Abstract

   Background: Liver cirrhosis is a life-threatening seqsuel of many
   chronic liver disorders of varying etiologies. In this study, we
   investigated protein targets of curcumin in liver cirrhosis based on a
   bioinformatics approach. Methods: Gene/protein associations with
   curcumin and liver cirrhosis were probed in drug–gene and gene–diseases
   databases including STITCH/DGIdb/DisGeNET/OMIM/DISEASES/CTD/Pharos and
   SwissTargetPrediction. Critical clustering groups (MCODE), hub
   candidates and critical hub genes in liver cirrhosis were identified,
   and connections between curcumin and liver cirrhosis-related genes were
   analyzed via Venn diagram. Interaction of hub genes with curcumin by
   molecular docking using PyRx-virtual screening tools was performed.
   Results: MCODE analysis indicated three MCODEs; the cluster (MCODE 1)
   comprised 79 nodes and 881 edges (score: 22.59). Curcumin database
   interactions recognized 318 protein targets. Liver cirrhosis genes and
   curcumin protein targets analysis demonstrated 96 shared proteins,
   suggesting that curcumin may influence 20 candidate and 13 hub genes,
   covering 81% of liver cirrhosis critical genes and proteins. Thirteen
   shared proteins affected oxidative stress regulation, RNA, telomerase
   activity, cell proliferation, and cell death. Molecular docking
   analysis showed the affinity of curcumin binding hub genes (Binding
   affinity: ΔG < −4.9 kcal/mol). Conclusions: Curcumin impacted on
   several critical liver cirrhosis genes mainly involved in extracellular
   matrix communication, focal adhesion, and the response to oxidative
   stress.

   Keywords: liver cirrhosis, curcumin, protein–protein interaction,
   biological process, gene expression

1. Introduction

   Liver cirrhosis is the terminal step of many chronic liver disorders
   with varied etiologies such as fatty liver disease and hepatitis
   [[32]1]. It has long been recognized as a considerable risk factor for
   hepatocellular cancer and a leading cause of liver disease-related
   morbidity and death [[33]2]. Cirrhosis has increased by 1.5-fold to
   two-fold in the last two decades, and chronic liver disease and
   cirrhosis are estimated to be responsible for two million deaths
   globally each year [[34]3]. The prognosis for decompensated liver
   cirrhosis is poor, with an overall survival rate of 2–4 years, lower
   than several oncological illnesses [[35]4]. In liver
   cirrhosis/fibrosis, hepatic stellate cells (HSCs) are critical in
   initiating and in the development of this late-stage complication, with
   the activation and transdifferentiation of HSCs into myofibroblasts in
   response to liver damage, resulting in the generation of abundant
   extracellular matrix (ECM) proteins such as smooth muscle actin (SMA)
   [[36]5,[37]6]. A wide range of genes/proteins and biological processes
   and pathways are involved in this process and the progression to liver
   cirrhosis [[38]7,[39]8,[40]9,[41]10,[42]11]. The progress of cirrhosis
   commonly occurs after a protracted period of inflammation. In this
   process, the healthy liver parenchyma is replaced with fibrotic tissue
   and renewing nodules, which causes portal hypertension. The condition
   progresses from an asymptomatic level (compensated cirrhosis) to a
   symptomatic level (decompensated cirrhosis), leading to
   hospitalization, reduced quality of life, and a high death rate.
   Disease outcomes are influenced by systemic inflammation, liver
   failure, and progressive portal hypertension. The treatment of the
   causes and effects of liver cirrhosis are the main focus of care, while
   liver transplantation may be necessary [[43]12]. However, different
   genes and pathways are related to inflammation and fibrosis such as
   tumor necrosis factor (TNF), interleukin 17 A (IL-17 A), interleukin 1
   beta (IL-1β), interleukin 6 (IL-6), transforming growth factor beta
   (TGF-β), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)
   signaling pathway, and toll-like receptor 4 (TLR4) mediated activation
   of the nuclear transcription factor κB (NF-κB) signaling pathway that
   are the targets of curcumin. In this case, a bioinformatics study was
   designed to determine curcumin’s possible effect on liver cirrhosis
   [[44]13,[45]14,[46]15,[47]16].

