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Citation: Ren, J.; Zhang, X.;
Heiyan-Perhat, S.; Yang, P.; Han, H.;
Li, Y.; Gao, J.; He, E.; Li, Y.
Therapeutic Role of Polyphenol
Extract from Prunus cerasifera Ehrhart
on Non-Alcoholic Fatty Liver. Plants
2024,13, 288. https://doi.org/
10.3390/plants13020288
Academic Editor: Sebastian Granica
Received: 8 December 2023
Revised: 4 January 2024
Accepted: 8 January 2024
Published: 18 January 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
plants
Article
Therapeutic Role of Polyphenol Extract from Prunus cerasifera
Ehrhart on Non-Alcoholic Fatty Liver
Jiabao Ren 1, †, Xing Zhang 1 ,† , SU Heiyan-Perhat 1, Po Yang 2,3, Helong Han 1, Yao Li 1, Jie Gao 1, Enpeng He 2,*
and Yanhong Li 1,*
1Key Laboratory of Special Environment Biodiversity Application and Regulation in Xinjiang,
College of Life Sciences, Xinjiang Normal University, Urumqi 830054, China; rjb331154180@163.com (J.R.);
zxyybh@163.com (X.Z.); 13579269571@163.com (S.H.-P.); hhl13779071291@163.com (H.H.);
l2031021@163.com (Y.L.); jiegao72@gmail.com (J.G.)
2Key Laboratory of Sports Human Sciences, Institute of Physical Education, Xinjiang Normal University,
Urumqi 830054, China; 15635295235@163.com
3College of Arts and Sports, Hebei Institution of Communication College, Shijiazhuang 051430, China
*Correspondence: hep0983@163.com (E.H.); liyh1330824@163.com (Y.L.)
†These authors contributed equally to this work.
Abstract: Prunus cerasifera Ehrhart (P. cerasifera) flourishes uniquely in the arid landscapes of Xinjiang,
China. Preliminary studies have revealed the therapeutic potential of its polyphenol extract (PPE)
in mitigating liver lipid accumulation in mice fed a high-fat diet. We established a mouse model
that was subjected to a continuous high-fat diet for 24 weeks and administered PPE to investigate
the effects of PPE on cholesterol and BA metabolism in NAFLD mice. The results showed that PPE
administration (200 and 400 mg/kg/day, BW) led to a reduction in liver TC, an increase in liver
T-BAs, and normalization of the disrupted fecal BA profile. Concurrently, it decreased levels of
lipotoxic BAs and inhibited hepatic cholesterol synthesis (evidenced by reduced HMGCR activity)
and intestinal cholesterol absorption (indicated by lower ACAT2 levels) while enhancing intestinal
cholesterol efflux (via LXR
α
, ABCA1, ABCG5, and ABCG8) and stimulating hepatic BA synthesis
(CYP7A1, CYP27A1) and secretion (BSEP). PPE thus led to a significant reduction in lipotoxic BAs
metabolized by gut microbiota and a downregulation of the BA secretion pathway under its influence.
Our findings reveal the therapeutic effect of PPE on NAFLD mice via regulating cholesterol and BA
metabolism, providing a theoretical basis for exploring the potential functions of P. cerasifera.
Keywords: Prunus cerasifera Ehrhart; drought environments; polyphenol extracts; non-alcoholic fatty
liver disease; bile acid metabolism
1. Introduction
Plants from extreme regions, particularly those from arid areas, often possess signifi-
cant biomedicinal properties. Non-alcoholic fatty liver disease (NAFLD), a prevalent global
metabolic disorder, primarily arises from obesity. This condition is typified by an exces-
sive accumulation of fat in the liver, a consequence of an imbalance where hepatic lipid
synthesis surpasses lipid degradation [
1
]. Epidemiological studies indicate that NAFLD
often represents the initial phase in the progression of more severe liver pathologies, in-
cluding non-alcoholic steatohepatitis (NASH), liver fibrosis, and hepatocellular carcinoma
(HCC). Despite its widespread nature and potential to advance to more serious diseases,
effective clinical treatments specifically targeting NAFLD remain elusive, with no currently
approved pharmaceutical interventions available [
2
,
3
]. This gap in treatment underscores
the critical need for ongoing research and development in this area.
The understanding of NAFLD pathogenesis has evolved significantly, transitioning
from the double-hit to the multiple-hit theory. This shift underscores the complexity
of NAFLD, linking its onset to a network of molecular pathways within the body [
4
].
Plants 2024,13, 288. https://doi.org/10.3390/plants13020288 https://www.mdpi.com/journal/plants
Plants 2024,13, 288 2 of 16
Central to these is the imbalance in cholesterol homeostasis, identified as a primary risk
factor in NAFLD development [
5
]. Cholesterol homeostasis is a tightly regulated process,
encompassing endogenous synthesis, exogenous uptake, efflux, and biotransformation.
Endogenous cholesterol, primarily synthesized by hepatic HMG-CoA reductase (HMGCR),
together with exogenous cholesterol, chiefly absorbed through the intestinal actions of
NPC1-like intracellular cholesterol transporter 1 (NPC1L1) and acetyl-CoA acetyltrans-
ferase 2 (ACAT2), constitute the dominant sources of cholesterol intake [
6
]. The regulation
of cholesterol efflux is mediated by the liver X receptor (LXR
α
), a pivotal member of the
nuclear hormone receptor superfamily of ligand-dependent transcription factors. LXR
α
orchestrates this process by controlling the activity of ABC family transporters, such as
ATP binding cassette subfamily A member 1 (ABCA1), ATP binding cassette subfamily G
member 5 (ABCG5), and ATP binding cassette subfamily G member 8 (ABCG8), thereby
maintaining cholesterol equilibrium [
6
,
7
]. Furthermore, a significant portion of cholesterol,
approximately 40%, undergoes metabolic conversion into bile acids (BAs) as part of its
degradation pathway [
8
]. This cholesterol-to-BA conversion is not just a degradation
route but also a crucial regulatory mechanism for maintaining cholesterol homeostasis,
underscoring the intricate balance and interplay of various biochemical pathways in the
pathogenesis of NAFLD. Understanding these processes is essential for developing targeted
therapeutic strategies for this increasingly prevalent disease.
