Journal of Functional Foods 75 (2020) 104264

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Effect of Lactobacillus plantarum KFY02 isolated from naturally fermented

yogurt on the weight loss in mice with high-fat diet-induced obesity via
PPAR-α/γ signaling pathway
Jianfei Mu a, b, c, d, e, Jing Zhang f, Xianrong Zhou b, c, d, e, Zsolt Zalan g, Ferenc Hegyi g,
Krisztina Taka´cs g, Amel Ibrahim h, Sameh Awad h, Yun Wu i, Xin Zhao c, d, e, Muying Du a,
b, *

a
College of Food Science, Southwest University, Chongqing 400715, China
b
Chinese-Hungarian Cooperative Research Centre for Food Science, Southwest University, Chongqing 400715, China
c
Chongqing Collaborative Innovation Center for Functional Food, Chongqing University of Education, Chongqing 400067, China
d
Chongqing Engineering Research Center of Functional Food, Chongqing University of Education, Chongqing 400067, China
e
Chongqing Engineering Laboratory for Research and Development of Functional Food, Chongqing University of Education, Chongqing 400067, China
f
Environment and Quality Inspection College, Chongqing Chemical Industry Vocational College, Chongqing 401228, China
g
Food Science Research Institute of National Agricultural Research and Innovation Center, Budapest H-1022, Hungary
h
Dairy Microorganisms and Cheese Research Laboratory (DMCR), Department of Dairy Science and Technology, Faculty Agriculture, Alexandria University, Egypt
i
College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China

A R T I C L E I N F O
A B S T R A C T
Keywords:
Lactobacillus plantarum KFY02 In this study, an in vivo lipid-lowering animal experiment was performed on Lactobacillus plantarum KFY02 (LP-
High fat diet KFY02) newly isolated from Xinjiang fermented yogurt. The study used a high-fat diet (HFD) to induce obese
Obesity mice. Serum and tissue samples from mice were examined by molecular biological detection. The results indi-
Weight loss cated that LP-KFY02 could inhibit increases in various organ indexes of mice caused by HFD. LP-KFY02 could
PPAR-α/γ signaling pathway reduce the levels of alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate aminotransferase
(AST), triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) in the serum and
liver of obese mice with HFD and improve high-density lipoprotein cholesterol (HDL-C) levels. Observation of
pathological changes also showed that LP-KFY02 could alleviate the damage caused by obesity on the tissue
samples from the liver of mice and reduce the expansion of fat cells. The results of qPCR experiments further
showed that LP-KFY02 could effectively up-regulate the mRNA expression of lipoprotein lipase (LPL), peroXi-
some proliferative-activating receptor α (PPAR-α), cholesterol 7α-hydroXylase (CYP7A1), and carnitine palmi-
toyltransferase 1A (CPT1A) in the liver and epididymal adipose tissues, as well as cut down the mRNA expression
of peroXisome proliferative-activating receptor γ (PPAR-γ) and CCAAT enhancer-binding proteins (C/EBP-α).
Furthermore, ELISA results showed LP-KFY02 obviously decreased the level of pro-inflammatory factors inter-
leukin (IL)-6, TNF-α, IL-1β and IFN-γ in serum, but increased the anti-inflammatory factors IL-4 and IL-10
content. LP-KFY02 could effectively treat obesity arose from HFD, and its effect is comparable to commer-
cially available L-carnitine, and superior to the Lactobacillus delbruechii subsp. bulgaricus commonly used in in-
dustry. LP-KFY02 is a health beneficial strain with probiotic potential.

1. Introduction
Obesity has become one of the global health problems and an
important factor affecting the health and living conditions of people
worldwide (Dayan, Sforzo, Boisseau, Pereira-Lancha, & Lancha, 2019).
The occurrence of obesity may be related to genetics, endocrine
dyspraxia, metabolic disturbance, and over nutrition. Over capacity
intake of high-sugar and high-fat foods leads to over nutrition,
increasing the severity of obesity (Finicelli et al., 2019).
Simultaneously, obesity is often along with a series of metabolic
syndromes, incorpo- rating type 2 diabetes as well as cardiovascular
and cerebrovascular diseases. Thus, prevention and treatment of
obesity is particularly

* Corresponding author at: College of Food Science, Southwest University, Chongqing 400715, China.
E-mail address: muyingdu@swu.edu.cn (M. Du).

https://doi.org/10.1016/j.jff.2020.104264
Received 23 August 2020; Received in revised form 23 October 2020; Accepted 26 October 2020
Available online 5 November 2020
1756-4646/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
 J. Mu et Journal of Functional Foods 75 (2020)