   Since ancient times, turmeric has been proposed as a natural cure for a
   variety of diseases. Curcumin, a natural chemical extracted from the
   rhizome of Curcuma longa (turmeric), has been demonstrated to have
   antioxidant, antiproliferative, anti-inflammatory, anticancer,
   cardioprotective, analgesic, antidiabetic, and hepatoprotective
   properties
   [[48]17,[49]18,[50]19,[51]20,[52]21,[53]22,[54]23,[55]24,[56]25,[57]26,
   [58]27,[59]28,[60]29,[61]30,[62]31,[63]32,[64]33]. Numerous in vitro,
   in vivo, and clinical studies have reported that curcumin may have a
   beneficial effect on liver cirrhosis
   [[65]2,[66]34,[67]35,[68]36,[69]37,[70]38,[71]39]. For example, two
   randomized clinical trials indicated that curcumin supplementation
   reduced disease activity ratings and cirrhosis severity in individuals
   with cirrhosis. According to the chronic liver disease questionnaire
   (CLDQ), The Liver Disease Symptom Index (LDSI), and the Health Survey
   (SF-36) categories, curcumin enhances quality of life (QoL) in liver
   cirrhotic patients [[72]2,[73]39]. Curcumin is a pleiotropic compound
   that may directly or indirectly influence multiple genes/proteins and
   related pathways involved in inflammatory cytokines, oxidative stress
   response, growth factors and their receptors, enzymes, adhesion
   molecules, and apoptosis-related cell cycle proteins; therefore,
   curcumin may ameliorate liver cirrhosis through a number of mechanisms
   that are not fully defined [[74]40,[75]41].

   Virtual screening based on systems biology methodology has recently
   garnered interest in target deconvolution studies and the exploration
   of therapeutic targets in various diseases
   [[76]41,[77]42,[78]43,[79]44].

   In this study, we hypothesized that the positive effect of curcumin on
   the end-late injury of the liver in various diseases (liver fibrosis,
   cirrhosis) reported in previous investigations was through curcumin
   exerting its effect through multiple genes/proteins and biological
   pathways. Therefore, we investigated the possible protein targets of
   curcumin in liver cirrhosis based on a bioinformatics approach. An
   overview of our research process is shown in [80]Figure 1.

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   A summary of the study process undertaken in the current study. This
   research was planned in three main sections including exploring the
   gene/protein target for curcumin and liver cirrhosis, analyzing the two
   obtained datasets, and investigating the biological process and
   pathways related to identified important intersection protein/genes
   (PPI = protein–protein interaction).

2. Methods

2.1. Exploring Liver Cirrhosis-Protein/Gene Associations

   We used open-access databases including DisGeNET including Online
   Mendelian Inheritance in Man (OMIM), DISEASES, Comparative
   Toxicogenomics Dataset (CTD), and Pharos to investigate proteins
   associated with liver cirrhosis. OMIM ([83]http://omim.org; accessed on
   24 April 2022) is a thorough and comprehensive database including
   information about human phenotypes and genes as well as their
   associations [[84]45]. DisGeNET ([85]https://www.disgenet.org; accessed
   on 24 April 2022) is a database containing a list of genes linked to
   various illnesses, detailing their associations. CTD
   ([86]http://ctdbase.org; accessed on 24 April 2022) is a manually
   maintained database that contains information on gene–chemical
   interactions and their links to disorders [[87]46]. DISEASES
   ([88]https://diseases.jensenlab.org; accessed on 24 April 2022) is a
   weekly updated database that contains diseases and gene relationships
   from computer text mining and manually selected literature [[89]47].
   Pharos is an intermediate web source of the Knowledge Management Center
   (KMC) for assaying the drug target genes and ligands, and it also
   retains valuable information about the relation of the genes with
   numerous diseases [[90]48]. We obtained and combined all of the
   proteins/genes connected to liver cirrhosis from these sources.

2.2. Protein–Protein Interaction (PPI) Network and MCODE Analysis of
Proteins/Genes Related to Liver Cirrhosis

   We created a PPI network with liver cirrhosis-related genes obtained
   from the databases noted above to find the most critical genes (hub
   genes) and Molecular Complex Detection (MCODE) related to liver
   cirrhosis. PPI networks were constructed with Cytoscape software
   (version 3.9.1) and stringApp (version 1.7.0) plugin. A high confidence
   score >0.7 and the limitation of species to “Homo sapiens” were
   considered. STRING is a web source containing validated and predicted
   protein associations comprising physical and functional protein–protein
   interactions [[91]49]. To achieve the most critical hub genes, we used
   the NetworkAnalyzer version 4.4.8 plugin on Cytoscape software to
   survey hub gene candidates and hub genes. The hub candidate was
   determined by exerting 20 percent filtering on the three critical
   centralities (DegreeCentrality, ClosenessCentrality, and
   BetweennessCentrality), and the hub genes were determined based on the
   highest score in these centralities. We selected the 20 highest
   proteins in each centrality and then assessed the intersection between
   them to obtain the hub genes. The Degree and Closeness/Betweenness are
   factors of the topological algorithms and shortest paths, respectively.
   The MCODE (version 2.0.0) plugin for Cytoscape was utilized to cluster
   the gene/protein in the PPI network (degree cut-off = 4, haircut off,
   k-core = 2, node score cut-off = 0.2, and maximum depth = 100).
   Subsequently, we displayed the specificity of hub candidates related to
   liver cirrhosis on Cytoscape software using the stringAPP plugin. An
   illustration of analyzing the PPI network to obtain hub genes and
   modular clusters in the PPI network by Cytoscape software is depicted
   in [92]Figure 2A,B.

