Front. Nutr.Frontiers in NutritionFront. Nutr.2296-861XFrontiers Media S.A.10.3389/fnut.2023.1076569NutritionOriginal ResearchNetwork pharmacology-based analysis of Resinacein S against non-alcoholic fatty liver disease by modulating lipid metabolismMaoFei-Fei1†GaoShan-Shan1†HuangYan-Jie2†ZhouNian1FengJin-Kai3LiuZong-Han3ZhangYu-Qing4YuanLu-Yun4WeiGang5*ChengShu-Qun13*1Tongji University Cancer Center, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China2Guangdong Cardiovascular Institute, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China3Department of Hepatic Surgery VI, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China4Cancer Center, Yue Yang Hospital of Integrative Traditional Chinese and Western Medicine, Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China5Beijing Key Laboratory of Diabetes Research and Care, Department of Endocrinology, Beijing Diabetes Institute, Beijing Tongren Hospital, Capital Medical University, Beijing, China
Edited by: Helda Tutunchi, Tabriz University of Medical Sciences, Iran
Reviewed by: Samira Pourmoradian, Tabriz University of Medical Sciences, Iran; Fatemeh Naeini, Tehran University of Medical Sciences, Iran
*Correspondence: Gang Wei, ✉ gangwei_2013@163.comShuqun Cheng, ✉ chengshuqun@aliyun.com
†These authors have contributed equally to this work
This article was submitted to Nutritional Epidemiology, a section of the journal Frontiers in Nutrition
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Background
Ganoderma lucidum is reportedly the best source of traditional natural bioactive constituents. Ganoderma triterpenoids (GTs) have been verified as an alternative adjuvant for treating leukemia, cancer, hepatitis and diabetes. One of the major triterpenoids, Resinacein S, has been found to regulate lipid metabolism and mitochondrial biogenesis. Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease that has become a major public health problem. Given the regulatory effects on lipid metabolism of Resinacein S, we sought to explore potential protective effects against NAFLD.
Methods
Resinacein S was extracted and isolated from G. lucidum. And mice were fed with high fat diet with or without Resinacein S to detect hepatic steatosis. According to Network Pharmacology and RNA-seq, we analyzed the hub genes of Resinacein S against NAFLD disease.
Results
Our results can be summarized as follows: (1) The structure of Resinacein S was elucidated using NMR and MS methods. (2) Resinacein S treatment could significantly attenuate high-fat diet (HFD)-induced hepatic steatosis and hepatic lipid accumulation in mouse. (3) GO terms, KEGG pathways and the PPI network of Resinacein S induced Differentially Expressed Genes (DEGs) demonstrated the key target genes of Resinacein S against NAFLD. (4) The hub proteins in PPI network analysis could be used for NAFLD diagnosis and treatment as drug targets.
Conclusion
Resinacein S can significantly change the lipid metabolism in liver cells and yield a protective effect against steatosis and liver injury. Intersected proteins between NAFLD related genes and Resinacein S-induced DEGs, especially the hub protein in PPI network analysis, can be used to characterize targets of Resinacein S against NAFLD.
Ganoderma resinaceumResinacein SNAFLDlipid metabolismnetwork pharmacology82002480National Natural Science Foundation of China10.13039/501100001809Introduction
Ganoderma lucidum is a medicinal mushroom that can prolong life and promote health and has a long history in traditional Chinese medicine. Given its growing consumption, it has been intensively planted and sold since the 1970s. It is widely thought to be effective in preventing and treating many diseases and has anti-cancer properties (1). G. lucidum is considered the best source of traditional natural bioactive components. It contains various compounds, including polyphenols, polysaccharides, steroids, triterpenes, nucleotides, amino acids, trace elements, and vitamins. Over the past few years, G. lucidum extract has been used as a dietary addition to treating various diseases (2). Among various active components of G. lucidum, polysaccharides (GL-PS) and terpenoids (GL-T) predominantly exert physiological activities. They can inhibit the cell cycle and yield cytotoxicity and anti-metastasis, immunomodulation, antioxidant, antibacterial, anti-inflammatory and other effects (3). Moreover, G. lucidum has been recognized as an alternative adjuvant for treating leukemia, cancer, hepatitis and diabetes (4). Current evidence suggests that Ganoderma triterpenes represent one of the main active ingredients of mushrooms and yield an inhibitory effect on adipogenesis, leading to decreased lipid synthesis and accumulation. Other studies revealed that G. lucidum extracts and ethanol extracts of chigger mites rich in triterpenes contribute to adipogenesis and adipocyte differentiation. Ganoderma resinaceum is generally utilized for treating hepatitis, hyperglycemia, and dysimmunity in China and Nigeria (5).
