Oral administration of Lactobacillus plantarum strains increases

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Bioactive Constituents, Metabolites, and Functions

Oral administration of Lactobacillus plantarum strains increases cadmium excretion by regulating enterohepatic circulation in mice Qixiao Zhai, Yang Liu, Chen Wang, Jianxin Zhao, Hao Zhang, Fengwei Tian, Yuan Kun Lee, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

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Journal of Agricultural and Food Chemistry

1

Oral

administration

of

Lactobacillus

2

strains increases cadmium excretion by regulating

3

enterohepatic circulation in mice

4

Qixiao Zhai†,‡, Yang Liu†,‡, Chen Wang

5

Tian†,‡, Yuan-kun Lee∥*, Wei Chen†,‡,§,⊥*

†,‡,

plantarum

Jianxin Zhao†,‡, Hao Zhang†,‡,§, Fengwei

6 7

†State

8

214122, China

9

‡School

Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu

of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.

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§National

11

⊥

12

Business University (BTBU), Beijing 100048, China

13

∥

14

117597, Singapore

Engineering Research Centre for Functional Food, Wuxi, Jiangsu 214122, China

Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology and

Department of Microbiology & Immunology, National University of Singapore, Singapore

15 16

*Corresponding author:

17

Yuan-kun Lee, Department of Microbiology & Immunology, National University of Singapore,

18

Singapore 117597, Singapore. E-mail: [email protected], Phone: (65)6516-3284

19

Wei Chen, State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

20

Jiangsu 214122, China. E-mail: [email protected], Phone: (86)510-85912155

21

1

ACS Paragon Plus Environment


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Abstract

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The heavy metal cadmium (Cd) is a contaminant widely distributed

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in the food-chain. In the present study, eight-week oral administration of

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a probiotic strain, Lactobacillus plantarum CCFM8610, markedly

26

decreased blood Cd levels in volunteers. Further animal study showed

27

that three L. plantarum strains administered orally exhibited significantly

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different effects on the regulation of bile acid (BA) metabolism and Cd

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excretion in mice. Among the strains, L. plantarum CCFM8610 showed

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the most significant effects on enhancing hepatic BA synthesis, biliary

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glutathione output, and fecal BA excretion. Biliary Cd output and fecal

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Cd excretion were markedly increased after L. plantarum CCFM8610

33

administration, resulting in a marked reduction in tissue Cd levels. The

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regulation of BA homeostasis and Cd excretion was due to the

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suppression of the enterohepatic farnesoid X receptor-fibroblast growth

36

factor 15 (FXR-FGF15) axis by L. plantarum CCFM8610 and could be

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abolished by treatment with the FXR agonist GW4064. The regulatory

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effects were also related to the gut microbiota, as antibiotic pre-treatment

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reversed L. plantarum CCFM8610-induced effects in BA and Cd

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metabolism.

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Keywords:

Lactobacillus

plantarum;

43

circulation; Bile acid; Gut microbiota

Cadmium;

2


Enterohepatic

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Introduction

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Cadmium (Cd) is a heavy metal and a significant food-chain

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contaminant. Besides inducing pathological lesions in tissues, Cd is also a

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carcinogen as a number of epidemiological and clinical studies indicated

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increased risk of renal cancer associated with Cd exposure.1 Several

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recent surveys showed that the dietary Cd intakes in adults in Europe and

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China were 7.6-12.0 and 15.3 μg/kg body weight (BW) /month,

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respectively.2,3 A survey based on 12523 individuals indicated a lower

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average dietary Cd consumption in the U.S. population (0.54 μg/kg body

53

weight/week).4 A study on 910 subjects from Southwest China showed

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that the dietary Cd intake of the residents in polluted areas reached

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88.80-113.10 μg/kg BW/month, indicating significant health hazards

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from this non-essential metal.5 Tobacco smoking is another major source

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of Cd exposure.6 Previous studies have indicated higher blood and tissue

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Cd contents in smokers than the non-smokers.7,8 It has been reported that

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people residing in Cd-polluted areas inhaled more than 30 µg of Cd/day

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from smoking locally grown tobacco.9 Based on a population-based study

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(n=994), the population-attributable risk of lung cancer was 73% for Cd

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inhalation via smoking.10

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As a cumulative element, Cd has a biological half-life of more than

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10 years in humans.11 A series of long-term follow-up studies from