Figure 2.

   [93]Figure 2
   [94]Open in a new tab

   The methodology for screening hub genes through analyzing centralities
   (A) and modular clusters via MCODE analysis (B). PPI—protein–protein
   interactions.

2.3. Curcumin and Target Search

   We probed the molecular targets of curcumin in five popular databases
   (STITCH, DGIdb, SwissTargetPrediction, Pharos, CTD). The STITCH
   database ([95]http://stitch.embl.de; accessed on 29 April 2022) is a
   tool for determining chemical–protein interactions including over
   9,640,000 proteins from 2031 species [[96]50]. A high confidence
   (0.700) cut-off and the limitation of the species to Homo sapiens were
   used. Drug–gene interaction databases (DGIdb) are web-based storage
   databases that include critical information about drug–gene/protein
   interactions acquired from a variety of sources including PharmGKB,
   Therapeutic Target Database (TTD), DrugBank, Drug Target Commons
   Chembl, and others [[97]51]. SwissTargetPrediction is a web service
   that uses a mix of 2D and 3D similarity metrics with known ligands to
   predict the targets of bioactive compounds [[98]52]. We selected all
   gene/protein targets for curcumin in those databases.

2.4. Estimation of Curcumin Targets with the Genes/Proteins Connected with
LIVER Cirrhosis

   To exclude spelling errors in gene symbols, we first converted all of
   the genes/proteins obtained from all databases to the UniProt Accession
   via [99]https://biodbnet-abcc.ncifcrf.gov/db/db2db.php; accessed on 25
   April 2022, then applied the Venn diagram.

   We evaluated the intersection of the datasets (gene association with
   liver cirrhosis and curcumin targets) in three phases to find important
   protein targets of curcumin in liver cirrhosis disease:
     * 1
       Evaluation of the curcumin targets with all achieved proteins/genes
       associated with liver cirrhosis (All liver cirrhosis connection
       genes ∩ curcumin targets).
     * 2
       Evaluation of the curcumin targets with important proteins/genes
       obtained based on PPI network analysis (hub/candidate hub genes
       connection liver cirrhosis ∩ curcumin targets).
     * 3
       Evaluation of the curcumin targets with important proteins/genes
       obtained based on MCODE analysis (MCODEs connection with liver
       cirrhosis ∩ curcumin targets). The intersection across these lists
       was conducted with online Venn diagram software
       ([100]https://bioinfogp.cnb.csic.es/tools/venny; accessed on 24
       April 2022).

   We also investigated the possible direct interaction of hub genes with
   curcumin by molecular docking. We first downloaded the 3D structure of
   the hub genes from the RCSB PDB web server. The 2D structure of
   curcumin (SDF) was determined from the PubChem source. For docking
   operations, we used PyRx-virtual screening tools. Before the docking,
   we first prepared the structure of macromolecules using UCSF Chimera
   software (version 1.8.1) and then we performed docking of the
   macromolecules and curcumin compound in PyRx software. The Protein Data
   Bank (PDB) is the first open-access digital data resource with
   information about the 3D structure of biological molecules in all
   organisms [[101]53]. PubChem is an open web server at the National
   Institutes of Health (NIH), which is a resource with information about
   the chemical structures [[102]54]. PyRx software is used for
   Computational Drug Discovery to probe compounds against potential
   biological targets [[103]55]. UCSF Chimera software is used to analyze
   the structures of the molecules [[104]56]. The results of molecular
   docking analysis are reflected in the binding energy (ΔGbind). Energy
   binding less than −5 kJ·mol^−1 (−1.19503 kcal/mol) refers to the
   certain binding activity of the ligand and the targets [[105]57].

2.5. Gene Ontology and KEGG Pathways Enrichment Analysis

   Functional enrichment analysis (GO) and biological pathway (KEGG) were
   accomplished using the freely available ClueGO plugin for Cytoscape
   software, and the charts are presented with Excel 2013 software. ClueGO
   was used to interpret the interrelations of the gene/protein sets and
   functional groups in biological networks [[106]54]. GO is defined in
   three stages: Biological Process, Cellular Component, and Molecular
   Function [[107]58]. Likewise, KEGG is a repository database that
   interprets genomic, diseases, pathways, chemical, and systemic
   functional data [[108]59].