important. Obesity is a chronic metabolic disease which is caused by medicine,

many reasons, and the fundamental difference lies in that energy
ingestion and consumption are unbalanced (Asano et al., 2019). The
induction of obesity in C57BL/6J mice by HFD is a classic model for
the construction of obesity, mainly to simulate the unhealthy eating
habits of humans by ingesting more high-fat high-sugar and high-
calorie foods and lack of exercise, resulting in body fat accumulation,
serum TC, TG elevation, glycolipid, protein metabolism disorder
produces insulin resistance (Appiakannan, Kestyus, & Weber, 2019).
Simultaneously, the levels of inflammatory cytokines, tumor necrosis
factor α and interferon- gamma increase, and the effects of
adipocytokines, immune responses, endoplasmic reticulum stress,
autophagy, and endotoXin lead to chronic inflammation (Corrˆea &
Rogero, 2019). Studies have shown that Lactobacillus kefiri DH5
can significantly regulate the expression of PPAR-α in the epididymal
adipose tissue and irritate fat cell differenti- ation and fatty acid
oXidation (FAO) via PPAR-α signaling, these pro- cesses result in
weight loss and reduction in size of adipose tissues and fat cells (Kim et
al., 2017). In addition, active substances have been reported to inhibit
lipid accumulation in mouse liver via the PPAR- α signaling pathway
and reduce lipid accumulation in hepatocytes induced by free fatty
acids (FFA). They are related to the regulation of lipid metabolism-
related genes, up regulation of the expression of PPAR- α, CPT1, and
acyl-CoA oXidase, and down regulation of sterol regulatory element-
binding protein expression-1 (SREBP-1), fatty acid synthase (FAS), and
acetyl-CoA carboXylase (ACC) (Maleki Kakelar, Barzegari, Hanifian,
Barar, & Omidi, 2019). Moreover, intake of probiotic or fer- mented
dairy products exhibit a good lipid-lowering effect, which can change
the composition of the intestinal flora, reduce intestinal inflammation
and regulate intestinal permeability, accordingly affecting viscera
function and dropping serum cholesterol, liver fat, and triglyc- eride
content (Ding et al., 2017).
Xinjiang is an important animal husbandry base for multiple eth-
nicities (Uygur, Kazak, Kirgiz, Manchu, Russian, etc.), with unique
geographical locations and large climate differences. For thousands of
years, fermented dairy products made in traditional ways have been
one of the main foods of ethnic minorities (Yi et al., 2016). Xinjiang
tradi- tional fermented yogurt has been one of the typical traditional
fer- mented diets in Xinjiang since ancient times. Yogurt is rich in
aromatic substances, extracellular polysaccharides, various types of
lactic acid, amino acids, minerals, vitamins, enzymes, and other
nutrients, and its nutritional value exceeds that of pure milk (Li,
Mutuvulla, Chen, Jiang, & Dong, 2012; Zhong et al., 2016). Traditional
peasant family yogurt is made from fresh milk and is naturally
fermented by traditional tech- niques. It is type-IV fermented milk
mainly produced using microor- ganisms for instance lactic acid
bacteria and yeast (Chen et al., 2017). The nutrients in milk fermented
with these special lactic acid bacteria and yeasts change significantly.
Small molecular substances, such as lactic acid, succinic acid,
unsaturated fatty acids, and low-molecular- weight fatty acids, have
increased. This increase improves the nutri- tion and health functions.
The lactic acid bacteria resource bank in Xinjiang, China protects the
lactic acid bacteria with high fermentation capacity because of its
distinct geographical environment and climatic conditions (less rain,
dryness, and more sunshine) (Zhao, Qian, et al., 2019).
Numerous studies reported that lactobacillus can effectively treat
intestinal diseases. Lactobacillus can improve food digestibility and uti-
lization, lower serum cholesterol, regulate toXins in the body, and
restrain the growth and reproduction of putrefaction bacteria and the
production of corrupt products in the intestines (Arena et al., 2018;
Hajavi et al., 2019). Lactobacillus perform the functions of maintaining
human micro ecological balance. When the probiotics in the body are
reduced and harmful bacteria are increased, the body can produce
immunosuppressive factors, inflammatory cells, and other reactions,
which can lead to metabolic disorders (Bell, Ferr˜ao, Pimentel,
Pintado, & Fernandes, 2018; McFarland, 2015). Owing to their special
physio- logic activity; Lactobacillus has been proverbially used in food,

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 J. Mu et Journal of Functional Foods 75 (2020)

and other industries. At present, there are many studies on microbial

diversity, separation and purification, partial characterization and
me- tabolites in traditional fermented milk yogurt in Xinjiang, but few
re- searches have been done on the identification and development of
Lactobacillus characteristics of isolated strains. Some scholars have
studied the AntioXidant effect of strain LP-KSFY02 isolated and purified
from Xinjiang natural fermented yogurt on D-galactose-induced
oXida- tive aging mice (Zhao, Yi, et al., 2019). Some scholars have
studied the structure and antioXidant activity of Lactobacillus MB2-1
extracellular polysaccharides (Li et al., 2015), but no scholars have
studied the weight loss effect of Lactobacillus isolated and purified
from Xinjiang natural fermented yogurt.
In the current study, we used LP-KFY02 to model obese mice induced
by high-fat diet. Various indicators in organ and serum were detected
to evaluate whether LP-KFY02 can regulate the lipid metabolism of
the liver by regulating the expression and anti-inflammatory effect of
PPARα/γ and its target genes in liver tissue and epididymal adipose
tissue, which determines the lipid-lowering effect and possible
mecha- nism of KFY02 on obese mice induced by high-fat diet, and
elucidates the weight-loss effect of LP-KFY02. In order to refrain from
the by-effect of chemical slimming drugs and develops natural and
effective lipid- lowering substances to provide direction.

2. Materials and methods

2.1. Animal experiments

LP-KFY02 was used as the experimental strain, which was isolated

from the naturally fermented milk yogurt in Korla of Xinjiang, and its
colony morphology characteristics and the gel electrophoresis
diagram of PCR amplification products was shown in Fig. 1. It was
deposited in the China General Microbiological Culture Collection
Center (CGMCC, Beijing, China) with an access code CGMCC No.
15638.
SiXty specific pathogen-free C57/BL6J mice aged 6 weeks (30
males and 30 females), which were purchased from the Animal
EXperimental Center of Chongqing Medical University (License
number: SCXK (yu) 2017–0001), were tamed for 1 week and were
randomly divided into 6 groups, the normal, model, L-carnitine, low-
dose LP-KFY02 gavage group (LKFY02), high-dose LP-KFY02 gavage
group (HKFY02), and Lactobacillus delbruechii subsp. bulgaricus
(China Center for Type Culture Collection, Wuhan, Hubei, China,
AB200048) gavage group (LDSB). Each group of 10 mice consisting
of 5 males and 5 females were fed for 8 weeks. The normal group was
fed 10% fat diet (AIN-93 M) (24.89% corn flour, 15.00% bran, 7.00%
wheat flour, 18.50% soybean meal, 16.38% sucrose, 1.02% lard, and
17.21% premiX) (Fotschki et al., 2017; Len- quiste et al., 2019). The
other groups were fed 45% HFD (7.79% corn flour, 7.00% bran,
22.00% soybean meal, 20.00% sucrose, 19.50% lard, and 23.71%
premiX). The mice in the LDSB group and HKFY02 group
were intragastrically administered with LDSB and KFY02 were sus-
pended in sterile saline to concentrations of 1.0 1010 CFU/kg. The ×
mice in the LKFY02 group were intragastrically administered
with
KFY02 at a concentration of 1.0 109 CFU/kg, and the amount of ×
gastric perfusion was 0.1 mL/10 g (body weight). The L-carnitine
group was administered with L-carnitine dissolved in water at a
concentration of 200 mg/kg/d (body weight). Feeding conditions
were as follows:
◦ ◦
light/dark cycle, 12 h/12 h; temperature, 22 C–25 C; moisture, 50% ±
10%; free feeding, water intake, weekly weight. After all mice were
fasted for 24 h, blood was collected by taking eyeballs. And the liver
and epididymal fat were quickly dissected and weighted and stored
for subsequent exeriments. Calculated the organ index according to
the following formula: Organ index Organ mass (g)/Mouse body =
mass (kg).

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 J. Mu et Journal of Functional Foods 75 (2020)

Fig. 1. Morphological characteristics of LP-KFY02; The 16S rDNA agarose gel electrophoresis of the PCR-amplified product of LP-KFY02. M: 2000 bp DNA Ladder; 0:
negative control group; 1: LP-KFY02.

