W.-L. Sun et al.
Bioorganic Chemistry 112 (2021) 104966
[5].
luteolin and composed the model group (MD) and the luteolin treatment
group (LT). In the 12th week of the experiment, the body weight and
food intake were measured, and the plasma and liver tissue were
collected for physiological and biochemical tests.
Recently, an increasing number of researches have indicated that the
gut microbiota is closely related to the occurrence and development of
NAFLD. The liver and intestine communicate in two directions via the
bile duct, portal vein and systemic circulation [6]. Intestinal perme-
ability has a vital impact on this two-way communication, especially by
directly affecting the systemic immune response and chronic inflam-
mation. When the gut microbiota becomes imbalanced, intestinal
permeability increases, which leads to an increase in antigens such as
lipopolysaccharide (LPS). LPS, an endotoxin, is the main component of
gram-negative bacteria. When LPS passes through the intestinal barrier
and enters the circulatory system, antibody responses to these antigens
occur, causing immune activation and various inflammatory reactions
[7,8]. Therefore, improving the intestinal barrier and reducing intestinal
permeability is one of the main methods to suppress inflammation from
SS to NASH [9].
2.2. Biochemical indicators
The levels of total cholesterol (TC), total triglycerides (TG), high-
density lipoprotein cholesterol (HDLC), low-density lipoprotein choles-
terol (LDLC), alanine aminotransferase (ALT) and aspartate amino-
transferase (AST) in plasma were measured by using commercial kits
from Nanjing Jiancheng Biological Co., Ltd. (Nanjing, Jiangsu, China).
The levels of TG and TC in the liver were also measured by using com-
mercial kits from Nanjing Jiancheng Biological Co., Ltd. The levels of
Interleukin- 6 (IL6) and tumor necrosis factor
α (TNFα) in the plasma,
liver and colon were measured by using commercial kits purchased from
CUSBIO Co., Ltd. (Wuhan, China). The level of LPS was also measured by
using a commercial kit purchased from CUSBIO Co., Ltd. (Wuhan,
China).
The search for drugs to treat NAFLD is in progress, but no significant
results have been achieved. There are currently no Food and Drug
Administration (FDA)-approved first-line drug for the treatment of
NAFLD or NASH, and the efficacy of indirect drugs is also very limited
[10]. Therefore, there is an urgent need to develop effective and safe
drugs and foods that could prevent the progression from SS to NASH.
Luteolin (3′,4′,5,7-tetrahydroxyflavone), a natural flavonoid compound,
exists in a variety of vegetables, fruits, and natural herbs, including
pepper, wild chrysanthemum, honeysuckle and Perilla [11]. Luteolin
has a variety of biological activities, such as anti-inflammatory, anti-
allergic, uric acid-lowering, antitumor, antibacterial, and antiviral ac-
tivities [12–17]. Chen et al. reported that luteolin suppresses
inflammation-associated gene expression in mouse alveolar macro-
phages [18]. However, luteolin shows low bioavailability (<5%) in vivo
[19,20]. The relationship between the low bioavailability and diverse
biological activities of luteolin remains elusive. Therefore, the under-
lying mechanism of the action of luteolin, including the anti-
inflammatory effect, remains to be further elucidated. A large amount
of luteolin could escape absorption in the intestine and exist in the in-
testine [21]. Meanwhile, many researchers have reported that luteolin
has a broad-spectrum of antimicrobial activity [22,23] and Li et al. re-
ported that luteolin modulate gut microbiota in ulcerative colitis rats
[24]. Besides, it has been reported that a variety of flavonoids could
significantly improve related diseases by regulating the gut microbiota
[25,26]. For example, baicalein could treat diabetes by regulating the
gut microbiota [25]. Therefore, based on these previous studies,
whether luteolin could improve NAFLD by regulating the inflammatory
response through the interaction between the liver and the intestine is
worthy of further study and how the gut microbiota modulation by
luteolin contributes to its effects needs to be further clarified.