3. Results

3.1. Gene Related to Liver Cirrhosis and PPI Network Analysis

   We found 1155 associated genes with liver cirrhosis by probing
   gene/protein-disease association databases. The PPI network constructed
   with the stringApp plugin possesses 906 nodes and 9647 edges with PPI
   enrichment p-value: <1.0 × 10^−16. The subsequent network analysis of
   centralities (DegreeCentrality, ClosenessCentrality, and
   BetweennessCentrality) on Cytoscape distinguished 28 candidate hub
   genes and 16 hub genes. The hub genes included tumor protein 53 (TP53),
   albumin (ALB), fibronectin 1 (FN1), serine/threonine kinase 1 (AKT1),
   non-receptor tyrosine kinase (SRC), actin beta (ACTB), tumor necrosis
   factor (TNF), epidermal growth factor receptor (EGFR), signal
   transducer and activator of transcription 3 (STAT3), catenin beta 1
   (CTNNB1), ras homolog family member A (RHOA), jun proto-oncogene (JUN),
   insulin (INS), CD44, E1A binding protein p300 (EP300), and heat shock
   protein 90AA1 (HSP90AA1). Candidate hub genes and hub genes are
   illustrated in [109]Figure 3A and described in [110]Table 1,
   respectively. The presentation of proteins/genes associated with liver
   cirrhosis is shown by color intensity in [111]Figure 3B (score 1 to 5)
   ([112]Figure 3); all of the genes/proteins described had a score of
   three or higher, and 19 proteins scored more than 4. The scoring of
   tissue specificity was achieved from TISSUES 2.0 databases as part of
   the STRING app in Cytoscape software from a score of 1 to 5. The
   scoring was based on integrating multiple sources of evidence:
   transcriptomics as well as manually curated and mined from the
   scientific literature [[113]60].

Figure 3.

   [114]Figure 3
   [115]Open in a new tab

   (A) Hub protein/gene candidate interaction network constructed by
   filtering proteins/genes by high-level centralities using Cytoscape
   software. Twenty-eight candidate hubs with 237 edges were identified.
   The Degree is depicted with the size of nodes, and Betweenness is
   depicted with the intensity of the node color. (B) The amount of
   presentation of proteins/genes related to liver cirrhosis liver tissue.
   The intensity of the color indicates the amount of presentation in the
   liver.

Table 1.

   The network analysis information of critical hub genes related to liver
   cirrhosis.
   Ensembl Gene ID Gene Symbol Gene Full Name Network Analyzer
   Protein Class Liver Specificity Degree Betweenness Closeness
   ENSG00000075624 ACTB Actin beta Plasma proteins 4.98 121 0.0332 0.4504
   ENSG00000142208 AKT1 serine/threonine kinase 1 Enzymes 4.88 156 0.0403
   0.4619
   ENSG00000163631 ALB Albumin Plasma proteins 5.00 88 0.0493 0.4336
   ENSG00000026508 CD44 CD44 Molecule Blood group antigen proteins 4.83 88
   0.0197 0.4219
   ENSG00000168036 CTNNB1 Catenin beta 1 Plasma proteins 4.77 113 0.0276
   0.4401
   ENSG00000146648 EGFR Epidermal growth factor receptor RAS pathway
   related proteins 4.75 149 0.0311 0.4498
   ENSG00000100393 EP300 E1A binding protein p300 Metabolic proteins 4.65
   88 0.0172 0.4166
   ENSG00000115414 FN1 Fibronectin 1 Plasma proteins 4.97 127 0.0408
   0.4293
   ENSG00000080824 HSP90AA1 Heat shock protein 90 alpha family class A
   member 1 Enzymes 4.98 95 0.0166 0.4285
   ENSG00000254647 INS Insulin RAS pathway related proteins 4.00 92 0.0219
   0.4295
   ENSG00000177606 JUN Jun proto-oncogene Transcription factors 3.88 111
   0.0224 0.4395
   ENSG00000067560 RHOA Ras homolog family member A Enzymes 4.40 103
   0.0239 0.4256
   ENSG00000197122 SRC non-receptor tyrosine kinase Enzymes 4.64 168
   0.0350 0.4612
   ENSG00000168610 STAT3 Signal transducer and activator of transcription
   3 Transcription factors 4.84 162 0.0279 0.4491
   ENSG00000232810 TNF Tumor necrosis factor Plasma proteins 3.82 140
   0.0314 0.4449
   ENSG00000141510 TP53 Tumor protein p53 Transcription factors 3.72 115
   0.0658 0.4412
   [116]Open in a new tab

   Furthermore, in MCODEs analysis, the three highest MCODEs were
   determined ([117]Figure 4). MCODE 1 had the highest score (22.59) with
   79 nodes and 881 edges. MCODE 2, with a score of 18.79, 44 nodes, and
   404edges was the second most crucial modular cluster in the PPI network
   related to liver cirrhosis. PTPN11 and SRC are the seed of MCODE 1 and
   2, respectively. The third score cluster comprises 59 nodes and 287
   edges with 9.89 score and seed of MAPK3.