2.2. Determination of AST, AKP, HDL-C, ALP, TC, TG, and LDL-C levels
in serum and liver tissue
◦
Mouse plasma was centrifuged in a refrigerated centrifuge at 4 C
and 1500g for 10 min, and the supernatant serum was used. AST, AKP,
HDL-C, ALP, TC, TG, and LDL-C contents in the mouse serum and
liver tissue were measured accordance with the kit instructions
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
template and 10 μL of SYBR Green PCR Master MiX (Thermo Fisher
Scientific, Waltham, USA) and miXed well, 1 μL of upstream and
downstream primers (Thermo Fisher Scientific, Waltham, USA)
(Table 1), and 7 μL of sterile distilled water were miXed. Detected the
relative expression of mRNA in the tissue by StepOnePlus Real-Time
PCR System (Thermo Fisher Scientific, Inc., Waltham, USA). The reac-
◦
tion conditions of the whole process were as follows: 95 C for 60 s;
◦ ◦
then 40 cycles at 95 C for 15 s; 55 C for 30 s; 72 C for 35 s.
◦

◦ ◦
Uultimately tested at 95 C for 30 s and 55 C for 35 s. Used
2.3. Detection of serum cytokine GAPDH as a

Detected serum cytokine levels according to ELISA kit instructions

Table 1
(Shanghai Enzymelink Biotechnology Co., Ltd., Shsanghai, China).
Sequences of primers.
Gene Name Sequence
2.4. Observation of pathological section of liver and epididymal fat tissue
′ ′
CYP7A1 Forward: 5 -AGCAACTAAACAACCTGCCAGTACTA-3
Liver and epididymal adipose tissues about 0.5 cm 2 were removed ′
Reverse: 5 -GTCCGGATATTCAAGGATGCA-3
′

′ ′
from the mice and immersed in tissue fiXative for 48 h. Tissues were PPAR-γ Forward: 5 -AGGCCGAGAAGGAGAAGCTGTTG-3
′ ′
Reverse: 5 -TGGCCACCTCTTTGCTGTGCTC-3
′ ′
PPAR-α Forward: 5 -CCTCAGGGTACCACTACGGAGT-3
′ ′
evaporated, pellucid, dipped in wax, inlayed, and sliced for hematoX- Reverse: 5 -GCCGAATAGTTCGCCGAA-3
′ ′
CPT1 Forward: 5 -AAAGATCAATCGGACCCTAGACA-3
ylin–oesin staining (H&E) staining, Morphological changes were ′ ′
Reverse: 5 -CAGCGAGTAGCGCATAGTCA-3
observed with optical microscope (BX43; Olympus, Tokyo, Japan). solution was taken out, and the cDNA template was obtained by reverse
tran- scription (Thermo Fisher Scientific, Waltham, USA). Taken 1 μL
2.5. Real time quantitative PCR assay cDNA

Liver and epididymal fat tissues were pulverized using a biological

sample homogenizer (Hangzhou Allsheng Instrument Co., Ltd.). The
total RNA in the tissue was extracted by TRIzol TM reagent, and the total
RNA concentration was diluted to 1 μg/μL. Hereafter, 1 μL RNA

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 J. Mu et Journal of Functional Foods 75 (2020)
′ ′
LPL Forward: 5 -AGGGCTCTGCCTGAGTTGTA-3
′ ′
Reverse: 5 -AGAAATCTCGAAGGCCTGGT-3
′ ′
C/EBP-α Forward: 5 -TGGACAAGAACAGCAACGAGTAC-3
′ ′
Reverse: 5 - GCAGTTGCCCATGGCCTTGAC-3
′ ′
GAPDH Forward: 5 -ACCCAGAAGACTGTGGATGG-3
′ ′
Reverse: 5 -ACACATTGGGGGTAGGAACA-3

CYP7A1: cholesterol 7α-hydroXylase; PPAR-γ: peroXisome proliferator activated

receptors gamma; PPAR-α: peroXisome proliferators-activated receptor alpha;
CPT1: carnitine palmitoyltransferase1; LPL: lipoprotein lipase; C/EBP-α:
CCAAT/enhancer binding protein-α; GAPDH: glyceraldehyde-3-phosphate
dehydrogenase.

5
 J. Mu et Journal of Functional Foods 75 (2020)

housekeeping gene, and calculated the relative gene expression by the groups fed with the same HFD had a obviously lower body weight than
—
2 ΔΔCt method (Zhai et al., 2018; Zhang et al., 2018). model group. The body weights of the overall growth trend of the
HKFY02 group tended to be lower than those of the L-carnitine and LDSB
groups. This finding indicates that a high concentration of LP-KFY02
2.6. Western blot analysis
can alle- viate the enhancement in body weight in mice fed with a HFD
and that it exerts a certain preventive effect on obesity.
Up to 100 mg liver and epididymal adipose tissue samples with 1
mL RIPA (Invitrogen, Carlsbad, CA, USA) and 10 μL
Phenylmethanesulfonyl fluoride (Beijing Suolai Bao Technology Co.
Ltd., Beijing, China) were homogenized (Hangzhou Allsheng
Instruments Co., Ltd.) at 6.00 m/s work for 30 s, intermittently for 30
s, cycle 6 times. After centrifugation
◦
at 12,000 r/min 4 C for 10 min, the intermediate protein layer solution
was taken out, and the BCA protein quantification kit (Tiangen
biothech (Beijing) Co. Ltd., Beijing, China) was used to determine the
concen- tration of extracted protein. Each group of sample protein
solutions was diluted to 30 μg/uL. Taken the diluted protein solution
and sample buffer (Thermo Fisher Scientific, Inc., Waltham, USA) and
miX at a ratio
◦
of 4:1, and denaturized at 100 C for 5 min and then quickly left it at
low
temperature for 5 min. Acrylamide, resolving buffer, stacking buffer,
distilled water, 10% ammonium persulfate, and a stabilizer, N,N,N’,N’-
Tetramethylethylenediamine (TEMED) (Thermo Fisher Scientific, Inc.,
Waltham, USA) were miXed proportionally to produce an SDS–PAGE
separation gel. Added the protein marker and sample to the sample wells
of the SDS-PAGE protein gel, and performed vertical gel
electrophoresis for 50 min (Chen et al., 2019). The poly vinylidene
fluoride (PVDF) membrane (Thermo Fisher Scientific, Inc., Waltham,
USA) was activated using methanol for 2 min and then transferred the
protein to the PVDF membrane. Subsequently, the PVDF membrane ×
was blocked by 1
TBST with 5% skim milk (Becton, Dickinson and Company, New York,
◦
USA) for 1 h. Then, the primary antibody was incubated at 4 C for
overnight; and then washed PVDF membrane 5 times with 1 × TBST, 5
min each time, and the secondary antibody was combined for 1 h at
◦
25 C. Finally, Supersignal West Pico PLUS (Thermo Fisher Scientific,
Inc., Waltham, USA) was used to infuse the PVDF membrane for 1 min
and placed in iBright FL1000 (Thermo Fisher Scientific, Inc.,
Waltham, USA) for observation (Gil-Iturbe, Castilla-Madrigal,
BarrenetXe, Villaro, & Lostao, 2019). The CYP7A1 (PA5-79135), PPAR-
γ (MA5-14889), PPAR-α (MA1-822), CPT1A (PA5-69347), LPL (PA5-
47033), C/EBP-α
(MA1-825), FAS (MA1-7623), and β-actin (PA1-183) antibodies were
purchased from Thermo Fisher Scientific. Proteins were visualized by
chemiluminescence, and the density of individual bands was
quantified using Image J with normalization to β-actin.