Previous researchers had separated four new triterpenoids and four identified triterpenoids with anti-obesity effects from G. resinaceum. What’s more, one of the triterpenoids, Resinacein S has been found to induce beige and brown phenotypes, which may be relevant to the activation of AMPK/PGC1α signaling pathway to inhibit and treat obesity and relevant diseases. At the molecular level, Resinacein S treatment could significantly induce the expression of genes and/or proteins related to thermogenesis, fatty acid oxidation and lipolysis (6). Resinacein S, as one of the major triterpenoids from G. resinaceum, provides a therapeutic strategy for lipid metabolic diseases such as NAFLD, but whether Resinacein S treatment could provide protective aspects against NAFLD remains unknown.
Nonalcoholic fatty liver disease (NAFLD) is currently recognized as the most common liver disease in the world, affecting about 25% of adults worldwide. It encompasses steatosis simplex to nonalcoholic steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The clinical manifestation of nonalcoholic steatohepatitis (NASH) is a serious form of nonalcoholic fatty liver disease (NAFLD) characterized by the accumulation (steatosis) of triglycerides in liver cells, inflammation, injury and apoptosis, which may lead to cirrhosis and liver cancer in extreme cases (7–9). According to the regional epidemiological model of NAFLD, economy, environment and lifestyle are the key factors of disease progression (10). Hepatic steatosis or fatty liver refers to the increase of lipid accumulation in liver cells caused by increased production or decreased clearance of hepatic triglycerides or fatty acids (11). It has been established that Resinacein S can reduce fat and triglyceride accumulation by inducing the expression of genes and/or proteins related to thermogenesis, fatty acid oxidation and lipolysis at the molecular level. Nevertheless, the protective aspects and molecular mechanisms underlying the ability of Resinacein S to target NAFLD development remain to be illustrated. Therefore, this study aimed to explore the protective effects of Resinacein S against NAFLD.
Materials and methodsGeneral experimental procedures
The NMR spectra were recorded on Bruker AV500 and AVIII600 instruments, with TMS used as an internal benchmark. Optical rotations were measured on an Autopol VI-91058 digital polarimeter. The UV spectra were detected by a UV-2700 spectrophotometer, while IR spectra were measured on a Nicolet iS10 spectrometer. HR-TOF ESI/MS was conducted on an Agilent 6,200 Q-TOF MS system spectrometer. Column chromatography was carried out with silica gel, reversed-phase C18 silica gel and Sephadex LH-20 as packing materials. Fractions were quantitated by thin-layer chromatography after spraying with sulphuric acid and heating. TLC was carried out on silica gel GF254. HPLC was performed on an Agilent 1,100 liquid chromatography system coupled with a diode-array detector and an Agilent Zorbax SB-C18 column (5 μm, 9.4 × 250 mm).
Fungal material
Fruiting bodies of G. resinaceum were collected from Vientiane, Laos, identified by Peigui Liu and a voucher specimen (accession number: KGR-201606) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, China (6).
Extraction and isolation procedures have been described before (6).
Animal treatments and histological evaluation
Four-week-old male C57BL/6 mice were kept under the standard environment. Mice were fed with high fat diet with or without Resinacein S (intraperitoneal injection, interval for 48 h) at a dose of 10 mg/kg/day for 15 weeks (9). Then mice were sacrificed and the liver were fixed in 4% formaldehyde for HE staining and Oil Red O (G1262, Solarbio, China) staining (abs7049, Absin Bioscience, China). Images were acquired with a light microscope (Zeiss, Germany). Triglycerides (TG) were detected by a commercial kit (E1025-105, Applygen, Beijing, China) according to manufacturer’s instructions. All the experiments were approved by the ethics committee of Shanghai tenth People’s Hospital, Shanghai, China.
