 7 week and immediately stored at −70 °C.

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Biochemical analysis of serum enzymes. Serum was obtained from blood by

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centrifugating at 5000 g for 5 min at 4°C. Serum ALT and AST were measured by

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using automated chemistry analyzer.

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Measurement of GSH, GSH-Px, SOD and MDA in liver tissue. Liver

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samples were weighed and homogenized with nine volumes of ice-cold buffer

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(1mmol/L EDTA, 0.32 mol/L sucrose and 10 nmol/L Tris-HCl, pH =7.4). The

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homogenates was centrifuged at 5000 g for 5 min. The activities of GSH-Px and

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SOD and the levels of GSH and MDA in liver tissues were determined using

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commercial assay kit according to the instructions.

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Assay of hepatic hydroxyproline in liver tissue. Hepatic hydroxyproline was

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detected using a commercial assay kit. The content of hepatic hydroxyproline was

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represented as µg of hydroxyproline per g of wet liver tissues.

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Histopathological examination and immunohistochemical staining. Liver

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samples from rats were fixed in 10% formalin. Hematoxylin-eosin (H&E) and Sirius

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red stains were performed using standard procedures. The Sirius red staining was

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used for analysis of hepatic fibrosis. A computerized image-analysis system was used

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to quantifiy the liver fibrosis (Image-Pro Plus version 6.0; Media Cybernetics, MD,

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USA). Data for liver fibrosis were showed as the mean percentage of the total

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hepatic area in the liver sections. For immunohistochemical examination, the liver

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tissues were incubated with monoclonal antibody of α-SMA and then incubated with

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streptavidin–peroxidase complex. The peroxidase conjugates were visualized by

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using diaminobenzidine (DAB) solution subsequently. The liver sections were

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counterstained by using hematoxylin, and then the liver sections were mounted on a

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cover slip.

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RNA extraction and real-time polymerase chain reaction (Real-time PCR)

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assay. The mRNA expression levels of Collagen I, α-SMA, TNF-α, IL-6, MCP-1,

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TGF-β, Bcl-2, Bcl-xL, Bax and TIMP1 were determined by Real-time PCR. The

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primers were designed by Primer 5.0 online software and shown in Table 1. The

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isolation of RNA and synthesis of cDNA were carried out as previously reported15.

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Quantitative realtime PCR was performed on an ABI Prism 7300 Detection System

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(Applied Biosystems, USA). Relative gene expression was determined by the ∆∆CT

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method using β-actin as an internal control. All reactions were performed in

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duplicate.

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Statistical analysis

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Data were analyzed statistically using SPSS 19.0 for Windows. Experimental

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results were expressed as mean ± SD. Statistical significance was tested with

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one-way ANOVA followed by the Student-Newman-Keuls post hoc test. P-values

Protective Effects of Selenium-Enriched Probiotics on Carbon

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