2.7. Statistical analysis

Each mouse serum and tissue indicators were subjected to 3

parallel experiments. SPSS ver. 19.0 (SPSS, Inc., Chicago, USA)
statistical soft- ware was used for statistical analysis. One-way analysis
of variance (one-way ANOVA) was used to define whether the
statistical data ob-
tained by each group was significantly different at the p < 0.05 level
(Chu-Sook Kim, 2018). All figures were drawn with GraphPad Prism 7
software.

3. Results

3.1. Change in weight in mice

The weight of the normal group was more moderate (Fig. 2), and
the change in weight of the model group was significantly faster than
that of the normal group. The L-carnitine, LKFY02, HKFY02, and LDSB

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 J. Mu et Journal of Functional Foods 75 (2020)
values with different letters under the same column are significantly different (p
< 0.05) in accordance with Duncan’s multiple range test. LDSB: Mice treated
with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02:
Mice treated with LP-KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with
LP- KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

Fig. 2. Effect of KFY02 on body weight gain in mice. LDSB: Mice treated with
Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice
treated with LP-KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with LP-
KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

3.2. Mouse organ indices

It could be seen from table 2 that the liver index, perirenal fat
index and epididymal fat index of the model group were evidently
increased
relative to that of the normal group (p < 0.05). The perirenal fat index
and epididymal fat index of the model group were 2.7 times and 10
times that of the normal group, respectively. This finding indicated
that compared with the model group, the liver index, the epididymal
fat index and the perirenal fat index of the KFY02 treatment
group, L-
carnitine group, and LDSB treatment group, two different concentra-
tions of KFY02 treatment group were uncommonly lower (p < 0.05). The
lipid-lowering effects on the HKFY02 and L-carnitine groups
were
evident, consistent with the trend in mouse body weight. LP-KFY02
could effectively inhibit the increase in organ index caused by a HFD
and relieve the increase in body tissue caused by excessive tissue fat
content.

3.3. Contents of ALT, AST, AKP, TC, TG, HDL-C, and LDL-C in
serum and liver tissue

Tables 3 and 4 showed that the content of ALT, AST, AKP, TC, TG,
and LDL-C in the serum and liver of the model group were the
highest, whereas the level of HDL-C is the lowest. After treatment
with LDSB, KFY02, and L-carnitine, the levels of ALT, AST, AKP, TC,
TG, HDL-C, and LDL-C in the HKFY02 and L-carnitine groups tend to be
close to those of the normal group. The results indicate that the
inhibitory effect of high- concentration KFY02 on obesity induced by
HFD was similar to that of L- carnitine and superior to those of LDSB
and low-concentration KFY02.

Table 2
Organ index of mice in each group (N = 10).
Group Liver index Epididymal fat index Perirenal fat index
d e
Normal 37.97 ± 0.60 10.76 ± 2.49 1.78 ±
d a a
0.24 Model 45.05 ± 1.41 27.78 ± 3.47 11.41 ±
a
1.60
cd cd
L-carnitine 38.49 ± 1.59 16.60 ± 3.64 5.08 ±
c bc bc
0.63 LKFY02 40.37 ± 1.21 21.59 ± 4.06 7.53 ±
b cd de
0.64 HKFY02 38.69 ± 0.63 14.60 ± 3.50 5.67 ±
c b ab
0.28 LDSB 40.13 ± 1.11 22.97 ± 3.79 7.66 ±
b
0.59
a–d
Values presented are the mean ± standard deviation (N = 10/group). Mean

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 J. Mu et Journal of Functional Foods 75 (2020)

Table 3
Levels of ALT, AST, AKP, TC, TG, HDL-C, and LDL-C in mouse serum (N = 10).
Group Normal Model L-carnitine LKFY02 HKFY02 LDSB
c a c b c b
AKP (U/L) 23.88 ± 2.32 61.57 ± 1.27 25.45 ± 3.72 37.46 ± 2.10 27.69 ± 6.01 38.36 ± 3.46
d a bc b cd b
ALT (U/L) 18.67 ± 1.69 47.16 ± 7.42 27.18 ± 5.26 30.21 ± 1.28 21.77 ± 0.46 32.98 ± 3.82
d a d b c ab
AST (U/gprot) 99.50 ± 3.53 303.60 ± 32.10 115.87 ± 6.07 250.04 ± 24.02 199.84 ± 29.10 286.91 ± 48.3
a d bc cd ab abc
HDL-C (mmol/L) 3.40 ± 0.13 2.46 ± 0.16 3.02 ± 0.12 2.77 ± 0.22 3.32 ± 0.23 3.12 ± 0.29
d a bc ab c bc
LDL-C (mmol/L) 1.04 ± 0.08 1.44 ± 0.03 1.28 ± 0.02 1.37 ± 0.02 1.24 ± 0.07 1.31 ± 0.05
d a c b c b
TC (mmol/L) 4.50 ± 0.03 6.33 ± 0.12 4.71 ± 0.07 5.71 ± 0.41 4.72 ± 0.26 5.30 ± 0.20

d a bc ab cd ab
TG (mmol/L) 1.65 ± 0.10 3.06 ± 0.60 1.76 ± 0.28 2.27 ± 0.85 1.76 ± 0.03 2.72 ± 0.11
a–d
Values presented are the mean ± standard deviation (N = 10/group). Mean values with different letters under the same column are significantly different (p < 0.05)
in accordance with Duncan’s multiple range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-
KFY02 (1.0 × 10 CFU/kg); HKFY02: Mice treated with LP-KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).
9