Collection and sorting of nonalcoholic fatty liver disease disease-related genes
Genes related to NAFLD were acquired from DisGeNET,1 GeneCards,2 and OMIM3 databases. The keyword “nonalcoholic fatty liver disease” was entered into the above three databases to search for genes related to NAFLD. Then, the intersection was obtained using the Venny Venn diagram tool (version 2.1.0).4
Determination of relevant target of Resinacein S
First, the canonical SMILES structure of Resinacein S was obtained through PubChem.5 Later, canonical SMILES information of Resinacein S was input into the SwissTargetPrediction6 database. Finally, the Uniprot ID obtained from the TargetNet database was transformed into Gene ID in UniProt7 database, yielding a total of 124 target genes associated with Resinacein S.
Protein–protein interactions network construction
The above Resinacein S targets of NAFLD were imported in String,8 with the organization parameter and threshold of combination score being set as Homo sapiens and 0.7 for obtaining protein–protein interactions (PPI). Then, the protein interaction network data from the STRING database was incorporated into Cytoscape 3.9.0 for classification and mapping according to the degree value.
RNA-seq analysis
TRIzol reagent was utilized for extracting total cellular RNA, which was later subject to spectrophotometry and agarose gel electrophoresis (AGE) with the NanoDrop ND-1000 instrument to analyze RNA integrity. Using a KAPA Stranded RNA-seq Library Prep Kit, this study built an RNA library, while a 2100 Bioanalyzer was employed for library quality assessment. Quantification of the library was performed by qRT-PCR. NCBI Gene Expression Omnibus was utilized to import raw RNA-seq information.
Functional analysis of identified genes
FastQC v0.11.8 was applied to analyze the raw RNA-seq data. Fragments per kilobase of gene/transcript model per million mapped fragments (FPKM) values of diverse genes and transcripts were determined by the Ballgown package of R v2.10.0 software. Additionally, R was used to generate volcano plots and heatmaps to further analyze the gene expression profiles.
Gene Ontology (GO) enrichment of Differentially Expressed Genes (DEGs) was conducted to identify significantly enriched GO-biological processes (BP), GO-molecular functions (MF) and GO-cellular components (CC). Meanwhile, DEG-related pathways were identified based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (12).
Statistical analysis
All data were expressed as mean ± standard deviation (SD). Experiments were repeated at least three times. The statistical significance of differences was evaluated using either the Student’s unpaired t-test, One-way analysis of variance (ANOVA), or two-way ANOVA as indicated. A p-value <0.05 was statistically significant.
ResultsStructure elucidation of Resinacein S
Resinacein S is a natural compound that has only been documented in G. resinaceum. We obtained Resinacein S white powder from the ethanol extracts of G. resinaceum through a series partition and column chromatography as previously described (Figure 1A) (13). Resinacein S was established as C30H44O8 based on the HRESIMS (Supplementary Figure S6), and the X-ray crystallographic structure of Resinacein S was obtained (Figure 1B). The 1H NMR and 13C NMR data of Resinacein S are shown in Table 1. The 1H NMR, 13C NMR and DEPT, HSQC, HMBC, 1H-1H COSY, and ROESY spectra are displayed in Figures 1B,C and Supplementary Figures S1–S6. Resinacein S was elucidated as (6R)-6-((3S,5R,7S,10S,13R,14R,17R)-3,7-dihydroxy-4,4,10,13,14-pentamethyl-11,15-dioxo-,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-3-hydroxy-2-methyl-4-oxo heptanoic acid.
Resinacein S was obtained from Ganoderma resinaceum(A), and 1H–1H COSY correlations, key HMBC, ROESY correlations for Resinacein S (B), and 1H NMR spectrum (600 MHz) of Resinacein S in CD3OD (C).
1H NMR and 13C NMR Data (δ) for Resinacein S (600 MHz) in CD3OD (δ in ppm, J in Hz).