Recent studies have illuminated a strong connection between bile acid (BA) metabolic
disorders [
9
,
10
] and gut microbiota imbalance [
11
,
12
] in the context of non-alcoholic fatty
liver disease (NAFLD). These findings underscore the complexity of NAFLD’s pathophysi-
ology. Notably, the farnesoid X receptor (FXR), a crucial modulator of BAs homeostasis,
plays a dual role in NAFLD’s progression [
13
]. In the liver, cholesterol undergoes transfor-
mation into primary BAs through two distinct pathways: the classical pathway involving
cholesterol 7a-hydroxylase (CYP7A1) and the alternative pathway via sterol-27-hydroxylase
(CYP27A1). These primary BAs then combine with taurine or glycine, forming conjugated
BAs. These conjugated BAs are secreted into the intestine under the influence of dietary
stimuli via the bile salt export pump (BSEP), playing a pivotal role in lipid digestion and
absorption in the body. However, a disruption in this process, either by inhibited BAs syn-
thesis or reduced BSEP activity (stemming from suppressed hepatic FXR-small heterodimer
partner (SHP) axis signaling), exacerbates NAFLD [14–16]. Concurrently, NAFLD is often
accompanied by a marked imbalance in gut microbiota. In the NAFLD context, there is
a significant enrichment of microbiota with bile salt hydrolase (BSH) activity and dehy-
droxylation capabilities. This enrichment leads to an increased conversion of conjugated
primary BAs into lipotoxic deconjugated secondary BAs. This conversion activates the
signaling of the intestinal FXR-fibroblast growth factor 15 (FGF15) axis, further aggravat-
ing the progression of NAFLD [
17
–
19
]. Thus, the interplay between gut microbiota, BAs
metabolism, and liver function emerges as a critical area in understanding and potentially
managing NAFLD.
Wild Prunus cerasifera Ehrhart (P. cerasifera) is a valuable wild fruit resource distributed
in the mountains of Central Asia. In China, it is only distributed in the narrow valleys of
Daxigou and Xiaoxigou in the arid area of northwest China. The survey found that local
residents who eat P. cerasifera fruits for a long time have a much lower rate of cardiovascular
disease than other people. At the same time, P. cerasifera is a unique medicinal germplasm
resource in Xinjiang, and its fruits are rich in polyphenols. These polyphenols, classified as
plant secondary metabolites, have garnered significant interest for their efficacy in com-
bating metabolic diseases. For instance, various polyphenolic compounds, such as those
found in apples, alongside berberine, hyperoside, and elexosaponin A1, have demonstrated
considerable potential in mitigating the progression of non-alcoholic fatty liver disease
(NAFLD) in animal models. This therapeutic effect is attributed to their ability to modulate
cholesterol metabolism, bile acids (BAs) metabolism, and gut microbiota [
13
,
20
–
22
]. These
findings not only highlight the therapeutic promise of polyphenols in metabolic disorders
Plants 2024,13, 288 3 of 16
but also underscore the intricate biological mechanisms through which these compounds
exert their beneficial effects.
In our previous research, we found that the polyphenol extract from P. cerasifera (PPE)
consists of 18 polyphenol monomers, including 3-O-caffeoylquinic acid, 5-O-caffeoylquinic
acid, 4-O-caffeoylquinic acid, and Apigenin-O-glucoside (refer to Table S1). Furthermore,
this extract has been shown to have a notable therapeutic effect on obesity [
23
,
24
]. Despite
these promising findings, it remains uncertain if PPE can effectively mitigate the develop-
ment of non-alcoholic fatty liver disease (NAFLD) through the modulation of cholesterol
and bile acids (BAs) metabolism. This area of uncertainty presents a crucial avenue for
further exploration to understand the full potential of PPE in treating metabolic disorders.
Addressing the aforementioned uncertainties, we have formulated two key research ques-
tions to guide our investigation: (1) Does PPE exert a significant impact on cholesterol
and bile acids (BAs) metabolism in NAFLD mouse models? (2) What are the specific
mechanistic pathways through which PPE modulates cholesterol and BAs metabolism?
These queries are fundamental in advancing our understanding of PPE’s potential role in
the management of NAFLD.
2. Results
2.1. Effects of PPE on TC, T-BAs, and Liver Damage in the Liver
To explore whether PPE is effective in NAFLD, different doses of PPE were adminis-
tered to mice with NAFLD induced by long-term high-fat feeding. The concentrations of
total cholesterol (TC) and total bile acids (T-BAs) in the liver are depicted in Figure 1A,B.
The TC and T-BAs in the HFD group were higher than those in the CK group, which were
12.06
±
2.35 mmol/g and 64.15
±
10.63
µ
mol/L, respectively. Remarkably, upon PPE
intervention, the hepatic TC levels were substantially reduced, while the T-BAs levels were
significantly elevated in comparison to those observed in the HFD group. This indicates a
notable effect of PPE on modulating these key metabolic parameters.
To ascertain the protective impact of PPE on liver and adipose tissues, sections of both
liver and fat were prepared (Figure 1C). Notably, the NAFLD activity score in the liver,
encompassing parameters like inflammation, ballooning, and steatosis, along with the size
of adipocytes in NAFLD mice, exhibited a significant reduction following PPE intervention.
This is evidenced in the detailed analyses presented in Figure 1D,E, highlighting the
effectiveness of PPE in mitigating the key pathological features associated with NAFLD.
2.2. Effects of PPE on Cholesterol Metabolism
HMG-CoA reductase (HMGCR) mRNA levels were significantly higher in the high-fat
diet (HFD) group compared to the control (CK), but they notably decreased in the high-dose
HFD (HFD+H) group. Liver X receptor alpha (LXR
α
) mRNA showed a similar trend, with
a lower expression in the HFD group and a significant increase in the HFD+H group. In
the ileum, expressions of ATP binding cassette transporters A1 (ABCA1), G5 (ABCG5),
G8 (ABCG8), and Niemann–Pick C1-Like 1 (NPC1L1) were significantly reduced in the
HFD group but increased in the HFD+H group. Conversely, acetyl-CoA acetyltransferase
2 (ACAT2) mRNA was higher in the HFD group but decreased in the HFD+H group. These
differential gene expressions in response to diet and treatment are detailed in Figure 2A.
The expression of HMGCR and LXR
α
proteins in the HFD group was notably higher
than in the CK group. However, in the HFD+H group, HMGCR protein expression
significantly decreased, while LXR
α
expression increased in comparison to the HFD group,
as detailed in Figure 2B,C.
Plants 2024,13, 288 4 of 16
Plants 2024, 13, x FOR PEER REVIEW 4 of 16
Figure 1. The levels of total cholesterol (TC), total bile acids (T-BAs), and indicators of liver damage
were meticulously assessed. Panels (A,B) illustrate the concentrations of hepatic TC and T-BAs, re-
spectively. Panel (C) displays representative histological images of liver and fat sections, stained
with hematoxylin and eosin (H&E) at 200× magnification and the scale bar is 200 μm. The NAFLD
Activity Score (NAS) of liver sections is depicted in panel (D), while panel (E) presents an analysis
of adipocyte size within fat sections. The data are presented as mean ± SD. Statistically significant
differences among the multiple groups (P < 0.05) are denoted by distinct letters, highlighting the
differential impacts of these treatments on liver health and adiposity. CK: mice fed a standard chow
diet with normal saline. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-
fat diet with high-dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE
(200 mg/kg/day). HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day).
2.2. Effects of PPE on Cholesterol Metabolism
HMG-CoA reductase (HMGCR) mRNA levels were significantly higher in the high-
fat diet (HFD) group compared to the control (CK), but they notably decreased in the high-
dose HFD (HFD+H) group. Liver X receptor alpha (LXRα) mRNA showed a similar trend,
with a lower expression in the HFD group and a significant increase in the HFD+H group.