Table 4
Levels of ALT, AST, AKP, TC, TG, HDL-C, and LDL-C in mouse liver (N = 10).
Group AKP (king unit/gprot) ALT (U/gprot) AST (U/gprot) LDL-C (mmol/gprot) TC (mmol/gprot) TG (mmol/gprot)
c c c d e c
Normal 23.88 ± 2.32 0.62 ± 0.11 0.41 ± 0.01 0.031 ± 0.0083 0.067 ± 0.012 0.11 ± 0.017
a a a a a a
Model 61.57 ± 1.27 1.36 ± 0.04 0.88 ± 0.07 0.104 ± 0.10 0.22 ± 0.035 0.22 ± 0.031
c b bc c d bc
L-carnitine 25.45 ± 3.72 0.79 ± 0.11 0.47 ± 0.02 0.068 ± 0.085 0.10 ± 0.014 0.13 ± 0.024
b b b c c b
LKFY02 37.46 ± 2.10 0.86 ± 0.06 0.75 ± 0.08 0.065 ± 0.0073 0.12 ± 0.0032 0.16 ± 0.010
c bc c d de bc
HKFY02 27.69 ± 6.01 0.76 ± 0.06 0.54 ± 0.06 0.036 ± 0.0094 0.078 ± 0.015 0.15 ± 0.027
b b b b b b
LDSB 38.36 ± 3.46 0.87 ± 0.04 0.70 ± 0.07 0.084 ± 0.0064 0.16 ± 0.010 0.16 ± 0.018
Values presented are the mean ± standard deviation (N = 10/group). a–d Mean values with different letters under the same column are significantly different (p < 0.05)
in accordance with Duncan’s multiple range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-
KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with LP-KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

3.4. Observation of pathological changes

In the normal liver tissue, synthesis and decomposition of hepatocyte
lipids keep a dynamic equilibrium. Generally, neither lipid cumulation
nor lipid droplets are formed. Nevertheless, when lipids are present in
the cytoplasm, lipid droplets of uneven sizes are taken shape, further
damaging the integral structure of liver cells and affecting the liver
function (Sharma, Gupta, & Abdullah, 2019). H&E staining illustrated
that the liver cells of the normal mice had no abnormal changes, for
instance, steatosis, the structure of the liver tissue was clear and com-
plete, the cell boundaries were evident, and the nucleus was in the
middle (Fig. 3). The model group showed hepatic tissue vesicular stea-
tosis, an increase in fat contents; lots of fat vesicles around the blood
vessels, cell swelling, and damage to cell integrity (Yamada et al.,
2019). After treatment with LP-KFY02, hepatic steatosis became
significantly less than that in the model group. The hepatocyte fat
vesicles were

Fig. 3. Observation of pathological changes in mouse liver by hemato Xylin–oesin staining. Magnification: 200×. LDSB: Mice treated with Lactobacillus delbruechii

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 J. Mu et Journal of Functional Foods 75 (2020)
subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP- KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with LP-KFY02 (1.0 × 1010 CFU/kg); L-
carnitine: L-carnitine (200 mg/kg).

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 J. Mu et
shrunk and the morphology was same as that in the normal mice,
particularly in the HKFY02 group and the L-carnitine group.
The upshots of epididymal adipose tissue staining are presented in
Fig. 4. In the normal group, the fat cells of the mice are smaller and more
nearly arranged; in the model group, the fat cells are larger, the cell
membrane is thinner, and two cells tend to fuse into one cell
(Adamcova et al., 2018). The adipose tissue samples from mice in the
HKFY02 group and the L-carnitine group were denser than the tissue
samples from normal mice, High-concentration KFY02 and L-
carnitine exerted better effects than low-concentration KFY02 and
LDBS, which could conspic- uously decrease fat cell hypertrophy
induced by HFD and the effects of both high-concentration KFY02 and
L-carnitine were similar.
3.5. Content of inflammatory factors and leptin in mouse serum
The content of proinflammatory cytokines IL-6, IFN-γ, TNF-α, and IL-
1β in the serum of the model mice with a HFD were uncommonly
higher than those in the normal mice, whereas the anti-inflammatory
factors IL- 4 and IL-10 were uncommonly decreased (Fig. 5). This
finding indicates that the mice were in an inflammatory state. The
presence of high- concentration KFY02 in the stomach inhibited the
inflammation draw in the HFD, as characterized by the down
regulation IL-6, TNF-α, IFN-γ, IL-1β and up regulation IL- 4 and IL-10,
with a tendency to rebound to the normal group status. The level of
adipokines in the mouse serum also changed. Leptin was a major
regulator of the balance between animal food and energy, synthesized
and released to regulate energy storage in adipose tissue (Clawson et
al., 2019). Fig. 5 shows that the serum leptin content in the model mice
were higher than those in the normal group,
whereas the leptin levels in the LDSB, HKFY02, LKFY02, and L-carnitine
groups are significantly lower (p < 0.05); the HKFY02 was more effec-
tive than any of the other groups. The results indicated that high-
concentration KFY02 exerted a regulating effect on the production of
adipokines.
Fig. 4. Observation of pathological changes in epididymal fat in mice by hemato Xylin–oesin staining. Magnification: 40×. LDSB: Mice treated with Lactobacillus
delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP- KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with LP- KFY02 (1.0 × 1010 CFU/
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Journal of Functional Foods 75 (2020)
3.6. Mouse liver tissue-related mRNA expression
Fig. 6 shows that the normal group exhibited the highest mRNA
expression of LPL, PPAR-α, CYP7A1, and CPT1 in the liver tissues and
the lowest expression of PPAR-γ and C/EBP-α. However, obese mice
got the opposite trend of expression completely. LDSB, HKFY02,
LKFY02, and L-carnitine significantly inhibited the decline in LPL,
PPAR-α, CYP7A1, and CPT1 expression and suppressed the increase in
PPAR-α and C/EBP-α expression in the mouse liver. Among the groups,
HKFY02 and L-carnitine exerted the strongest and equivalent effect,
which inhibited the effect of HFD-induced obesity on the mRNA
expression in the liver tissue samples.
3.7. Mouse epididymal adipose tissue-related mRNA expression
As could be seen from Fig. 7, the mRNA expression trend in the
epididymis adipose tissue of mice in each group was same as that in
the liver tissue. After HKFY02 and L-carnitine acted on mice, each
gene’s expression in the epididymis adipose tissue of mice was closest
to that of normal group. LPL, PPAR-α, CYP7A1 and CPT1 expression in
the epididymal adipose tissue of mice in HKFY02 and L-carnitine
groups were stronger than that of LDSB and LKFY02 groups, but PPAR-γ
and C/ EBP-α expression were weaker than that of LDSB and LKFY02
groups.
HKFY02 obviously inhibited (p < 0.05) the effect of high-fat diet on
epididymal adipose tissue-related gene expression in mice.
3.8. Analysis of mouse liver protein level
The model group exhibited the lowest protein expression levels of
LPL, CPT1A, PPAR-α, and, CYP7A1 in the liver tissue but the highest
expression of FAS, PPAR-γ, and C/EBP-α (Fig. 8). The aforementioned
expression in the tissue samples in the liver of mice in the HKFY02 and L-
carnitne groups were similar to those in the normal group and stronger
than those of the LDSB and LKFY02 groups.
 J. Mu et Journal of Functional Foods 75 (2020)
kg); L-carnitine: L-carnitine (200 mg/kg).