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1979-2008 on a Cd-polluted hamlet in Japan showed that half-life of 3



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urinary Cd in residents were 11.4-23.4 years and the values were affected

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by creatinine-adjustion.12,13 Another study indicated that the threshold

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safety levels of urinary Cd were 2.4 μg/g and 3.3 μg/g creatinine in men

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and women, respectively.14 The half-life of Cd is also related with the

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abundance of essential metals in humans, since the deficiency of iron and

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zinc has been reported increase the expression of metal uptake

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transporters in the gut and induce a higher uptake of Cd.15,16 Chelators are

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commonly used to increase Cd excretion, but they are reported to induce

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the loss of zinc, iron and manganese.17

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Our previous studies have shown that Lactobacillus plantarum

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CCFM8610 is protective against acute and chronic Cd toxicity in

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mice.18,19 This probiotic is able to reduce tissue Cd accumulation and

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alleviate Cd-induced tissue histopathology. The protective mechanism

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may be due to the intestinal Cd sequestration (due to the good Cd binding

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ability of this strain) and gut barrier protection of L. plantarum

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CCFM8610.20,21 In these studies, the probiotic was used as a dietary

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intervention and was administered simultaneously with oral Cd exposure.

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The “enterohepatic circulation” of heavy metals, including Cd, has

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been well demonstrated by a number of studies. After intestinal

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absorption, Cd is transported to the liver, where it induces the production

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of metallotheinein (MT) and accumulates as a Cd-MT complex.22 The

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Cd-MT complex can be transported from liver to kidneys via systemic 4


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circulation and induces necrosis or apoptosis of renal tubular cells.23

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Some hepatic Cd is secreted into the intestines via bile as S-conjugates of

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GSH and cysteine and then reabsorbed by the enterocytes.23-26 Based on

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these analyses, we hypothesized that the regulation of enterohepatic

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circulation may protect against Cd accumulation in the host. Based on a

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global transcriptomic analysis of the mouse liver (unpublished data), we

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noted that oral administration of L. plantarum CCFM8610 can induce

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gene expression changes in hepatic bile acid (BA) neosynthesis-related

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pathways. Some previous studies also demonstrated that Lactobacillus

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strains can modulate BA metabolism by sequestering unconjugated BAs,

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hydrolyzing bile salts and regulating gut microbiota.27-29

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In this study, three L. plantarum strains (CCFM8610, CCFM11, and

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CCFM309) with similar Cd binding abilities21 were used to bypass the

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intestinal Cd sequestration activity of the strains. We sought to gain

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insight into the protective mechanisms of probiotics against Cd

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accumulation in the host, with a focus on their regulation of enterohepatic

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circulation.

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Materials and Methods

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Bacterial strains

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Three L. plantarum strains (CCFM8610, CCFM11, and CCFM309)

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were obtained from the in-house Culture Collections of Food 5



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Microbiology, Jiangnan University (Wuxi, China). These strains have

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been previously reported to have good Cd binding abilities in vitro.21 For

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the animal experiments, the strains were cultured in de Man, Rogosa, and

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Sharpe (MRS) broth (Hope Bio-technology Company, Qingdao,

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Shandong, China) at 37°C for 18 h and lyophilized with skim milk as a

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protectant.19 For the human trial, L. plantarum CCFM8610 was cultured,

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lyophilized, and packaged into small aluminum foil sachets by a probiotic

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strain manufacturer (Jiangsu Wecare Biotechnology Co. Ltd., Suzhou,

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Jiangsu, China).

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Human Trial

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The human trial followed a double-blind, placebo-controlled

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randomized design with two parallel arms. The project was approved by

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the medical ethics committee of the First People’s Hospital of Guiyang

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City, Hunan Province, China (GYH-201801), and registered in the

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Chinese Clinical Trial Registry (ChiCTR1800015066). All participants

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received written and verbal information about the project where upon

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informed consent was obtained.

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A total of 108 participants were recruited from four towns in

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Guiyang City, Hunan province of China (Fig. 1). These four towns are

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close to nonferrous metals mining districts abundant in zinc, cadmium

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and lead. High levels of Cd were detected in the soil (10.31 ± 0.98 Cd 6


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mg/kg) and rice (0.43 ± 0.21 Cd mg/kg) grown near the towns. The

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participants underwent a medical examination at the First People’s

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Hospital of Guiyang City prior to the study. The inclusion criteria

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included local residents aged 45–60, with a body mass index of 18.4–28.