In the ileum, expressions of ATP binding cassette transporters A1 (ABCA1), G5 (ABCG5),
G8 (ABCG8), and Niemann–Pick C1-Like 1 (NPC1L1) were significantly reduced in the
Figure 1. The levels of total cholesterol (TC), total bile acids (T-BAs), and indicators of liver damage
were meticulously assessed. Panels (A,B) illustrate the concentrations of hepatic TC and T-BAs,
respectively. Panel (C) displays representative histological images of liver and fat sections, stained
with hematoxylin and eosin (H&E) at 200
×
magnification and the scale bar is 200
µ
m. The NAFLD
Activity Score (NAS) of liver sections is depicted in panel (D), while panel (E) presents an analysis
of adipocyte size within fat sections. The data are presented as mean
±
SD. Statistically significant
differences among the multiple groups (P< 0.05) are denoted by distinct letters, highlighting the
differential impacts of these treatments on liver health and adiposity. CK: mice fed a standard chow
diet with normal saline. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a
high-fat diet with high-dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose
PPE (200 mg/kg/day). HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day).
Plants 2024,13, 288 5 of 16
Plants 2024, 13, x FOR PEER REVIEW 5 of 16
HFD group but increased in the HFD+H group. Conversely, acetyl-CoA acetyltransferase
2 (ACAT2) mRNA was higher in the HFD group but decreased in the HFD+H group.
These differential gene expressions in response to diet and treatment are detailed in Figure
2A.
The expression of HMGCR and LXRα proteins in the HFD group was notably higher
than in the CK group. However, in the HFD+H group, HMGCR protein expression signif-
icantly decreased, while LXRα expression increased in comparison to the HFD group, as
detailed in Figure 2B,C.
Figure 2. Impact of PPE on cholesterol metabolism in mice: (A) mRNA levels of key cholesterol
metabolism genes (HMGCR, LXR, ABCA1, ABCG5, ABCG8, NPC1L1, ACAT2) in liver and ileum. (B)
Western blot analysis of HMGCR and LXRα protein expression in liver. (C) Quantitative results of
protein bands. Data are shown as mean ± SD (n = 3), with different letters indicating significant
differences among groups (P < 0.05). CK: mice fed a standard chow diet with normal saline. HFD:
mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day). HFD+S: mice
fed a high-fat diet with statin (10 mg/kg/day).
2.3. Effects of PPE on Fecal BAs Profile in Mice
In the high-fat diet (HFD) group, fecal total bile acids (T-BAs) and other BA propor-
tions were significantly elevated compared to the control (CK) group, with decreased un-
conjugated and glycine-conjugated BAs. PPE intervention notably reversed these altera-
tions (Figure 3A,B). Additionally, the HFD group showed a lower secondary/primary BA
ratio than the CK group. Concentrations of primary BAs (cholic acid (CA), chenodeoxy-
cholic acid (CDCA), β-cholic acid (βCA), β-muricholic acid (βMCA), ω-muricholic acid
(ωMCA), cholic acid-3-sulfate (CA-3S)) and secondary BAs (deoxycholic acid (DCA), 23-
nordeoxycholic acid (23norDCA), lithocholic acid (LCA)) were higher in the HFD group.
Figure 2. Impact of PPE on cholesterol metabolism in mice: (A) mRNA levels of key cholesterol
metabolism genes (HMGCR, LXR, ABCA1, ABCG5, ABCG8, NPC1L1, ACAT2) in liver and ileum.
(B) Western blot analysis of HMGCR and LXR
α
protein expression in liver. (C) Quantitative results
of protein bands. Data are shown as mean
±
SD (n= 3), with different letters indicating significant
differences among groups (P< 0.05). CK: mice fed a standard chow diet with normal saline. HFD:
mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day). HFD+S:
mice fed a high-fat diet with statin (10 mg/kg/day).
2.3. Effects of PPE on Fecal BAs Profile in Mice
In the high-fat diet (HFD) group, fecal total bile acids (T-BAs) and other BA pro-
portions were significantly elevated compared to the control (CK) group, with decreased
unconjugated and glycine-conjugated BAs. PPE intervention notably reversed these alter-
ations (Figure 3A,B). Additionally, the HFD group showed a lower secondary/primary
BA ratio than the CK group. Concentrations of primary BAs (cholic acid (CA), chen-
odeoxycholic acid (CDCA),
β
-cholic acid (
β
CA),
β
-muricholic acid (
β
MCA),
ω
-muricholic
acid (
ω
MCA), cholic acid-3-sulfate (CA-3S)) and secondary BAs (deoxycholic acid (DCA),
23-nordeoxycholic
acid (23norDCA), lithocholic acid (LCA)) were higher in the HFD group.
PPE intervention significantly adjusted the primary and secondary BAs concentrations and
their ratio (Figure 3C,D).
Plants 2024,13, 288 6 of 16
Plants 2024, 13, x FOR PEER REVIEW 6 of 16
PPE intervention significantly adjusted the primary and secondary BAs concentrations
and their ratio (Figure 3C,D).
Figure 3. PPE’s impact on mice fecal BAs profile: utilizing UHPLC-MS/MS for targeted metabolom-
ics, we analyzed mouse feces to assess (A) fecal total bile acid (T-BA) concentrations, (B) ratios of
conjugated and unconjugated BAs, (C) secondary/primary BAs ratio, and (D) levels of various pri-
mary and secondary BAs. Data are presented as mean ± SD (n = 5), with different letters indicating
significant differences across groups (p < 0.05). CK: mice fed a standard chow diet with normal sa-
line. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-
dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day).
HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day). CA: cholic acid. CDCA: chenodeoxy-
cholic acid. βCA: β-cholic acid. βMCA: β-muricholic acid. ωMCA: ω-muricholic acid. CA-3S: cholic
acid-3-sulfate. DCA: deoxycholic acid. LCA: lithocholic acid. 23norDCA: 23-nordeoxycholic acid.
7,12-diketoLCA: 7,12-diketolithocholic acid. THDCA: taurohyodeoxycholic acid.
2.4. Effects of PPE on BAs Metabolism
Gene Expression in BAs Metabolism (Figure 4A): In the HFD group, hepatic
CYP27A1 and BSEP showed no significant changes compared to CK, but the expressions
of CYP7A1, FXR, SHP, and Na+/taurocholate cotransporter (NTCP) were markedly re-
duced. Compared with the HFD group, the HFD+H group exhibited increased levels of
liver CYP7A1, CYP27A1, FXR, SHP, BSEP, and NTCP. Ileal Gene Expression: Compared
to CK, the HFD group had higher ileal FXR and lower Apical sodium-dependent bile acid
Figure 3. PPE’s impact on mice fecal BAs profile: utilizing UHPLC-MS/MS for targeted metabolomics,
we analyzed mouse feces to assess (A) fecal total bile acid (T-BA) concentrations, (B) ratios of
conjugated and unconjugated BAs, (C) secondary/primary BAs ratio, and (D) levels of various
primary and secondary BAs. Data are presented as mean
±
SD (n= 5), with different letters indicating
significant differences across groups (p< 0.05). CK: mice fed a standard chow diet with normal saline.
HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day). HFD+S:
mice fed a high-fat diet with statin (10 mg/kg/day). CA: cholic acid. CDCA: chenodeoxycholic
acid.
β
CA:
β
-cholic acid.
β
MCA:
β
-muricholic acid.
ω
MCA:
ω
-muricholic acid. CA-3S: cholic
acid-3-sulfate. DCA: deoxycholic acid. LCA: lithocholic acid. 23norDCA: 23-nordeoxycholic acid.
7,12-diketoLCA: 7,12-diketolithocholic acid. THDCA: taurohyodeoxycholic acid.
2.4. Effects of PPE on BAs Metabolism
Gene Expression in BAs Metabolism (Figure 4A): In the HFD group, hepatic CYP27A1
and BSEP showed no significant changes compared to CK, but the expressions of CYP7A1,
FXR,SHP, and Na+/taurocholate cotransporter (NTCP) were markedly reduced. Com-
pared with the HFD group, the HFD+H group exhibited increased levels of liver CYP7A1,
CYP27A1,FXR,SHP,BSEP, and NTCP. Ileal Gene Expression: Compared to CK, the HFD
group had higher ileal FXR and lower Apical sodium-dependent bile acid transporter
(ASBT) expressions, with no effect on FGF15. PPE notably downregulated FXR and FGF15
and upregulated ASBT in the ileum relative to HFD (Figure 4A). Protein Expression in BAs
Metabolism (Figure 4B,C): In the liver, the HFD+H group significantly increased FXR, SHP,
Plants 2024,13, 288 7 of 16
and CYP7A1 expressions compared to HFD. Post-PPE, the ileal ASBT expression decreased
notably compared to the HFD group.
Plants 2024, 13, x FOR PEER REVIEW 7 of 16
transporter (ASBT) expressions, with no effect on FGF15. PPE notably downregulated
FXR and FGF15 and upregulated ASBT in the ileum relative to HFD (Figure 4A). Protein
Expression in BAs Metabolism (Figure 4B,C): In the liver, the HFD+H group significantly
increased FXR, SHP, and CYP7A1 expressions compared to HFD. Post-PPE, the ileal ASBT
expression decreased notably compared to the HFD group.
Figure 4. Impact of PPE on BAs metabolism in mice: (A) mRNA levels of crucial BAs metabolism
genes (CYP7A1, CYP27A1, FXR, SHP, BSEP, NTCP, FGF15, ASBT) in liver and ileum. (B) Western
blot analysis of ABST, SHP, FXR, CYP27A1, and CYP7A1 proteins in liver and ileum. (C) Quantita-
tive results of protein bands. Data are shown as mean ± SD (n = 3), with different letters indicating
significant differences across groups (P < 0.05). CK: mice fed a standard chow diet with normal
saline. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-
dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day).
HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day).
2.5. Correlation Analysis between Gut Microbiota and BAs Metabolism
The above studies have confirmed that PPE alleviates NAFLD by regulating choles-
terol metabolism and BAs metabolism, but it remains unclear whether BAs metabolism is
related to PPE-driven structural changes in gut microbiota. Therefore, a correlation anal-
ysis was conducted on the structural changes in gut microbiota and changes in BAs me-
tabolism in NAFLD mice after PPE intervention. At the genus level, Spearman correlation
analysis was performed on the top 30 differential microorganisms and 15 differential BAs
Figure 4. Impact of PPE on BAs metabolism in mice: (A) mRNA levels of crucial BAs metabolism
genes (CYP7A1, CYP27A1, FXR, SHP, BSEP, NTCP, FGF15, ASBT) in liver and ileum. (B) Western blot
analysis of ABST, SHP, FXR, CYP27A1, and CYP7A1 proteins in liver and ileum. (C) Quantitative
results of protein bands. Data are shown as mean
±
SD (n= 3), with different letters indicating
significant differences across groups (P< 0.05). CK: mice fed a standard chow diet with normal saline.
HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose PPE (200 mg/kg/day). HFD+S:
mice fed a high-fat diet with statin (10 mg/kg/day).
2.5. Correlation Analysis between Gut Microbiota and BAs Metabolism
The above studies have confirmed that PPE alleviates NAFLD by regulating cholesterol
metabolism and BAs metabolism, but it remains unclear whether BAs metabolism is related
to PPE-driven structural changes in gut microbiota. Therefore, a correlation analysis was
conducted on the structural changes in gut microbiota and changes in BAs metabolism in
NAFLD mice after PPE intervention. At the genus level, Spearman correlation analysis was
performed on the top 30 differential microorganisms and 15 differential BAs between the
HFD and HFD+H groups, and data without significant correlation were filtered out. There
were negative correlations between taurohyodeoxycholic acid (THDCA) and isohyodeoxy-
cholic acid (isoHDCA) with Mucispirillum, Erysipelatoclostridium, and Bifidobacterium
and positive correlations with Lactobacillus, Akkermansia, Parabacteroides, Neglecta, Turi-
cibacter, Lachnospiraceae_NK4A136_group, Monoglobus, Candidatus_Saccharimonas, and
Plants 2024,13, 288 8 of 16
Clostridia_UCG
−
014_unclassified (Figure 5A). Gut microbiota with BSH activity (Turi-
cibacter, Streptococcus, Lactobacillus, Alloprevotella and Bacteroides) were enriched in the
HFD+H group compared to the HFD group (Figure 5B,C), but the ratio of DCA/(DCA+CA)
was significantly reduced (Figure 5D).
Plants 2024, 13, x FOR PEER REVIEW 8 of 16
between the HFD and HFD+H groups, and data without significant correlation were fil-
tered out. There were negative correlations between taurohyodeoxycholic acid (THDCA)
and isohyodeoxycholic acid (isoHDCA) with Mucispirillum, Erysipelatoclostridium, and
Bifidobacterium and positive correlations with Lactobacillus, Akkermansia, Parabac-
teroides, Neglecta, Turicibacter, Lachnospiraceae_NK4A136_group, Monoglobus, Candi-
datus_Saccharimonas, and Clostridia_UCG−014_unclassified (Figure 5A). Gut microbiota
with BSH activity (Turicibacter, Streptococcus, Lactobacillus, Alloprevotella and Bac-
teroides) were enriched in the HFD+H group compared to the HFD group (Figure 5B,C),
but the ratio of DCA/(DCA+CA) was significantly reduced (Figure 5D).
Figure 5. Correlation analysis between gut microbiota and BAs metabolism. (A) Spearman correla-
tion analysis of differential gut microbiota and differential BAs at the genus level. (B) Differential
BSH-producing gut microbiota at the genus level. (C) Sum of BSH-producing gut microbiota at the
genus level. (D) DCA/(DCA + CA) and LCA/(LCA + CDCA) at the genus level. Data are presented
as the mean ± SD (n = 5). Different letters represent a significant difference among multiple groups
(P < 0.05) and * P ≤ 0.05, ** P ≤ 0.01. CK: mice fed a standard chow diet with normal saline. HFD:
mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day).