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 J. Mu et
Fig. 5. Cytokine levels in mouse serum. a–e Mean values with different letters in the same bar are significantly different (p < 0.05) in accordance with Duncan’s multiple-
range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-KFY02 (1.0 × 109 CFU/kg); HKFY02:
Mice treated with LP- KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).
3.9. Analysis of epididymal adipose tissue protein level in mice
The expression content of LPL, CPT1A, PPAR- α and CYP7A1 in the
epididymal adipose tissue of the normal mice were obviously higher
than those of the other groups (Fig. 9). KFY02 could up regulate the
decrease in the expression levels of LPL, PPAR- α, CYP7A1, and CPT1A in
epididymal adipose tissue in mice. The results indicated that the
expression levels of LPL, PPAR- α, CYP7A1, and CPT1A in the epididymal
adipose tissue of the HKFY02 and L-carnitine groups were higher than
those of the LDSB and LKFY02 groups, whereas the expression levels
of FAS, PPAR-γ, and C/EBP-α were lower than those of the LDSB and
LKFY02 groups.
4. Discussion
Body weight is one of the most intuitional indicators for evaluating
obesity in mice. In addition, one of the basic indicators commonly used
in biomedical research is the organ index. Obesity in mice is usually
accompanied by an augment in white fat. Epididymal adipose tissue is
a common white adipose tissue. Weighting the white adipose tissue
and contrast to the body weight to obtain the liposome ratio; this can
be used to manifest the degree of obesity in mice (Unamuno et al.,
2018). Long- term exposure to a HFD can result in stress of the body,
and lipid accumulation in the liver can lead to hepatomegaly and
liver function
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Journal of Functional Foods 75 (2020)
damage. In normal liver tissue, compound and egestion of hepatocyte
lipids sustain a kinetic equilibrium. Whereas, when lipids are in the
cytoplasm, lipids of different sizes are formed, and drops destroy the
Original structure of liver cells, affecting liver function. Thus, the organ
index of a mouse can immediately present structural changes in the
organ and its function and provides a reference value for assessing the
success of constructing the obese mouse model. The results of this
study revealed that HFD significantly augmented the organ index of
mice. HKFY02 and l-carnitine could availably alleviate the increase in
organ index, delay weight gain in mice under high-fat conditions, and
lead to organ index of obese mice verge on that of normal mice.
The liver is the largest detoXification organ and lipid metabolism
center of the body. Thus, determining whether the liver is normal is
vital to the human body (Min et al., 2018). ALT, AST, AKP are crucial
in- dicators of liver function and can reflect the extent of liver damage.
ALT and AST are prevailingly existed in hepatocytes. When
hepatocytes are necrotic, ALT and AST are flowed into the blood
circulation, leading to increases in serum enzymes. The level of
increase is consistent with the level of hepatocyte injury. Thus, these
enzymes are currently the most common indicators of liver function.
The World Health Organization (WHO) recommends ALT as the most
sensitive indicator of liver function damage. When 1% of hepatocytes
become necrotic, the serum ALT levels increase once (Al Zarzour et al.,
2017; Tung, Huang, Lin, & Yen, 2018). WHO defines blood lipid
 J. Mu et Journal of Functional Foods 75 (2020)
abnormalities (including triglycerides and high

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 J. Mu et Journal of Functional Foods 75 (2020)

Fig. 6. mRNA expression in mouse liver. a–e Mean values with different letters in the same bar are significantly different (p < 0.05) in accordance with Duncan’s
multiple-range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP- KFY02 (1.0 × 109 CFU/
kg); HKFY02: Mice treated with LP- KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

Fig. 7. mRNA expression in the epididymal adipose tissue in mice. a–e Mean values with different letters in the same bar are significantly different ( p < 0.05) in
accordance with Duncan’s multiple-range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-
KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with L. plantarum KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

and low density lipoproteins) are among the common signs of

metabolic syndrome, and blood lipid levels can reflect systemic lipid
metabolism (Panelli et al., 2018). Accordingly, the present study also
measured the lipid levels in mouse serum, including triglyceride (TG),
total cholesterol (TC), and high and low density lipoprotein cholesterol
(HDL-c, LDL-c).
Obesity is the primary cause of nonalcoholic fatty liver disease; 30%
~50% of obese people have fatty liver, which is repairable if diagnosed
and treated early (Lee et al., 2019). To explore the regulatory effect of
LP-KFY02 on hepatic enlargement, the extent of liver enlargement was
determined using the liver organ index, as well as the contents of TG, TC,

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 J. Mu et Journal of Functional Foods 75 (2020)

Fig. 8. Protein expression in mouse liver. a–e Mean values with different letters in the same bar are significantly different (p < 0.05) in accordance with Duncan’s
multiple-range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-KFY02 (1.0 × 109 CFU/kg);
HKFY02: Mice treated with LP-KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).