Position
Resinacein S
Position
Resinacein S
δC, type
δH, (J in Hz)
δC, type
δH, (J in Hz)
1
35.9, CH2
2.78, overlapped; 1.01, overlapped
16
41.8, CH2
2.10, dd (19.3, 9.3); 2.74, dd (19.3, 7.8)
2
28.2, CH2
1.65, m; 1.56, overlapped
17
46.8, CH
2.17, dd (18.5, 9.3)
3
78.9, CH
3.14, dd (11.7, 4.5)
18
17.8, CH3
0.99, s
4
39.7, C
19
18.8, CH3
1.20, s
5
50.3, CH
0.96, d (13.0)
20
32.6, CH
2.23, overlapped
6
28.0, CH2
1.56, overlapped; 2.17, overlapped
21
20.2, CH3
0.98, d (6.8)
7
67.9, CH
4.83, overlapped
22
46.4, CH2
2.55, dd (17.7, 8.5); 2.62, overlapped
8
158.9, C
23
212.5, C
9
144.1, C
24
78.7, CH
4.37, d (5.0)
10
39.9, C
25
43.3, CH
2.87, overlapped
11
200.4, C
26
177.7, C
12
51.5, CH2
2.92, d (16.7); 2.62, d (16.7)
27
11.6, CH3
1.09, d (7.0)
13
46.7, C
28
28.7, CH3
1.01, s
14
60.4, C
29
16.2, CH3
0.83, s
15
218.3, C
30
24.9, CH3
1.36, s
Resinacein S alleviated liver damage and hepatic lipid accumulation
To investigate the benefits of Resinacein S against the HFD-induced extraphysiological process of NAFLD, we employed Resinacein S to treat HFD-diet mice and found that Resinacein S could effectively reduce liver damage and hepatic steatosis in HFD-fed mice (Figures 2A,B). In addition, Oil Red O staining of livers in Resinacein S-treated HFD-fed mice yielded few lipid droplets of small size. Compared to the control, the livers of Resinacein S-treated mice displayed an almost normal phenotype (Figures 2C–E). Consistently, significantly lower TG levels were found in the livers of the Resinacein S-treated group (Figure 2F).
Resinacein S reduced liver damage and hepatic steatosis in HFD-fed mice. (A,B) H&E staining of livers in HFD-fed mice (A) and Resinacein S treated HFD-fed mice (B). (C–E) Oil Red O staining of livers in HFD-fed mice (C) and Resinacein S treated HFD-fed mice (D). (E) is the quantitative analysis of Oil Red O staining. (F) TG levels in livers of Resinacein S treated or not HFD-fed mice were detected. Values were means ± SD, and for statistical analysis, one-way ANOVA were performed between indicated groups.
Target recognition results of Resinacein S and nonalcoholic fatty liver disease
Twenty and 101 gene targets in human related to Resinacein S were obtained from SwissTargetPrediction and TargetNet databases, respectively. Moreover, 61, 395 and 183 gene targets related to NAFLD disease were obtained from DisGeNET, GeneCards and OMIM, respectively. The Venny 2.1.0 online system was used for target recognition (Figure 3A). One hundred nineteen action targets of Resinacein S were acquired, along with 576 NAFLD related genes. Finally, 20 action targets of Resinacein S against NAFLD come to light. Detailed information on the action targets is provided in Table 2.
(A) 119 drug treatment targets based on the examination and combination of Swiss Target Prediction and TargetNet databases. Five hundred and seventy six genes related to NAFLD based on the examination and combination of DisGeNET, GeneCards and OMIM databases. There are 20 intersection targets between NAFLD and Resinacein S. (B) PPI protein interaction network diagram, in which the innermost circle degree value is 0–6, and the outer circle degree value is 7–16.
Twenty key target genes of Resinacein S related to NAFLD.