Figure 4.

   [118]Figure 4
   [119]Open in a new tab

   The three top score clusters of genes/proteins related to liver
   cirrhosis based on MCODE analyses.

3.2. Obtained Curcumin Targets and Exploring the Impact of Curcumin on
Proteins/Genes Related to Liver Cirrhosis

   Screening curcumin targets in the STITCH, DGIdb, SwissTargetPrediction,
   Pharos, and CTD databases identified 318 capable protein/gene targets.

   Intersection analysis with a Venn diagram of all proteins/genes
   associated with liver cirrhosis and the protein targets of curcumin
   exhibited 96 shared proteins ([120]Figure 5A).

Figure 5.

   [121]Figure 5
   [122]Open in a new tab

   Intersection analysis of the curcumin targets and liver
   cirrhosis-related genes/proteins using a Venn diagram with (A) curcumin
   targets and all liver cirrhosis association genes, (B) curcumin targets
   and hub candidate/hub genes, (C) curcumin targets and MCODEs analysis
   of liver cirrhosis association genes/proteins.

   The Venn diagram for hub candidates and hub proteins/genes associated
   with liver cirrhosis demonstrated that 20 and 13 proteins/genes,
   respectively, connected with curcumin ([123]Figure 5B). The 13 hub
   genes that curcumin could interact with included AKT1, ALB, CTNNB1,
   EGFR, EP300, FN1, HSP90AA1, INS, JUN, SRC, STAT3, TNF, and TP53. This
   result indicated that although curcumin covered only 8.31% of all liver
   cirrhosis protein targets, it could cover 81.25% of the most critical
   proteins/genes based on PPI network analysis.

   Comparing the curcumin targets with the three top-scored MCODE
   clustering of liver cirrhosis-related proteins/genes revealed that
   curcumin targeted 32 crucial proteins/genes of MCODE1, the highest
   covering of proteins/genes among other MCODEs. Interestingly, MCODE3
   and 2 targeted 22 and 12 proteins/genes, respectively, suggesting that
   curcumin targets critical proteins/genes that belong to the most
   important clustered proteins/genes associated with liver cirrhosis
   ([124]Figure 5C).

   To analyze the molecular docking of curcumin against 16 hub
   genes/protein targets, we first identified the 3D structure of the
   targets ([125]Figure 6). Then, in UCSF Chimera, other chains, ligands,
   non-complexed ions, and solvent were removed. The structure of curcumin
   was obtained in PubChem with CID: 969516. As shown in [126]Figure 7,
   the docking result indicated that curcumin is a pleiotropic and
   flexible compound that could interact with most of the targets with a
   binding affinity within the minimum ΔG energy.

Figure 6.

   [127]Figure 6
   [128]Open in a new tab

   The structure of proteins encoded by 16 hub genes obtained from the
   RCSB PDB databases.

Figure 7.

   [129]Figure 7
   [130]Open in a new tab

   The possible interaction of curcumin with proteins encoded by 16 hub
   genes using the molecular docking method.

3.3. GO and KEGG Enrichment Analyses of Shared Proteins

   Functional enrichment analysis of 13 shared proteins (intersection of
   hub genes associated with liver cirrhosis ∩ curcumin targets) at the
   biological process level suggested the regulation of nitric-oxide
   synthase activity with 9.09% of associated genes percent and p-value of
   1.26 × 10^−8, the regulation of monooxygenase activity with 7.24% of
   associated genes percent and p-value of 3.97 × 10^−8, and the
   regulation of the production of small RNA involved in gene silencing by
   RNA 4.04% of associated genes percent and p-value of 8.91 × 10^−8
   ([131]Figure 8A). In addition, the analysis demonstrated that these
   shared proteins were mainly applied in enzyme binding with 5.13% of
   associated genes percent and p-value of 2.78 × 10^−7; oxidoreductase
   activity with 5.05% of associated genes percent and p-value of 4.23 ×
   10^−7; nitric-oxide synthase regulator activity with 42.85% associated
   genes percent and p-value of 7.93 × 10^−7, and the regulation of
   telomerase activity with 7.27% of associated genes percent and p-value
   of 3.83 × 10^−6 ([132]Figure 8B). Moreover, vesicle lumen with 1.36% of
   associated genes percent and p-value: 1.01 × 10^−4, intracellular
   organelle lumen with 0.21% of associated genes percent and p-value of
   2.03 × 10^−4, membrane microdomain with 1.09% of associated genes
   percent and p-value of 0.0026, and secretory granule lumen with 1.10%
   of associated genes percent and p-value of 0.0026 were detected under
   cellular components ([133]Figure 8C).

Figure 8.