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The exclusion criteria were (1) a history of chronic disease or critical

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illness, (2) intake of probiotics or antibiotics during the 3 months before

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recruitment for the study; (3) respiratory tract infection, intestinal

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infection, or other diseases of the digestive tract during the previous 2

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weeks; and (4) intake of any drugs during the previous 7 days.

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Randomization was performed by an investigator from the First

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People’s Hospital of Guiyang City who was blinded to the study protocol.

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Participants were randomly assigned to two experimental groups: a

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probiotic group (n = 37) and a placebo group (n = 38). The participants

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were instructed to take one sachet of probiotic or placebo daily for two

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months. Each probiotic sachet contained L. plantarum CCFM8610 at a

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dose of 1 × 109 cfu. The placebo was identical in composition and

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packaging but without the addition of the bacterial strains. The sachets

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were packaged according to the randomization code and the code was

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kept from the participants and investigators until the analyses were

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concluded. Fasting blood samples were collected from the participants at

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baseline and at the end of the study. Blood samples were stored at -80 ℃

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for the assessment of Cd levels. 7



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Animals and experimental design

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Animals were purchased from the Shanghai Laboratory Animal

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Center (Shanghai, China). C57BL/6 mice (male, 4–6 weeks old, weighing

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14-20 g) were kept in plastic cages (5 per cage) with stainless steel grid

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lids in a temperature- and humidity-controlled room. The mice had free

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access to commercial mouse food (Xietong Organism Inc., Nanjing,

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Jiangsu, China) and drinking water and were monitored every 24 hours

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by the lab assistant. Mice were acclimatized for 7 days before the

160

experiment. All of the protocols for the animal trials in the study were

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approved by the Ethics Committee of Jiangnan University, China (JN. No

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20170310-0902[25] and JN. No 20180615c0400930[149]). The trial

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procedures for the care and use of experimental animals were carried out

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in accordance with the European Community guidelines (directive

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2010/63/EU).

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Mice were randomly assigned to one of the following six groups and received treatments as follows.

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Cd (n = 16): mice were first given Cd-containing drinking water at

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100 mg/L CdCl2 (Sinopharm Chemical Reagent Company, Shanghai,

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China) for 8 weeks.19 Then the mice were given plain drinking water and

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received 0.5 ml of skim milk (as the vehicle control) via gavage once

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daily for 4 weeks.

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Cd+CCFM8610 (n = 16): after an identical Cd exposure via drinking 8


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water to the Cd group for 8 weeks, mice were given plain drinking water

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and received a oral dose of L. plantarum CCFM8610 at 1 × 109 cfu each

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day for 4 weeks.19

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Cd+CCFM11 (n = 6): after an identical Cd exposure via drinking

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and received a single oral dose of L. plantarum CCFM11 at 1 × 109 cfu

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each day for 4 weeks.

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Cd+CCFM309 (n = 6): after an identical Cd exposure via drinking

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and received a single oral dose of L. plantarum CCFM309 at 1 × 109 cfu

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each day for 4 weeks.

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Cd+CCFM8610+GW4064 (n = 6): besides an identical treatment to

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that of the Cd+CCFM8610 group, mice were given a gavage of a

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synthetic FXR agonist,30 GW4064 (Sigma-Aldrich, St. Louis, MO, USA),

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at 75 mg/kg/body weight each day for the last 2 days of the experiment.29

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Cd+Antibiotics(AN)+CCFM8610 (n = 6): besides an identical

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treatment to that of the Cd+CCFM8610 group, mice were treated with a

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cocktail of antibiotics in drinking water, as previously described, for 3

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days prior to the administration of L. plantarum CCFM8610.31

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During the last 4 weeks of the experiment, fecal samples were

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collected weekly as previously described.20 Mice were fasted for 12 h and

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sacrificed under light ether anesthesia at the end of the experiment. 9



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Blood, gallbladder bile, and tissue samples were collected and stored

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either at -80℃ or in liquid nitrogen.

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Determination of Cd contents in feces, blood, bile and tissues

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After digestion by a Microwave Digestion System (MARS; CEM,

200

Matthews, NC, USA), Cd levels in the samples were measured using

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atomic absorption spectrophotometry (Spectr AAS or AA; Varian, Palo

202

Alto, CA, USA) or inductively coupled plasma mass spectrometry

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(NexIon-300X; PerkinElmer, Spokane, WA, USA).