HDL-c, and LDL-c. After the liver tissue of mice was homogenized, (Shi, Hou, Liu, Guo, & He, 2019). Lee et al. (2006) indicated that the
colorimetric analysis was conducted using a centrifugal absorption kit to expression of FAS in the white adipose tissue of mice
determine the degree of liver fat accumulation and liver function dam-
age in mice. The aforementioned indicators of liver and serum were
measured. As expected, the levels of ALT, AST, ALP, TG, TC, and LDL-
c in the experimental mice fed with a HFD were significantly
elevated,
resulting in serious vacuolation and severe lipid deposition in the liver.
However, KFY02 and drug treatment in HFD mice obviously (p < 0.05)
decreased the concentrations of liver ALT, AST, ALP, TG, TC, and
LDL-c
in the liver, whereas lipid deposition increased the HDL-c content.
In recent years, more and more research data have shown that
PPAR makes an important impact in the development and progression
of nonalcoholic fatty liver, especially in steatohepatitis, and may be a
po- tential target for drug therapy. PPAR, including α, β, γ subtypes, is
a kind of ligand-activated nuclear transcription factor. After activation,
it binds to the specific nucleotide sequence of the response element of
the target gene promoter region, adjusts the transcription of the target
gene, and makes an important impact in regulating glycolipid
metabolism and inflammation (Khajebishak, Payahoo, Alivand, &
Alipour, 2019; Wang et al., 2019; Wu, Xie, Morrison, Bucher, &
Farmer, 1998). PPAR-α is highly expressed in the liver and participates
in regulating the expres- sion of lipid-producing genes, including fatty
acid synthase (FAS), li- poprotein lipase (LPL), carnitine
palmitoyltransferase-1A (CPT1A) and so on. Fatty acid synthetase
(FAS), a key gene for fatty acid biosynthesis and the main rate-limiting
enzyme in the ability of organisms to regenerate fatty acid, its quantity
and activity play an important role in fat deposition (Naeini et al.,
2019). Different diets exert varying effects on the expression of FAS.
HFD can increase the expression of FAS in the adipose tissue of mice

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 J. Mu et Journal of Functional Foods 75 (2020)
fed with HFD was significantly increased; meanwhile, Lactobacillus
rhamnosus PL60 observably decreased the expression level of FAS and
controlled the body weight of mice. In this study, the relative
expression levels of FAS mRNA and protein in the epididymal adipose
tissue and liver samples from mice in the obese model group induced
by HFD were significantly increased. After 8 weeks of administration
of LPKFY02 via gavage, the high-fat diet obesity model group
exhibited significantly decreased mRNA and protein expression levels
relative to that of FAS; HKFY02 reduced the relative expression of
FAS to the normal level.
CPT1A is widely expressed in various tissues of the human body,
mainly existed in the outer membrane of mitochondria. It is the rate-
limiting enzyme and key regulatory enzyme of β-oXidation of long-
chain fatty acids in liver tissue cells (Freitag, 2016; Lundsgaard, Frit-
zen, & Kiens, 2018). PPAR-α is the upstream transcription factor in fatty
acid oXidation, and CPT-1 is the cruX to its downstream target gene.
The expression of CPT-1 in liver is adjusted by its upstream factor
PPAR-α (Dihingia, Bordoloi, Dutta, Kalita, & Manna, 2018). The main
mecha- nism of action is that PPAR-α facilitates the carry of fatty
acids to mitochondria and promotes the o Xidation of fatty acids by
prompting the expression of CPT1 in muscle and liver. In addition,
PPAR-α can regulate mitochondrial β oXidation and ω oXidation by
adjusting the expression of acetyl-CoA oXidase and cytochrome P450,
thereby regu- lating lipid metabolism in mitochondria (Colom et al.,
2018; Lee, 2010; Li, Tan, Hou, & Zhao, 2018). The results of the
current study indicates that the expression of PPAR-α and CPT1A in
the epididymal adipose tissue and liver in the normal mice were
significantly more than those in the HFD and treatment with LP-
KFY02 mice. The expression levels of PPAR-α and CPT1A in the HFD
mice were significantly increased to similar levels in the normal
group. This outcome is consistent with those of previous studies,
confirming that PPAR-α and CPT1A exhibit a mutual