   [134]Figure 8

   [135]Figure 8

   [136]Figure 8

   [137]Figure 8
   [138]Open in a new tab

   Gene Ontology enrichment analysis of the 13 shared protein targets (hub
   associated with liver cirrhosis ∩ curcumin targets) using ClueGO. (A)
   The ten highest adjusted p-value biological process; (B) the ten
   highest adjusted p-value molecular function; (C) the ten highest
   adjusted p-value cellular compound; (D) the ten highest adjusted
   p-value biological pathway (KEGG pathway) enrichment. Associated genes
   % indicates the percentage of gene sets present in each functional
   term. For example, if 50 genes are involved in a pathway, and ten genes
   of our gene set are involved in this pathway, the associated genes %
   equals 5%.

   KEGG enrichment revealed that the pathways with the highest adjusted
   p-value were associated with 13 shared proteins including proteoglycans
   in cancer with 3.90% of associated genes percent and p-value of 1.39 ×
   10^−8, the AGE-RAGE signaling pathway in diabetic complications with
   5.00% of associated genes percent and p-value of 2.23 × 10^−5, the
   focal adhesion with 2.99% of associated genes percent and p-value of
   2.29 × 10^−5, and the HIF-1 signaling pathway with 4.59% of associated
   genes percent and p-value of 3.28 × 10^−5 ([139]Figure 8D).

4. Discussion

   Hepatic cirrhosis is a global and increasing health care problem with
   high morbidity and mortality. There is a need for anti-fibrotic
   treatments to modulate and prevent disease [[140]61]. In hepatic
   fibrosis, the level of extracellular matrix constituents such as
   fibronectin, proteoglycans, and laminin increased in reaction to liver
   damage. Leukocyte recruitment occurs after the injury and needs
   multiple adhesion molecules. By generating nitric oxide and
   inflammatory cytokines, macrophages and monocytes play a role in
   inflammatory responses. Stellate cells and extracellular
   matrix-producing cells undergo a process of activation due to chronic
   tissue injury, distinguished by motility, proliferation, contractility,
   and extracellular matrix synthesis [[141]62]. Curcumin is a potent
   compound that significantly impacts numerous genes/proteins in various
   biological and pathway processes [[142]41] and we show here that
   several important genes implicated in liver cirrhosis based on
   topological algorithms and the shortest paths analysis of the PPI
   network were identified. We also showed that these targets could
   interact with and are potentially modulated by curcumin. Molecular
   docking also indicated that curcumin has a high affinity in interaction
   with hub genes. The biological processes analysis of the acquired
   targets is mainly concerned with oxidative stress regulation, small RNA
   production, telomerase activity, cell proliferation, and necrotic cell
   death. In our study, various important pathways were highlighted based
   on enrichment analysis, and some of them have been widely studied for
   liver cirrhosis [[143]63,[144]64,[145]65,[146]66].

   In this study, the proteoglycans in the cancer pathway were
   significantly identified with a set of genes that included AKT1,
   CTNNB1, EGFR, FN1, SRC, STAT3, TNF, and TP53, which was the highest
   pathway in biological pathways analysis that was influenced by curcumin
   in liver cirrhosis. Proteoglycans are macromolecules made up of a
   protein core with sugar chains linked covalently. They can be found on
   the cell surface and in the extracellular matrix (ECM) of living
   organisms. Syndecan-1 is the most common transmembrane proteoglycan in
   normal liver [[147]63]. During the fibrogenesis process, proteoglycans
   may play a role in controlling cellular processes and preserving the
   supramolecular architecture of the extracellular matrix in both normal
   and diseased livers [[148]67]. Furthermore, it documented that
   proteoglycans increase in liver cirrhosis [[149]63]. Transforming
   growth factor β1 (TGF-β1) is a potent pro-fibrotic cytokine [[150]68]
   that in our analysis was found to be a hub candidate that might be
   influenced by curcumin. Decorin is a short leucine-rich proteoglycan
   that binds to TGF-1 with high affinity and blocks it from interacting
   with pro-fibrotic receptors. Decorin has previously been demonstrated
   to have a protective function in liver fibrogenesis since the genetic
   ablation of decorin in mice results in increased poor matrix
   degradation, matrix deposition, and the activation of hepatic stellate
   cells, the major indices of fibrotic tissue [[151]61]. Furthermore,
   proteoglycans such as endocan and syndecan-1 in the serum of patients
   with alcoholic cirrhosis are potential prognostic biomarkers for liver
   cancer [[152]69]. The previous research by Kovalszky et al. in 1998 on
   animal models with liver fibrosis indicated that proteoglycans
   participate in fibrogenesis. Hepatocytes synthesize the bulk of
   liver-specific heparan sulfate proteoglycans such as fibroglycan and
   syndecan-1. Perlecan and decorin are extracellular matrix proteoglycans
   produced by non-parenchymal liver cells. In a healthy liver, the amount
   of proteoglycans is relatively modest, but it rises rapidly in liver
   fibrosis [[153]70]. On the other hand, in vitro studies indicate that
   curcumin could inhibit the apoptosis of chondrocytes, repress the
   induction of proteoglycans and metal metalloproteases, cyclooxygenase
   expression, prostaglandin E-2, and inflammatory factors in chondrocytes
   [[154]71]. In mesenchymal stem cell progenitors, curcumin markedly
   increases cartilage-specific proteoglycans (CSPGs), collagen type II
   production, activates MAPKinase signaling, β1-integrin, and represses
   cyclooxygenase-2 and caspase-3. It is suggested that curcumin may
   assist in establishing a microenvironment [[155]72].