Genes
Protein names
UniProt ID
NR1H4
Bile acid receptor
Q96RI1
CYP17A1
Cytochrome P450 17A1
P05093
MMP1
Matrix metalloproteinase 1
P03956
FDFT1
Squalene synthetase
P37268
TNF
TNF-alpha
P01375
AGTR1
Type-1 angiotensin II receptor (by homology)
P30556
AKT1
Serine/threonine-protein kinase AKT
P31749
AKT2
Serine/threonine-protein kinase AKT2
P31751
CYP3A4
Cytochrome P450 3A4
P08684
DNMT1
DNA (cytosine-5)-methyltransferase 1
P26358
ESR1
Estrogen receptor alpha
P03372
HMGCR
HMG-CoA reductase
P04035
MMP2
Matrix metalloproteinase 2
P08253
MMP9
Matrix metalloproteinase 9
P14780
MTOR
Serine/threonine-protein kinase mTOR
P42345
NOS2
Nitric oxide synthase, inducible (by homology)
P35228
PIK3CA
PI3-kinase p110-alpha subunit
P42336
PTPN11
Protein-tyrosine phosphatase 2C
Q06124
SERPINA6
Corticosteroid binding globulin
P08185
SHBG
Testis-specific androgen-binding protein
P04278
Protein–protein interactions network results
The top 20 action targets of Resinacein S for NAFLD were put into the STRING database. Moreover, the PPI network was obtained according to the method described in 2.5. Data from the STRING database was put into Cytoscape 3.9.0 for beautification and classification according to the degree value. The degree values were classified into 0–6 and 7–16, and the protein–protein interaction network was generated (Figure 3B).
Resinacein S induced gene expression profiles in human liver cells
In order to further investigate the hub regulated genes of Resinacein S against NAFLD in human liver cells, we treated the human normal liver cell line L02 with Resinacein S and detected the gene expression profiles by RNA-Seq. According to log2FC > 0.585 or < −0.585 and FDR < 0.05, we obtained 172 DEGs (111 up-regulated and 61 down-regulated) in Resinacein S treated group compared to DMSO group (Figure 4, Supplementary Table 1).
(A) The heatmap of the DEGs between Resinacein S treated group and DMSO group. (B) The volcano plot of the DEGs between Resinacein S treated group and DMSO group.
Functional enrichment analysis of Resinacein S induced genes in human liver cells
To study the biological function of Resinacein S induced genes in human liver cells, GO annotation was conducted. The significantly enriched GO terms in cellular component (CC), molecular function (MF) and biological process (BP) are shown in Figure 5A and Table 3. Significantly enriched GO terms associated with cellular components included mitochondrial oxoglutarate dehydrogenase complex, tRNA methyltransferase complex, mitochondrial oxoglutarate dehydrogenase complex, nuclear lumen, nuclear outer membrane, cytoplasmic microtubule, microtubule-associated complex, intermediate filament cytoskeleton, cytoskeleton, and nucleus. Significantly enriched GO terms associated with molecular function (MF) included N-acylglucosamine 2-epimerase activity, glycogenin glucosyltransferase activity, kinase activator activity, glucokinase activity, mannokinase activity, fructokinase activity, hexokinase activity, glucose binding, MAP kinase kinase activity, and MAP kinase kinase kinase activity. Significantly enriched GO terms associated with biological process (BP) consisted of fatty acid omega-oxidation, extracellular matrix constituent secretion, inflammatory cell apoptotic process, miRNA catabolic process, regulation of fatty acid oxidation, steroid catabolic process, reactive oxygen species metabolic process, oligosaccharide metabolic process, dolichol-linked oligosaccharide biosynthetic process, and positive regulation of the apoptotic process.
GO and KEGG pathway enrichment analysis was conducted with Resinacein S induced genes in human liver cells. (A) The categories in GO terms included cellular components (CC), molecular function (MF), and biological process (BP) were analyzed. (B) The top 20 significant GO terms of the overlapped DEGs were chosen based on the order of p value from small to large. (C) KEGG pathway enrichment analysis was performed with Resinacein S-induced genes. The top 20 significant KEGG pathways of the DEGs were chosen based on the order of p value from small to large.
Top 20 significant GO terms of the overlapped DEGs in livers.