2.6. Effects of PPE on Serum Metabolic Profile in Mice
The OPLS-DA model indicated significant metabolic differences between the HFD
and HFD+H groups, with a total of 179 differential metabolites identified in HFD vs.
HFD+H (Figures 6A and S1). Among these differential metabolites, there were 10 signifi-
cantly upregulated, 10 significantly downregulated, and 159 insignificantly differential
metabolites (Figure 6B). Notably, cholic acid was among the 10 significantly downregu-
lated differential metabolites in the HFD+H group. Pathway enrichment analysis demon-
strated a significant enrichment of the bile secretion pathway, with a proportion of 7.27%
of detected metabolites participating in this pathway (Figure 6C–E).
Figure 5. Correlation analysis between gut microbiota and BAs metabolism. (A) Spearman correlation
analysis of differential gut microbiota and differential BAs at the genus level. (B) Differential BSH-
producing gut microbiota at the genus level. (C) Sum of BSH-producing gut microbiota at the genus
level. (D) DCA/(DCA + CA) and LCA/(LCA + CDCA) at the genus level. Data are presented as
the mean
±
SD (n= 5). Different letters represent a significant difference among multiple groups
(P< 0.05)
and * P
≤
0.05, ** P
≤
0.01. CK: mice fed a standard chow diet with normal saline. HFD:
mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day).
2.6. Effects of PPE on Serum Metabolic Profile in Mice
The OPLS-DA model indicated significant metabolic differences between the HFD and
HFD+H groups, with a total of 179 differential metabolites identified in HFD vs. HFD+H
(Figures 6A and S1). Among these differential metabolites, there were 10 significantly
upregulated, 10 significantly downregulated, and 159 insignificantly differential metabolites
(Figure 6B). Notably, cholic acid was among the 10 significantly downregulated differential
metabolites in the HFD+H group. Pathway enrichment analysis demonstrated a significant
enrichment of the bile secretion pathway, with a proportion of 7.27% of detected metabolites
participating in this pathway (Figure 6C–E).
Plants 2024,13, 288 9 of 16
Plants 2024, 13, x FOR PEER REVIEW 9 of 16
analysis demonstrated a significant enrichment of the bile secretion pathway, with a
proportion of 7.27% of detected metabolites participating in this pathway (Figure 6C–E).
Figure 6. Effects of PPE on serum metabolic profile in mice. (A) Veen plot of differential metabolites.
(B) Matchstick plot of differential metabolites. (C) KEGG pathway enrichment analysis matchstick
plot. (D) KEGG pathway enrichment analysis bubble chart. (E) Detected metabolite proportion
histogram. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. (B–E) red frames: metabolites and pathways related
to BA in serum metabolic profile. CK: mice fed a standard chow diet with normal saline. HFD: mice
fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE (400
mg/kg/day).
3. Discussion
NAFLD is considered an epidemic affecting approximately 1/4 of the world’s
population, and its pathogenesis is complex. Currently, research on this disease is mainly
conducted using mouse models. Hepatic lipid accumulation plays a pivotal role in the
pathogenesis of NAFLD, contributing significantly to the development and progression
of the disease [5]. Our experimental findings provide compelling evidence that PPE
treatment effectively mitigated the excessive hepatic accumulation of TC, a hallmark of
NAFLD, in the murine model. Importantly, these observations were further substantiated
Figure 6. Effects of PPE on serum metabolic profile in mice. (A) Veen plot of differential metabolites.
(B) Matchstick plot of differential metabolites. (C) KEGG pathway enrichment analysis matchstick
plot. (D) KEGG pathway enrichment analysis bubble chart. (E) Detected metabolite proportion
histogram. * P
≤
0.05, ** P
≤
0.01, *** P
≤
0.001. (B–E) red frames: metabolites and pathways related
to BA in serum metabolic profile. CK: mice fed a standard chow diet with normal saline. HFD:
mice fed a high-fat diet with normal saline. HFD+H: mice fed a high-fat diet with high-dose PPE
(400 mg/kg/day).
3. Discussion
NAFLD is considered an epidemic affecting approximately 1/4 of the world’s pop-
ulation, and its pathogenesis is complex. Currently, research on this disease is mainly
conducted using mouse models. Hepatic lipid accumulation plays a pivotal role in the
pathogenesis of NAFLD, contributing significantly to the development and progression of
the disease [
5
]. Our experimental findings provide compelling evidence that PPE treatment
effectively mitigated the excessive hepatic accumulation of TC, a hallmark of NAFLD, in the
murine model. Importantly, these observations were further substantiated by histopatho-
logical analyses, which unequivocally demonstrated the hepatoprotective properties of
PPE in NAFLD-afflicted mice. The histopathological assessments revealed a notable re-
duction in hepatic steatosis, inflammation, ballooning, and overall NAFLD activity score
in the PPE-treated group when compared to untreated NAFLD mice. These histological
Plants 2024,13, 288 10 of 16
improvements underscore the potential therapeutic efficacy of PPE in ameliorating the liver
damage associated with NAFLD.
Numerous studies have provided compelling evidence that the metabolism of bile
acids (BAs) not only contributes to the development of NAFLD [
9
,
10
,
13
,
25
] but also rep-
resents a promising target for NAFLD treatment [
26
]. In the context of high-fat-induced
NAFLD in mice, there is a noticeable tendency for hepatic total BAs (T-BAs) to increase,
accompanied by a significant rise in fecal T-BAs. This suggests that the rate of lipid degrada-
tion is constrained in NAFLD [
1
]. Intervention with PPE leads to a decrease in fecal T-BAs
and an increase in liver T-BAs, indicating that the intervention facilitates the conversion of
cholesterol into BAs and their excretion into the intestine. To substantiate this hypothesis,
we further investigated the activity of CYP7A1 and CYP27A1, the rate-limiting enzymes
responsible for the synthesis of BAs from cholesterol, particularly cholic acid (CA) and chen-
odeoxycholic acid (CDCA), in the liver through the classical and alternative pathways [
10
].
Our findings demonstrate that NAFLD mice do convert a portion of cholesterol into BAs
via CYP7A1 and CYP27A1, but the rate of lipid degradation is considerably lower than
that observed with a high-dose PPE intervention. Of particular interest is the conversion
of most CDCA into
α
-/
β
-murine cholic acid (
α
-/
β
-MCA) through 6
α
-/
β
-hydroxylation
in rodents [
27
]. Among these, CA, CDCA, and
α
-/
β
-MCA are conjugated with taurine
or glycine in the liver to form conjugated BAs, which then flow into the gallbladder. Sub-
sequently, these conjugated BAs are discharged into the intestine via the bile salt export
pump (BSEP) under the coordination of the FXR-SHP axis to participate in the digestion
process [
13
,
14
]. At both the gene and protein levels, PPE intervention led to a significant
upregulation in the expression of liver FXR, SHP, and BSEP. However, low-dose PPE had no
discernible effect on FXR. These data collectively suggest that PPE enhances BAs synthesis
in the liver and promotes BAs efflux by activating the FXR-SHP axis signaling pathway.