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 J. Mu et
Fig. 9. Protein expression levels in the epididymal fat of mice. a–e Mean values with different letters in the same bar are significantly different (p < 0.05) in
accordance with Duncan’s multiple-range test. LDSB: Mice treated with Lactobacillus delbruechii subsp. bulgaricus (1.0 × 1010 CFU/kg); LKFY02: Mice treated with LP-
KFY02 (1.0 × 109 CFU/kg); HKFY02: Mice treated with LP-KFY02 (1.0 × 1010 CFU/kg); L-carnitine: L-carnitine (200 mg/kg).
promoting effect on mRNA and protein expression. LPL is a proteolytic
enzyme and a key enzyme in the lipid metabolic pathway. Its main
obligation is to catalyze glycerol in chylomicrons (CMs) and very low-
density lipoproteins (VLDLs) in plasma. Triacetin (TG) decomposes
into free fatty acids and promotes the vehicle of proteins,
phospholipids, and apolipoproteins, thereby improving HDL levels.
Thus, it is also referred to as TG hydrolase (Su, Kong, & Peng, 2018).
Reports have suggested that if the activity of this enzyme decreases,
TG-depleted VLDL and CM decomposition are reduced, clearance is
delayed, plasma TG is elevated, and HDL–cholesterol formation is
reduced. These processes lead to TG hyperemia and low HDL–C levels,
resulting in increased blood lipid levels and inducing obesity (Cruciani-
Guglielmacci & Magnan, 2017). This is consistent with the results of
this study. The LPL of liver and epididymal fat in mice was
significantly increased in the normal and high-concentration KFY02
treated groups by Western blot and qPCR experiments, while the LPL
levels in the obese model group were obviously reduced. The serum
content of TG, TC and LDL-c in the normal and high-concentration
KFY02 treatment groups was also significantly reduced and the HDL-C
content was enhanced, indicating that the high concentration of
KFY02 has a significant lipid-lowering
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Journal of Functional Foods 75 (2020)
function.
PPAR-γ is strongly expressed in tissues such as fat and liver. It acti-
vates glucose and lipid metabolism, adipocyte differentiation, insulin
resistance and inflammatory response. The molecular mechanisms of
adipocyte proliferation and differentiation have not been fully eluci-
dated; however, two major transcription factors have been determined
and identified to have a direct effect on the proliferation and differen-
tiation of preadipocytes: PPAR-γ and C/EBPs families (Wu et al.,
1998). PPAR-γ mainly expressed in adipose tissue, and is an important
factor in inducing adipocyte-specific gene expression and regulating
adipocyte differentiation. C/EBP-α is the first transcription factor shown
to directly regulate adipocyte differentiation. Furthermore, a synergy is
present between C/EBP-α and PPAR-γ. When PPAR-γ is activated, it
can trigger C/EBP-α gene expression, which exerts a positive feedback
effect on PPAR-γ (Kang et al., 2016; Kim et al., 2018); the two genes
are involved in activation and differentiation-related gene expression.
The expression of PPAR-γ mRNA and C/EBP-α mRNA can directly reflect
the adipogenic differentiation status of bone marrow mesenchyme stem
cells, which is are more specific markers (Bhatta et al., 2016). During
adipocyte dif- ferentiation, fat formation, the expression of transcription
factors PPAR-
 J. Mu et
γ and C/EBP-α enhanced. Qiao et al. (2015) determined the effect of
Lactobacillus reuteri L3 on fat storage in mice with diet-induced obesity;
they concluded that a HFD improved the expression of PPAR-γ in mouse
adipose tissue by 4.1 times, whereas L. reuteri L3 significantly reduced its
expression. This finding indicates that intervention using Lactobacillus in
mice regulates the expression of PPAR-γ. In the current research, Real-
time fluorescence quantification and western blotting were used to
detect the expression of adipokines in liver and adipose tissue. HFD
significantly increased the expression of PPAR-γ and C/EBP-α, and LP-
KFY02 interfered with its expression. Therefore, LP-KFY02 inhibits
the formation of fat by inhibiting the expression of the fat-forming
tran- scription factors thereby controlling obesity. In addition, more and
more studies have shown that PPAR-γ can play an anti-inflammatory
effect by directly adjusting the transcription of NF-κB and its
downstream in- flammatory factors, and PPAR-γ agonists can inhibit
monocyte activa- tion and release of inflammatory factors (Gadang et
al., 2011; Minxuan et al., 2019). Studies have shown that PPAR-γ
activation can drop the inflammatory response by inhibiting the
expression of NF-κB, which reduces the production of TNF- α and IL-6.
TNF-α plays an important regulatory role in the occurrence and
progression of fatty liver. It can participate in the pathological changes
of fatty liver through various pathways, such as inducing IR, and
increasing free fatty acid levels is the most common way. IFN-γ, IL-6
and IL-1β also play a vital role in the
progression of fatty liver, and their level are closely related to steatosis,
inflammation, necrosis and the degree of fibrosis (Barroso et al., 2019;
de Souza TeiXeira et al., 2018; Yu et al., 2019). From the results of this
study, it can be seen that the model group, non-alcoholic fatty liver
caused by the HFD, was caused dysregulation of cytokines in the body,
TNF-α, IFN-γ, IL-6 and IL-1β were obviously higher than other groups,
and the HKFY02 group mice displaied a significant reduce in inflam-
matory cytokine levels. The above dates indicate that KFY02 has the
effect of L-carnitine, and it has obvious anti-inflammatory effect, and
high concentration of KFY02 can regulate glycolipid metabolism and
improve liver inflammation and fat accumulation caused by a HFD.
Cholesterol 7-alpha hydroXylase (CYP7A1), the rate-limiting
enzyme in the classical pathway of bile acid synthesis, promotes the
breakdown of cholesterol into bile acids in the liver. CYP7A1 is a liver-
specific microsomal cytochrome P450 enzyme system (Yang et al.,
2019). Almost average of the cholesterol in the human body is excreted
through CYP7A1 catalysis into bile (Tanaka et al., 2018). In our study,
the expression levels of CYP7A1 mRNA and protein in liver and fat of
the model mice were lower than those in the normal group. Cholesterol
and fatty acids have been reported to induce CYP7A1 expression;
however, there is an effective range. Within this range, dietary
cholesterol and fatty acids induce CYP7A1 expression, cholesterol is
converted to bile acid, and bile acid exerts feedback inhibition on
CYP7A1 (She, MacKay, House, & Jones, 2018; Tanaka et al., 2018).
Overall, CYP7A1 mRNA and protein expression increases with dietary
cholesterol and fatty acids. As bile acids continue to increase beyond
this range, CYP7A1 expression varies. In the high-fat model group, the
gene and protein expression of CYP7A1 decreased, and the possible
mechanism was that the hyperlip- idemia induced by the HFD far
exceeded the carrying capacity of the liver. On the one hand, CYP7A1
was decompensated owing to over- expression; on the other hand, the
feedback suppression of CYP7A1 by bile acids exceeds the induction of
CYP7A1 by cholesterol and fatty acids. Moreover, mRNA and protein
expression of CYP7A1 is decreased, leading to cholesterol metabolism
disorders, which further result in liver fat accumulation and obesity.
5. Conclusions
In summary, LP-KFY02 isolated from traditional fermented yogurt
exhibits potential probiotic characteristics. Feeding mice with a high-
fat diet containing LP-KFY02 not only effectively reduces body weight,
serum cholesterol, LDL–C, and TG levels but also improves the intestinal
flora. Determination of lipogenic genes and lipid-lowering proteins in
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Journal of Functional Foods 75 (2020)
mouse liver and epididymal fat based on PPAR α/γ pathway, the obese
model mice exhibited the lowest mRNA and protein expression of the
obesity-related genes LPL, PPAR-α, CPT1A, CYP7A1, and the highest
PPAR-γ, FAS and C/EBP-α. After high-concentration LP-KFY02 treat-
ment of mice, the expression of related genes was adjusted, and the
final expression level tended to be close to the normal group of mice.
These results indicate that the LP-KFY02 strain has a good regulatory
potential for the intestinal flora and helps reduce hyperlipidemia and
excessive weight gain. Simultaneously, judged the regulation of
cytokines TNF-α, IL-6, IL-10 and IL-1β by KFY02 from hepatic steatosis
to steatohepatitis, liver fibrosis and cirrhosis. Through two aspects of
research, the regu- lation is linked to the PPAR-α/γ pathway, revealing
the mechanism of action of LP-KFY02 on the PPAR- α pathway in
obese mice and eluci- dating the effect of LP-KFY02 on weight loss.
Ethics statement
SiXty specific pathogen-free C57/BL6J mice aged 6 weeks (30
males and 30 females), which were purchased from the Animal
EXperimental Center of Chongqing Medical University (License
number: SCXK (yu)
2017–0001), the mice were housed in an air-conditioned room with the
◦
laboratory temperature maintained at 25± 1 C and relative humidity
(50 ± 5%) with a 12/12 h light/dark cycle and provided diet and water
ad libitum.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgment
This work was supported by the Foundation of Chongqing Science
and Technology Bureau (Grants NOs: cstc2017shms-kjfp80011;
cstc2017shms-kjfp80053) and Venture & Innovation Support Program
for Chongqing Overseas Returnees (project numbe: cX2018095) and
the Fundamental Research Funds for the Central Universities (No.
XDJK2017B039, Southwest University), China. All authors have read
and approved the final manuscript.
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