Category
GO ID
Term
List
p value
MF
GO:0019209
Kinase activator activity
2
0.000981
BP
GO:0006488
Dolichol-linked oligosaccharide biosynthetic process
4
0.002334
MF
GO:0005536
Glucose binding
2
0.0035
BP
GO:0072593
Reactive oxygen species metabolic process
1
0.00383
CC
GO:0005881
Cytoplasmic microtubule
3
0.007664
CC
GO:0009353
Mitochondrial oxoglutarate dehydrogenase complex
1
0.007916
CC
GO:0043527
tRNA methyltransferase complex
1
0.007916
MF
GO:0050121
N-acylglucosamine 2-epimerase activity
1
0.008203
CC
GO:0005634
Nucleus
64
0.013785
MF
GO:0004708
MAP kinase kinase activity
2
0.013861
CC
GO:0045111
Intermediate filament cytoskeleton
3
0.014839
MF
GO:0008466
Glycogenin glucosyltransferase activity
1
0.016339
BP
GO:0010430
Fatty acid omega-oxidation
1
0.016551
CC
GO:0005640
Nuclear outer membrane
2
0.016571
BP
GO:0009311
Oligosaccharide metabolic process
2
0.019557
BP
GO:0043065
Positive regulation of apoptotic process
7
0.020125
MF
GO:0004709
MAP kinase kinase kinase activity
2
0.020507
CC
GO:0031981
Nuclear lumen
1
0.023562
BP
GO:0070278
Extracellular matrix constituent secretion
1
0.024724
BP
GO:0006925
Inflammatory cell apoptotic process
1
0.024724
The top 20 GO terms associated with Resinacein S induced genes in human liver cells were selected according to the order of value of p in ascending order (Figure 5B; Table 3). It was found that the DEGs were mainly enriched in the fatty acid omega-oxidation, dolichol-linked oligosaccharide biosynthetic process, glucose binding, inflammatory cell apoptotic process, glycogenin glucosyltransferase activity, and fatty acid omega-oxidation, etc. These functions were closely related to the physiological regulation of liver metabolism, especially the metabolic homeostasis of lipids and cholesterol.
KEGG enrichment analysis was carried out to explore the biological pathways of the Resinacein S-induced DEGs (Figure 5C; Table 4). We found that the DEGs were significantly enriched in KEGG pathways, including T cell receptor signaling pathway, N-Glycan biosynthesis, Amino sugar and nucleotide sugar metabolism, Glycosphingolipid biosynthesis-globo series, MAPK signaling pathway, and TNF signaling pathway, etc.
Top 20 significant enriched pathways of the overlapped DEGs in livers.
Category
Pathway ID
p value
Genes
Fc gamma R-mediated phagocytosis
PATH:04666
0.028775
VAV3/SCIN/LIMK2
Butirosin and neomycin biosynthesis
PATH:00524
0.035794
HKDC1
T cell receptor signaling pathway
PATH:04660
0.044183
MAP3K8/VAV3/MALT1
TNF signaling pathway
PATH:04668
0.045195
MAP3K8/FAS/MAP2K6
Amino sugar and nucleotide sugar metabolism
PATH:00520
0.047149
RENBP/HKDC1
MAPK signaling pathway
PATH:04010
0.047367
MAP3K8/FAS/MAP2K5 MAP2K6/CACNA1H
N-Glycan biosynthesis
PATH:00510
0.048932
ALG9/DOLPP1
Caffeine metabolism
PATH:00232
0.049758
NAT1
Axon guidance
PATH:04360
0.069072
LIMK2/ABLIM3/NTN4
Taurine and hypotaurine metabolism
PATH:00430
0.070332
CSAD
Regulation of actin cytoskeleton
PATH:04810
0.071639
VAV3/SCIN/LIMK2 IQGAP2
Natural killer cell mediated cytotoxicity
PATH:04650
0.076769
FAS/VAV3/ULBP3
Non-homologous end-joining
PATH:03450
0.09047
POLM
Glycosphingolipid biosynthesis - globo series
PATH:00603
0.097087
ST3GAL1
Glycosaminoglycan biosynthesis - keratan sulfate
PATH:00533
0.103657
ST3GAL1
Glycosphingolipid biosynthesis - ganglio series
PATH:00604
0.103657
ST3GAL1
B cell receptor signaling pathway
PATH:04662
0.104971
VAV3/MALT1
Fc epsilon RI signaling pathway
PATH:04664
0.121474
VAV3/MAP2K6
Influenza A
PATH:05164
0.133377
FAS/RAE1/MAP2K6
Nitrogen metabolism
PATH:00910
0.154559
CA5B
Overall, these metabolic pathways are closely related to liver metabolic balance, which substantiates the protective effect of Resinacein S against NAFLD.