   The AGE-RAGE signaling pathway in the diabetic complications signaling
   pathway was significantly found in an enrichment analysis with the
   AKT1, FN1, JUN, STAT3, and TNF genes indicated as hub genes influenced
   by curcumin. These results indicate that curcumin is involved in the
   pathway associated with liver cirrhosis. The activation of advanced
   glycation end products (AGEs) through their receptors (RAGE) causes an
   increase in reactive oxygen species (ROS), which is hypothesized to
   contribute to hepatic fibrosis through hyperglycemia [[156]73]. Hepatic
   fibrosis was linked to the activation of the AGE receptor (RAGE) in
   hepatic stellate cells. The RAGE signaling system is activated by NADPH
   oxidase, which causes a rise in reactive oxygen species (ROS), matrix
   metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (alpha-SMA), and
   RAGE [[157]64]. The liver is one of the principal organs engaged in
   inactivating AGEs and this function is impaired in individuals with
   hepatic cirrhosis [[158]74]. Furthermore, a previous study on patients
   with hepatitis c-related fibrosis supported that AGEs stimulate
   autophagy and activation of HSCs that are involved with fibrosis
   [[159]75]. Curcumin has been shown to suppress fibrosis by hindering
   the RAGE-mediated effects of AGEs such as raised oxidative stress,
   inflammation, and cell proliferation in hepatic stellate cells via
   PPAR-γ activation [[160]76]. PPAR- is a central transcriptional
   regulator that inhibits multiple transcription factors linked to α-SMA
   production and hepatic fibrosis including activated protein-1 (AP-1)
   and specialized protein-1, and nuclear factor-kappa B (NF-κB)
   [[161]77]. Curcumin also inhibits the effects of AGEs in hepatic
   stellate cells by disrupting leptin signaling and triggering the
   transcription factor NF-E2 p45-related factor 2 (Nrf2), which resulted
   in an increase in cellular glutathione and a decrease in oxidative
   stress [[162]78]. A recent transcriptomic analysis reported that
   curcumin suppresses the extracellular matrix (ECM)–receptor interaction
   and AGE-RAGE signaling pathway in the diabetic retina [[163]79].
   Furthermore, a recent bioinformatic analysis proposed that curcumin
   could ameliorate fatty liver diseases by impacting the AGE-RAGE
   signaling pathway [[164]41].

   The focal adhesion pathway encompasses several genes, and KEGG analysis
   demonstrated that AKT1, CTNNB1, EGFR, FN1, JUN, and SRC were strongly
   related to curcumin compounds that might be influenced by liver
   cirrhosis in our study.

   Cell–matrix adhesions are critical in a variety of biological
   activities including cell motility, differentiation, proliferation,
   cell survival, and gene modulation. Focal adhesions arise at the
   cell-extracellular matrix contact sites, where bundles of actin
   filaments are tethered to transmembrane receptors of the integrin
   family via a multi-molecular complex of protein junctions. Focal
   adhesion includes signaling molecules such as phosphatases and protein
   kinases or structural links between the actin cytoskeleton and membrane
   receptors [[165]80,[166]81,[167]82]. Focal-adhesion-kinase (FAK) is one
   of the most significant targets for hepatic stellate cells and liver
   fibrosis development in vivo. In fibrotic living tissues, FAK
   activation is linked to enhanced expression of α-SMA and collagen.
   TGF-β1 induces FAK activation in a dose and time-dependent manner.
   Inhibiting FAK activation prevents collagen and α-SMA expression and
   decreases the production of stress fibers, which reduces liver fibrosis
   [[168]65]. An animal study in 2020 reported that animals lacking FAK in
   liver epithelial cells compared to the controls had more severe liver
   damage and fibrosis. They also showed that the activation of numerous
   pro-fibrotic pathways including the hedgehog/smoothened pathway was
   associated with FAK-deficient animals and promoted matrix stiffness
   independently [[169]83]. A proteomic and transcriptomic study reported
   that focal adhesion and actin cytoskeleton pathways were significantly
   involved in rats with liver fibrosis [[170]66]. Through the focal
   adhesion kinase pathway, curcumin may control cellular processes such
   as migration and proliferation. Curcumin could modulate this pathway by
   alternating phosphorylation kinases such as MAPK12, AXL, FRK, and TNK2
   as well as phosphatases such as INPPL1, PTPRK, and PTPN6 [[171]84].
   Others have reported that curcumin treatment hinders the expression of
   integrin, Nm23, E-cadherin, TIMP-2, metalloproteinase activity,
   adhesion, and focal adhesion kinase [[172]85], and previous studies
   have shown that curcumin could specifically downregulate MMP-2,
   integrin receptors, and FAK activity [[173]86].