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Determination of bile acid (BA) levels in feces, tissues, and bile

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During the last 3 days of the experiment, feces were collected

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according to a previous report for the analysis of fecal BA contents.32

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Liver, small intestine, and gallbladder bile samples were collected after

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the mice were sacrificed. The total contents of BA was extracted from the

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samples and the levels were measured with a kit purchased from

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Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The BA

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pool size was determined as the total contents of BA in the bile, liver and

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small intestines.29

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Determination of bile flow

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Gallbladder cannulations were performed on a proportion of the

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animals (n = 3-5 from each group) according to a previous report at the

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end of the experiment.33 Briefly, bile was collected in pre-weighed tubes 10


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for 30 min, and bile flow was determined gravimetrically assuming a

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density of 1.0 g/ml and normalized to liver weight.34

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Determination of glutathione (GSH) in bile

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Levels of biliary GSH were measured with a kit purchased from

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Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

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Analysis of real-time quantitative PCR

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Liver and ileum were collected and immediately stored in liquid

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nitrogen. Total RNA in these tissues was extracted the with Trizol reagent

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(Ambion, Austin, TX, USA) and NanoDrop and gel electrophoresis were

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used to assess RNA purity and integrity.35 Gene expression was

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determined on a real-time quantitative PCR system (CFX Connect;

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Bio-Rad, Hercules, CA, USA). The primers for the studied genes (Table

229

S1) were selected as previously reported.32,36 Relative quantiﬁcation of

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the target genes was analyzed by comparison with the expression level of

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the Gapdh gene and calculated according to the 2-△△Ct method.37

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Statistical analysis

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Data are expressed as the mean ± standard deviation (SD).

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Differences between means were analyzed using two-tailed Student’s t

235

tests or one-way analysis of variance, followed by Tukey’s post-hoc test.

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A P value of < 0.05 was considered to be statistically signiﬁcant.

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Statistical analyses of the obtained data were performed and visualized 11



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using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA).

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Results

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L. plantarum CCFM8610 administration reduced blood Cd levels in

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humans and decreased tissue Cd levels in mice

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Seven and eight participants discontinued in probiotic and placebo

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groups, respectively, due to the personal issues, onset of diseases or

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antibiotic treatment during the trial. Therefore, a total of 60 participants

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completed the study (Fig. 1). No significant differences in blood Cd

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levels were observed between the placebo and L. plantarum CCFM8610

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groups at the baseline (Fig. 2A). Eight-week oral administration of the

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probiotic markedly decreased blood Cd levels from 6.32 ± 4.44 μg/L to

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4.15 ± 3.11 μg/L (P < 0.05), while placebo treatment did not show the

250

same effect. As shown in Fig. 2B, the hepatic Cd contents were markedly

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lower in the L. plantarum CCFM8610-treated (9.81 ± 1.30 μg/g) and

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CCFM11-treated (15.48 ± 1.66 μg/g) groups than that in the vehicle

253

treated group (20.41 ± 1.96 μg/g). L. plantarum CCFM8610 also reduced

254

Cd accumulation in the kidney of mice (P < 0.05), while CCFM11 and

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CCFM309 failed to provide similar protection.

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L. plantarum CCFM8610 administration increased the hepatic BA

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synthesis and biliary output of Cd in mice

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The BA levels in the gallbladder and small intestines and the BA 12


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pool size in mice were up-regulated by L. plantarum CCFM8610

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administration (P < 0.05), while the hepatic BA contents remained

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unchanged after L. plantarum CCFM8610 treatment (Fig. 3A-D). Along

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with the induction of hepatic BA synthesis, oral administration of L.

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plantarum CCFM8610 significantly up-regulated bile flow and biliary

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GSH output (Fig. 4A and B), consistent with enhanced biliary Cd

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excretion (P < 0.05, Fig. 4C). Compared with L. plantarum CCFM8610

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treatment, other two strains had less significant effects on hepatic BA

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synthesis and biliary Cd excretion.

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L. plantarum CCFM8610 administration enhanced fecal excretion of

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BA and Cd in mice

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Compared with vehicle treatment (7.58 ± 0.43 μmol/day/100 g body

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weight), fecal BA excretion was prominently enhanced after L. plantarum

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CCFM8610 treatment (13.99 ± 0.71 μmol/day/100 g body weight, P

Oral administration of Lactobacillus plantarum strains increases

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