The hub proteins of Resinacein S regulated against nonalcoholic fatty liver disease
To determine the hub proteins that mediate the protective role of Resinacein S against NAFLD in human liver cells, we analyzed the interaction relationships of 172 DEGs of Resinacein S regulated in human normal liver cell line L02 according to RNA-Seq (Figure 3), and 20 target genes of Resinacein S against NAFLD from public database (Figure 4). The relationships were obtained by the STRING database, including evidence from experiments, databases, and co-expression data, and binding scores greater than 0.7 (high confidence). Therefore, according to the STRING analysis, the Resinacein S-mediated junction proteins against NAFLD were TNF, PIK3CA, AKT1, AKT2, ESR1, CYP3A4, CYP17A1, and PTPN11 (Figure 6). Among them, AKT1 and AKT2 play an important role in the interaction relationships which indicated the regulation of AKT pathway by Resinacein S against NAFLD.
The hub proteins of Resinacein S regulated against NAFLD. The interaction relationship analysis was performed with the DEGs of Resinacein S regulated in human liver cells and the target genes of Resinacein S against NAFLD from public database. DEGs of Resinacein S regulated in human normal liver cells were shown in red circle, and the 20 target genes of Resinacein S against NAFLD from public database were shown in green. The analysis was generated by Cytoscape 3.9.0.
Discussion
NAFLD is widely thought to be the most common chronic liver disease, concomitant with the global obesity epidemic. During the development and progress of NAFLD, liver steatosis is considered a benign state, while NASH exhibits chronic progressive liver damage. Fatty degeneration is related to inflammation and fibrosis and gradually develops into cirrhosis. The pathogenesis of NAFLD is closely related to dysregulated metabolism in obese patients, excess free fatty acid (FFA) accumulation and the excessive secretion of several cellular inflammatory mediators (14). There is an increasing consensus that NAFLD is a heterogeneous disease associated with multiple-hit pathogenesis yielding different phenotypes. Although the clinical presentation of NAFLD can be heterogeneous, insulin resistance plays a leading role in NAFLD (15).
In this study, we focused on the role of the Resinacein S compound extracted from G. resinaceum in treating NAFLD patients. First, we identified the extracted triterpenoid compounds and obtained the molecular structure of Resinacein S through mass spectrometry analysis. By interaction relationship analysis of the DEGs of Resinacein S regulated in human liver cells and the target genes of Resinacein S against NAFLD from public database, we found 8 proteins TNF, PIK3CA, AKT1, AKT2, ESR1, CYP3A4, CYP17A1, and PTPN11 may be the hub proteins of Resinacein S regulated against NAFLD.
According to former studies, Resinacein S treatment can dramatically induce the expression of thermogenesis related genes such as Ucp1 and Pgc1α, fatty acid oxidation related genes such as Pparα and Cpt1α, lipolysis related genes such as Hsl and Atgl, and mitochondriogenesis-related genes. Resinacein S may be involved in activation of AMPK/PGC1α signaling pathway (6). Now we obtained eight Resinacein S targeting genes with potential therapeutic effects against NAFLD through RNA seq analysis and PPI network analysis.