2.3. Liver tissue pathological sections
The obtained liver tissue was immersed in formalin for more than 24
h, embedded in paraffin and sliced into 3-μM slices. The sections were
stained with hematoxylin-eosin (HE) or Sirius red and observed under a
240 × microscope.
2.4. Immunoblotting
Primary antibodies against nuclear factor of kappa light polypeptide
gene enhancer in B cells inhibitor alpha (IκB-α; 1:1,000, 9242S), and
Toll-like receptor 4 (TLR-4, 1:1000, 9242S) were purchased from Cell
Signaling Technology Co. Ltd., Massachusetts, USA. Primary antibody
against zonula occludens-1 tight junction (ZO-1 1:1000, Ab190085) was
purchased from Abcam Co. Ltd., USA. Primary antibody against β-actin
was purchased from Solarbio Co. Ltd., Beijing, China. Secondary anti-
bodies against rabbit and goat were purchased from Solarbio Co. Ltd.,
Beijing, China.
The protein was extracted from the colon tissue or liver tissue by
using a commercial kit from Nanjing Jiancheng Biological Co., Ltd.
(Nanjing, Jiangsu, China). After preliminary separation by 12% SDS-
PAGE electrophoresis, the protein was transferred to a polyvinylidene
difluoride (PVDF) membrane and then blocked with 5% skimmed milk
for 4 h. The PVDF membrane was incubated with 1:6000 anti-β-actin or
1:1000 ZO-1 antibody overnight at 4 ◦C. The next day, the PVDF
membrane was washed 3 times with TBST for 10 min each time. After
incubating the PVDF membrane with a secondary antibody of 1:6000 at
room temperature for 1 h, the PVDF membrane was washed 3 times with
tris-buffered saline (TBST) for 10 min each time again. We used an ECL
kit for color development and grayscale analysis software for protein
grayscale analysis. Moreover, fresh liver tissue was taken for determi-
nation of TLR4 (1:1000) and IκB (1:1000) using a method similar to that
described above. Each immunoblotting experiment was repeated 3
times.
In this study, we performed a trial with 12-week supplementation
with luteolin in high-fat diet (HFD)-fed rats to determine the effect of
luteolin on the gut microbiota and the progression from SS to NASH.
2. Materials and methods
2.1. Animal experiments
As male rats tend to store more fat in the visceral adipose tissue than
female rats, male SPF-grade SD rats (160 g ꢀ 200 g) were used to induce
NAFLD, which were purchased from Shandong Provincial Animal
Experiment Center, China [27]. Luteolin (greater than98%, structural
information in Table S1, Table S2, and Fig. S1) was purchased from
Nantong Feiyu Co., Ltd. (Nantong, Jiangsu, China). The animals were
placed in a thermoneutral housing under a 12-h day/night alternating
cycle [28]. The study protocol was approved by the Institutional Animal
Care and Use Committee of Institute of Biomedical Research at Shan-
dong University of Technology. Six rats were fed a standard diet and
composed the normal group (NM). Twelve rats were fed a HFD (D16492,
SYSE Co., Ltd., Changzhou, Jiangsu, China) and a HFD containing 0.5%
2.5. Fecal DNA extraction, PCR amplification and illumina MiSeq
sequencing
Genomic DNA from stool samples was extracted by a soil DNA kit
(Omega Bio-Tek Co. Ltd., Norcross, GA, USA). Primers 338F (5′-barcode-
ACTCCTACGGGAGGCAGCA-3′)
and
806R
(5′-GGAC-
TACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 region of
the bacterial 16S rRNA gene by polymerase chain reaction (PCR)
(denaturation at 95 ◦C for 30 s, annealing at 55 ◦C for 40 s and extension
at 72 ◦C for 1 min, amplification was performed in 27 cycles). The PCR
system was as follows: 4 μL 5 × FastPfu buffer, 2 μL 2.5 mM dNTP, 0.8 μL
2
Sun, Wen-Long