Nevertheless, it is noteworthy that serum metabolic profiles predicted a downregulation
of the bile secretion pathway, while liver BSEP was upregulated. This discrepancy might
be attributed to inconsistencies in gene and protein expression patterns and warrants
further investigation.
In contrast to the activation of hepatic FXR, the activation of intestinal FXR in NAFLD
mice has been associated with the promotion of NAFLD through the FXR-FGF15 signaling
pathway [
17
–
19
]. However, emerging evidence suggests that polyphenols exert a down-
regulatory effect on intestinal FXR-FGF15 signaling by modulating the composition of gut
microbiota, particularly those involved in bile acid metabolism, in the context of metabolic
diseases [
28
–
30
]. Our study yielded intriguing findings, revealing that PPE intervention
effectively attenuated ileal FXR-FGF15 signaling in NAFLD-afflicted mice. Notably, the
inhibitory effect was more pronounced in the high-dose PPE intervention group. This
observation sheds light on a potentially novel mechanism by which PPE may exert its
therapeutic effects in NAFLD. It appears that PPE’s influence on NAFLD extends beyond
hepatic factors as it impacts intestinal FXR-FGF15 signaling, offering a multifaceted ap-
proach to mitigate the development and progression of this complex metabolic disorder.
Further investigations into the precise mechanisms underlying this effect are warranted
and could hold significant implications for NAFLD treatment strategies.
Bile acid biosynthesis serves as a pivotal pathway for cholesterol efflux, complement-
ing the other pathway primarily mediated by the ileal ABC family under the regulatory
influence of hepatic LXR
α
. Following PPE intervention, there was a substantial increase in
the expression of hepatic LXR
α
and ileal ABCA1, ABCG5, and ABCG8 at both gene and
protein levels. These findings provide compelling evidence that PPE effectively facilitates
cholesterol efflux from the liver. Excessive cholesterol levels have been shown to inhibit the
functionality of ileal NPC1L1, thereby promoting cholesterol uptake by ileal ACAT2 [
6
,
31
].
Remarkably, PPE intervention resulted in the reversal of this cholesterol uptake mechanism.
HMGCR, a key rate-limiting enzyme responsible for endogenous cholesterol synthesis in
the liver, is known to be competitively inhibited by statins [
32
]. Our study unequivocally
confirmed that PPE exerts an inhibitory effect on the expression of HMGCR. These insights
Plants 2024,13, 288 11 of 16
into the regulatory effects of PPE on key players in cholesterol metabolism shed light on its
potential therapeutic role in mitigating cholesterol-related disorders.
Alterations in the composition of gut microbiota can exert a profound impact on
the fecal BAs profile and, conversely, changes in the fecal BAs profile can reciprocally
influence gut microbiota. These intricate interactions between gut microbiota and the
fecal BAs profile play a pivotal role in regulating host health through enterohepatic axis
circulation [
11
,
12
,
19
]. In our study, we observed noteworthy changes in the fecal BAs
profile of NAFLD mice following PPE intervention. Specifically, there was a significant
increase in the low-level secondary/primary BA ratio and the proportion of unconjugated
BAs, while the DCA/(DCA + CA) ratio exhibited a significant decrease. This phenomenon
can be attributed to the remarkable enrichment of gut microbiota with bile salt hydrolase
(BSH) activity, including Lactobacillus [
33
] and Turicibacter [
34
], in the intestine upon
PPE intervention. These microbiota play a crucial role in converting conjugated primary
BAs into deconjugated primary BAs, which are subsequently transformed into secondary
BAs through 7-
α
-dehydroxylation. Notably, secondary BAs such as DCA and LCA have
been linked to the development of various metabolic disorders [
19
]. In the context of
NAFLD, DCA and LCA further exacerbate the progression of the disease by activating
intestinal FXR-FGF15 axis signaling [
17
,
18
,
35
]. The reduction in elevated levels of DCA
and LCA in the intestines of NAFLD mice following PPE intervention is indicative of a
potential mechanism through which PPE inhibits the development of NAFLD. To validate
the intricate interplay between fecal BAs and gut microbiota, Spearman correlation anal-
ysis was conducted. The results revealed significant negative correlations between DCA
and LCA in feces and certain gut microbiota species, including Turicibacter [
34
] and Ne-
glecta, while positive correlations were observed with others such as Mucispirillum [
36
,
37
]
and Erysipelatoclostridium [
38
]. Additionally, secondary BAs THDCA and isoHDCA
exhibited significant enrichment [
39
,
40
] following PPE intervention and displayed positive
correlations with Turicibacter, Lactobacillus, Candidatus_Saccharimonas, Parabacteroides,
Akkermansia, and Neglecta. These findings provide robust evidence that PPE alleviates
NAFLD in mice by reshaping gut microbiota composition, reducing lipotoxic secondary
BAs (DCA and LCA), and enhancing THDCA and isoHDCA levels to suppress intestinal
FXR-FGF15 axis signaling.
4. Materials and Methods
4.1. Preparation of PPE
In the same batch, we harvested a sufficient amount of fresh wild P. cerasifera fruits
in Yili, Xinjiang, China, on 20 August 2022 and immediately quick-froze them to
−
20
◦
C.
We made slight modifications to the PPE extraction process based on previously reported
methods [
41
]. The process involved the following steps: First, fresh P. cerasifera fruits were
carefully deseeded and homogenized. Subsequently, the homogenized fruit material was
subjected to extraction using a mixture of methanol, water, and formic acid (in a ratio of
90:9:1, v/v) at room temperature for a duration of 4 h. This extraction process was repeated
twice to ensure thorough extraction. The resulting extract solution underwent a purification
step using AB-8 macroporous adsorption resin. After complete adsorption onto the resin,
the extraction solution was meticulously washed with a solution containing 1% formic
acid. Subsequently, the target compounds were effectively eluted from the resin using
60% ethanol. The eluent obtained from this process was carefully collected and subjected to
concentration and freeze-drying, ultimately yielding a fine powder. The total polyphenol
content of P. cerasifera polyphenol extract was 439.17 mg/g, and the yield after purification
was 68.54%.
4.2. Animals Experimental Design
A total of fifty-six 4-week-old ICR mice were procured from Xinjiang Medical Uni-
versity in Xinjiang, China. The mice underwent an initial period of standardized adaptive
feeding, which involved maintaining them in controlled environmental conditions with a
Plants 2024,13, 288 12 of 16
temperature of 25
±
2
◦
C, relative humidity of 60
±
5%, and a 12 h light and dark cycle.
During this phase, the mice had unrestricted access to both food and water.