Obesity is related to the increase of circulating Tumor Necrosis Factor (TNF, TNF-α), a pro-inflammatory cytokine that induces the death of liver cells (16). In the process of inflammation, TNF is one of the main pro-inflammatory cytokines, which regulates innate immunity and adaptive immune response (17). Clinical evidence shows that the level of circulating TNF-α in patients with nonalcoholic steatohepatitis (NASH) is highly correlated with the degree of liver fibrosis (18). AKT Serine/Threonine Kinase 1 (AKT1) is a serine/threonine protein kinase, which is called an important downstream target signal pathway of insulin and has anti-apoptosis and peripheral metabolic effects. Studies have shown that the inhibition of ROS production mediated by AKT1 inhibits the fibrosis transformation from NAFLD to liver (19). AKT1 deficiency led to the inhibition of AKT/mTOR/S6K signaling pathway in hepatocytes, which was crucial for the development of hepatic steatosis and provided a new scheme for the development of NAFLD therapy (20). Studies have shown that AKT Serine/Threonine Kinase 2 (AKT2) is essential for lipid synthesis in the liver (21). It was found that hepatocyte-specific Phosphatase and Tensin Homolog (PTEN) deficiency in mice showed liver steatosis due to over-activation of AKT2 (22). As the precursor of NAFLD, deleting AKT2, the downstream target of PTEN signal, can block the development of NASH and reduce the development of liver fibrosis, which also reduces the occurrence of NAFLD from another development process (23). Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) is an important component of PI3K/AKT/mTOR pathway. PIK3CA mutated cells showed inflated induction of the de novo lipogenesis transcriptional regulator SREBP1 and elevated exogenous FA uptake capacity both of which can lead to lipid-enriched phenotype (24, 25). ESR1 is a nuclear and membrane hormone receptor. Previous studies showed that ESR1 could negatively regulate hepatocyte pyroptosis by directly interacting with gasdermin D (GSDMD). ESR1 deficiency could induce pyroptosis, impaired glucose tolerance, and reduce lipid accumulation in hepatocytes (26–28). Cytochrome P450 Family 3 Subfamily A Member 4 (CYP3A4) is involved in the metabolism of sterols, retinoids and fatty acids. The activity of CYP3A4 is usually reduced in mouse and cell models of NAFLD, also in human. In NAFLD-model mice, the luciferase activity of CYP3A4 from livers was about 38% lower than the normal ones (29, 30). Cytochrome P450 Family 17 Subfamily A Member 1 (CYP17A1) is an important enzyme for Dehydroepiandrosterone (DHEA) synthesis. Former evidences showed that DHEA can modulate oxidative stress, insulin resistance, and fibrosis observed in serious NAFLD (31, 32). Protein Tyrosine Phosphatase Non-Receptor Type 11 (PTPN11) is the first identified oncogenic tyrosine phosphatase which can cooperate with PTEN to maintain the liver homeostasis and function. PTPN11 in hepatocytes could induce early-onset non-alcoholic steatohepatitis (NASH) (33).
Besides, according to our KEGG enrichment analysis, we revealed that the targets of Resinacein S mainly focused on T cell receptor signaling pathway, N-Glycan biosynthesis, Amino sugar and nucleotide sugar metabolism, Glycosphingolipid biosynthesis-globo series, MAPK signaling pathway, and TNF signaling pathway.
In conclusion, we have revealed the structure of Resinacein S and demonstrated that Resinacein S could significantly attenuate high-fat diet induced hepatic steatosis and hepatic lipid accumulation. We have also developed a new gene expression feature related to NAFLD by using Resinacein S-dependent DEGs, especially hub proteins in PPI network analysis, which can assist in diagnosing and treating NAFLD populations as well as drug discovery and development in the near future. Nevertheless, although the extracts of Ganoderma are usually safe and most of the side effects are very mild, the side effect of Resinacein S deserves further studies especially on human. Besides, the molecular mechanisms of Resinacein S against NAFLD and in vivo models for further proving the metabolic phenotypes of Resinacein S need to be further investigated.
Conclusion
Overall, Resinacein S yields a protective effect against steatosis and liver injury and can significantly change the gene expression profile in fatty liver cells. Functional enrichment analysis showed significant enrichment in lipid and glucose metabolism. In addition, intersected genes between NAFLD and Resinacein S-induced DEGs, especially the hub protein in PPI network analysis, can be used to characterize NAFLD-related gene expression characteristics for NAFLD diagnosis, treatment and drug development.
Data availability statement
The data presented in the study are deposited in the GEO repository, accession number GSE223990.
Ethics statement
The animal study was reviewed and approved by Animal Care and Use Committee of Shanghai Tenth People’s Hospital of Tongji University.
Author contributions
S-QC, GW, and F-FM conceived the project. The data analysis was done by FM, S-SG, and Y-JH. F-FM and S-SG wrote the drafts of the manuscript. Y-JH checked and revised the manuscript. NZ and J-KF participated in collection of the published datasets. Z-HL, Y-QZ, and L-YY gave many suggestions about the manuscript writing. All authors contributed to the article and approved the submitted version.
Funding
This work was partially supported by grants from the National Natural Science Foundation of China (Nos. 82002480, 8200032).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
We would like to thank NovelBio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for their assistance on RNA-seq data analysis.
Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2023.1076569/full#supplementary-material
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