Following one week of adaptive feeding, the mice were randomly assigned to two
main groups: a control group (referred to as the CK group, consisting of n= 13 mice)
that was fed a standard chow diet and a high-fat group (referred to as the HFD group,
consisting of n= 43 mice) that received a high-fat (60 FDC) purified rodent diet (HF60, Dyets
Biotechnology, Wuxi, China). After a period of 11 weeks on their respective diets, three
mice from each group were selected for assessment to confirm the successful establishment
of the NAFLD model. This confirmation was achieved by evaluating serum lipid levels
and assessing liver damage.
Subsequently, the NAFLD-afflicted mice were randomly divided into four distinct
groups, as outlined in Figure 7, and subjected to an additional 24 weeks of intervention. The
group divisions were as follows: (1) High-fat diet + normal saline (oral administration of
equal volume of normal saline, HFD group, n= 10), (2) high-fat diet + high-dose PPE (oral
administration, 400 mg/kg/day, BW, HFD+H group, n= 10), (3) high-fat diet + low-dose
PPE (oral administration, 200 mg/kg/day, BW, HFD+L group, n= 10), and (4) high-fat
diet + statin (oral administration, 10 mg/kg/day, BW, HFD+S group, n= 10).
Prior to the conclusion of the intervention period, fecal samples were collected for
subsequent analysis of the targeted bile acids profile. At the conclusion of the experiment,
all mice were euthanized via cervical dislocation. Various biological samples, including
blood, adipose tissue, liver, and ileum, were harvested for further analysis.
The study was conducted in accordance with the Declaration of Helsinki and approved
by the Animal Experimental Ethical Inspection of College of Life Sciences, Xinjiang Normal
University (No. XJNU2022-08).
Plants 2024, 13, x FOR PEER REVIEW 12 of 16
4.2. Animals Experimental Design
A total of fifty-six 4-week-old ICR mice were procured from Xinjiang Medical Uni-
versity in Xinjiang, China. The mice underwent an initial period of standardized adaptive
feeding, which involved maintaining them in controlled environmental conditions with a
temperature of 25 ± 2 °C, relative humidity of 60 ± 5%, and a 12 h light and dark cycle.
During this phase, the mice had unrestricted access to both food and water.
Following one week of adaptive feeding, the mice were randomly assigned to two
main groups: a control group (referred to as the CK group, consisting of n = 13 mice) that
was fed a standard chow diet and a high-fat group (referred to as the HFD group, consist-
ing of n = 43 mice) that received a high-fat (60 FDC) purified rodent diet (HF60, Dyets
Biotechnology, Wuxi, China). After a period of 11 weeks on their respective diets, three
mice from each group were selected for assessment to confirm the successful establish-
ment of the NAFLD model. This confirmation was achieved by evaluating serum lipid
levels and assessing liver damage.
Subsequently, the NAFLD-afflicted mice were randomly divided into four distinct
groups, as outlined in Figure 7, and subjected to an additional 24 weeks of intervention.
The group divisions were as follows: (1) High-fat diet + normal saline (oral administration
of equal volume of normal saline, HFD group, n = 10), (2) high-fat diet + high-dose PPE
(oral administration, 400 mg/kg/day, BW, HFD+H group, n = 10), (3) high-fat diet + low-
dose PPE (oral administration, 200 mg/kg/day, BW, HFD+L group, n = 10), and (4) high-
fat diet + statin (oral administration, 10 mg/kg/day, BW, HFD+S group, n = 10).
Prior to the conclusion of the intervention period, fecal samples were collected for
subsequent analysis of the targeted bile acids profile. At the conclusion of the experiment,
all mice were euthanized via cervical dislocation. Various biological samples, including
blood, adipose tissue, liver, and ileum, were harvested for further analysis.
The study was conducted in accordance with the Declaration of Helsinki and ap-
proved by the Animal Experimental Ethical Inspection of College of Life Sciences, Xin-
jiang Normal University (No. XJNU2022-08).
Figure 7. Animal experimental design. PPE: P. cerasifera polyphenol extracts. CK: mice fed a stand-
ard chow diet with normal saline. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice
fed a high-fat diet with high-dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-
dose PPE (200 mg/kg/day). HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day). NAS:
NAFLD activity score.
4.3. Liver Total Cholesterol (TC) and Total Bile Acids (T-BAs) Determination
Figure 7. Animal experimental design. PPE: P. cerasifera polyphenol extracts. CK: mice fed a standard
chow diet with normal saline. HFD: mice fed a high-fat diet with normal saline. HFD+H: mice fed a
high-fat diet with high-dose PPE (400 mg/kg/day). HFD+L: mice fed a high-fat diet with low-dose
PPE (200 mg/kg/day). HFD+S: mice fed a high-fat diet with statin (10 mg/kg/day). NAS: NAFLD
activity score.
4.3. Liver Total Cholesterol (TC) and Total Bile Acids (T-BAs) Determination
The concentration of TC and T-BAs in the liver was measured following the recom-
mended instructions provided by the kit manufacturer (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China). Liver tissue homogenates were subjected to centrifugation at
4
◦
C, 4000 rpm for 5 min, and the resulting supernatant was collected. Subsequently, all the
collected samples were assayed in accordance with the specifications outlined by the kit.
Plants 2024,13, 288 13 of 16
4.4. Histopathological Observation of the Liver and Fat
Following the previously established procedure, liver and adipose tissues were pro-
cured and subsequently fixed using 4% paraformaldehyde. These tissues underwent
dehydration using ethanol, were embedded in paraffin, sliced into sections, and ultimately
stained with hematoxylin and eosin (H&E). The alterations in liver and adipose tissues
were examined under a light microscope, enabling a visual assessment of the extent of liver
lesions and the dimensions of adipocytes. Details of NAS semi-quantitative scoring are
provided in Supplementary Materials and Methods S1.
4.5. Determination of Targeted BAs Metabolism Profile in Feces and Non-Targeted Metabolic
Profile in Serum
Mouse feces and serum samples, collected under aseptic conditions, were rapidly
frozen using liquid nitrogen. Subsequently, these frozen samples were dispatched to Shang-
hai biotree Biomedical Technology Co., Ltd. (Shanghai, China) for comprehensive targeted
and non-targeted metabolic profile analysis. The fecal and serum supernatants, prepared for
analysis, were subjected to identification utilizing UHPLC-MS/MS. The raw data obtained
were meticulously filtered and subsequently employed for the quantitative assessment of
the fecal bile acids (BAs) profile and the serum metabolic profile. For further insights into
the methodologies employed, please refer to the Supporting Information section.
4.6. Real-Time PCR Analysis
Liver and ileum tissue samples were removed from a
−
80
◦
C refrigerator, and the total
RNA was extracted using Trizol reagent; the RNA was reverse-transcribed to cDNA using
a cDNA synthesis kit (Tiangen Biochemical Technology, Beijing, China). Then, the cDNA
was used to amplify the target genes using SYBR Green PCR Master Mix on a StepOnePlus
Real-time PCR detection system (4376600, ABI StepOnePlus, Carlsbad, CA, USA). The
relative transcription of mRNA was calculated by the 2
−∆∆Ct
method with
β
-actin for
normalization, and the primer sequences are shown in Table S1.
4.7. Western Blot Analysis
The liver and ileum tissue homogenates were treated with a lysis buffer containing
protease inhibitors. Following homogenization, the resulting supernatant was separated by
centrifugation, and the protein concentration was determined using the BCA protein assay
kit from Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China. Subsequently, the
proteins were resolved through 12% SDS-PAGE, transferred onto PVDF membranes, and
subjected to overnight incubation at 4
◦
C with primary antibodies targeting proteins such
as CYP7A1, SHP, HMGCR, CYP27A1, LXR-
α
, FXR, ASBT, and GAPDH. The membranes
were then washed thrice with TBST, followed by incubation with secondary antibodies for
2 h at room temperature, and then subsequent washing with TBST took place. Visualization
of the blots was achieved using the Clarity Western enhanced chemiluminescence (ECL)
substrate kit (P1050, Applygen Technologies, Beijing, China). Band density values were
quantified and analyzed using Image J2x 2.1.5.0 software.
4.8. Statistical Analysis
Statistical analysis was conducted using SPSS 21.0 software, and the experimental data
were expressed as mean
±
SD, providing a comprehensive representation of the results.
To evaluate differences between groups, an ANOVA Dunnett’s multiple-comparison test
was employed, with statistical significance set at P< 0.05. The graphical illustrations were
created using either GraphPad Prism 8.0 software or R Studio (R version 4.0), ensuring a
visually clear and informative presentation of the findings.
5. Conclusions
This study has effectively demonstrated that PPE exerts a pronounced inhibitory effect
on liver cholesterol biosynthesis and intestinal cholesterol absorption. This results in an
Plants 2024,13, 288 14 of 16
increased conversion of liver cholesterol into bile acids (BAs) and enhanced intestinal
cholesterol efflux. Furthermore, PPE attenuates the transformation of conjugated primary
BAs into lipotoxic BAs, including LCA and DCA. These findings strongly suggest that
PPE has the potential to alleviate NAFLD by modulating cholesterol and BAs metabolism
in mice, as illustrated in Figure 8. This research lays the groundwork for considering
P. cerasifera fruit as a functional food for NAFLD alleviation. However, the precise mecha-
nisms underlying the alleviating effects of PPE intervention on NAFLD warrant further
in-depth exploration.
Plants 2024, 13, x FOR PEER REVIEW 14 of 16
Statistical analysis was conducted using SPSS 21.0 software, and the experimental
data were expressed as mean ± SD, providing a comprehensive representation of the re-
sults. To evaluate differences between groups, an ANOVA Dunnett’s multiple-compari-
son test was employed, with statistical significance set at P < 0.05. The graphical illustra-
tions were created using either GraphPad Prism 8.0 software or R Studio (R version 4.0),
ensuring a visually clear and informative presentation of the findings.
5. Conclusions
This study has effectively demonstrated that PPE exerts a pronounced inhibitory ef-
fect on liver cholesterol biosynthesis and intestinal cholesterol absorption. This results in
an increased conversion of liver cholesterol into bile acids (BAs) and enhanced intestinal
cholesterol efflux. Furthermore, PPE attenuates the transformation of conjugated primary
BAs into lipotoxic BAs, including LCA and DCA. These findings strongly suggest that
PPE has the potential to alleviate NAFLD by modulating cholesterol and BAs metabolism
in mice, as illustrated in Figure 8. This research lays the groundwork for considering P.
cerasifera fruit as a functional food for NAFLD alleviation. However, the precise mecha-
nisms underlying the alleviating effects of PPE intervention on NAFLD warrant further
in-depth exploration.
Figure 8. Potential molecular mechanisms of PPE affecting NAFLD. HMGCR: HMG-CoA reductase.
NPC1L1: NPC1-like intracellular cholesterol transporter 1. ACAT2: acetyl-CoA acetyltransferase 2.
LXRα: liver X receptor. ABCA1/5/8: ATP binding cassette subfamily A member 1/5/8. FXR: farnesoid
X receptor. CYP7A1: cholesterol 7a-hydroxylase. CYP27A1: sterol-27-hydroxylase. BSEP: bile salt
export pump. SHP: small heterodimer partner. FGF15: fibroblast growth factor 15. ASBT: apical so-
dium-dependent bile acid transporter. NTCP: Na+/taurocholate cotransporter. BAs: bile acids.
Supplementary Materials: The following supporting information can be downloaded at:
www.mdpi.com/xxx/s1, Supplementary Materials and Methods: 1. NAS semi-quantitative scoring
Figure 8. Potential molecular mechanisms of PPE affecting NAFLD. HMGCR: HMG-CoA reductase.
NPC1L1: NPC1-like intracellular cholesterol transporter 1. ACAT2: acetyl-CoA acetyltransferase 2.
LXR
α
: liver X receptor. ABCA1/5/8: ATP binding cassette subfamily A member 1/5/8. FXR: farne-
soid X receptor. CYP7A1: cholesterol 7a-hydroxylase. CYP27A1: sterol-27-hydroxylase. BSEP: bile
salt export pump. SHP: small heterodimer partner. FGF15: fibroblast growth factor 15. ASBT: apical
sodium-dependent bile acid transporter. NTCP: Na+/taurocholate cotransporter. BAs: bile acids.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants13020288/s1, Supplementary Materials and Methods: S1. NAS
semi-quantitative scoring system, S2. Detailed methods for detection of targeted BAs metabolic
profiling in feces, S3. Detailed methods for detection of non-targeted metabolic profile in serum,
Table S1: Analysis of main components of PPE polyphenol by mass spectrometry, Table S2: The
primer sequences related to cholesterol metabolism and BAs metabolism, Figure S1: Effect of PPE on
serum metabolic profile. (A) PCA score plot and (B) PCA 3D score plot of serum metabolic profile of
CK group, HFD group, and HFD+H group. (C,D) PCA score plot and OPLS-DA score plot of serum
metabolic profiles between CK group and HFD group. (E,F) PCA score plot and OPLS-DA score plot
of serum metabolic profiles between HFD group and HFD+H group.
Plants 2024,13, 288 15 of 16
Author Contributions: Y.L. (Yanhong Li) and E.H.: planning, supervision, and funding acquisition.
J.R.: conceptualization, research, experimentation, writing—original draft, and organizing data
optimization. S.H.-P., Y.L. (Yao Li), H.H. and P.Y.: experimentation and data curation. J.G.: original
draft revision. J.R. and X.Z. contributed equally to this work. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was supported by Xinjiang Uygur Autonomous Region university research
project (XJEDU2023J031) and the Natural Science Foundation project of Xinjiang Uygur Autonomous
Region (2022D01A100).
Institutional Review Board Statement: The animal study protocol was approved by the Animal
Experimental Ethical Committee of College of Life Sciences, Xinjiang Normal University (protocol
code No. XJNU2022-08 and 10 August 2022).
Data Availability Statement: Data are available in a publicly accessible repository.
Acknowledgments: Thanks to Figdraw (www.figdraw.com) for technical support (YWTPRbb57c).
Conflicts of Interest: The authors declare no conflicts of interest.
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