   Hypoxia-inducible factor 1 (HIF-1) is a heterodimer (Hif-1β and Hif-1α)
   and is considered to be a pivotal regulation factor that activates
   several hypoxia-responsive genes and is responsible for cell survival
   in hypoxia [[174]87]. Previous investigations focused on the role of
   HIF-1α in the pathogenesis of tumors; however, several reports proposed
   its role in liver fibrosis by the modulation of a set of genes involved
   in collagen synthesis and angiogenesis
   [[175]88,[176]89,[177]90,[178]91]. The HIF-1 signaling pathway was
   significantly found in enrichment analysis with the AKT1, EGFR, EP300,
   INS, and STAT3 genes, the hub genes that were also identified as a
   target of curcumin. The analysis of liver fibrosis in rats induced by
   carbon tetrachloride (CCl4) indicated that the HIF-1 signaling pathway
   in a KEGG analysis showed that HIF-1 is significantly involved in liver
   fibrosis [[179]66]. Another study reported that the development
   mechanism of liver fibrosis is implicated in the upregulation of HIF-1α
   transcriptional activity and its interconnected factors, TGF-β and
   NF-κB, in bile duct ligation-stimulated hepatocytes fibrosis in rats
   [[180]92].

   HIF-1 activates HSCs in a hypoxic environment caused by liver fibrosis
   and inflammation [[181]93]. It was recently discovered that HIF-1 is
   controlled by a variety of signaling mechanisms. HIF-1 transcription
   and synthesis are regulated by the phosphoinositol 3-kinase (PI3K)/Akt
   pathway, whereas HIF-1 transcription is induced by the
   mitogen-activated protein kinase (MAPK) pathway [[182]91]. In our
   study, these two pathways were also observed as the significant
   pathways in liver cirrhosis regulated with curcumin. Inhibition of PI3K
   signaling in HSCs reduces collagen production, ECM deposition, and
   profibrogenic factor expression [[183]94]. Others report that MAPK
   inhibition inhibits eNOS phosphorylation during hypoxia, resulting in
   decreased HIF-1 expression [[184]95]. This pathway is also at a
   crossroads with the focal adhesion pathway, influencing HSC activation
   and proliferation, which is important for hepatic fibrogenesis
   [[185]96,[186]97]. Evidence suggests a relation between the HIF-1 and
   Nrf2. The Nrf2 pathway plays a key role in activating the HIF-1-related
   activities [[187]98]. Curcumin can affect transcription factors such as
   NF-ĸB, Nrf2, STAT3, and protein kinases such as Akt and MAPKs
   [[188]99]; therefore, it might impact the HIF1 signaling pathway. The
   curcumin regulation of the ubiquitin-proteasomal system (UPS) has been
   linked to the regulation of cancer-linked apoptotic proteins,
   inflammatory proteins, cell cycle proteins, transcription factors such
   as HIF-1, and growth factors related to the inhibition of cancer
   processes [[189]100]. Curcumin has been demonstrated to downregulate
   Notch1 expression, hindering hypoxia-induced HIF-1 expression and
   acting as a possible anticancer drug for treating osteosarcoma
   [[190]101]. HIF-1 interacts with the intracellular domain of Notch1,
   which is produced by γ-secretase cleavage and translocates to the
   nucleus to activate Notch target genes in hypoxia. Furthermore, other
   research found that curcumin decreased angiogenesis and HIF-1
   expression in osteosarcoma cells. Curcumin also inhibits HIF-1
   expression by interfering with the p38 MAPK and Akt/mTOR pathways.
   According to these studies, Akt/mTOR is also a significant upstream
   regulator of NF-B and GSK3, and as a result, GSK3 and NF-B may play a
   role in the curcumin inhibition of HIF-1 [[191]102].

   Our study also illustrated that curcumin may strongly modulate SRC,
   which had a strong interaction with low ΔG energy: −8.4 kcal/mol. SCR
   was recognized as a key protein interaction based on the PPI network
   analysis with the highest degree score of 168, high Betweenness score
   of 0.0350, and Closeness score of 0.4612, introducing this gene as one
   of the vital therapeutic targets for liver cirrhosis. We also observed
   that SRC was markedly expressed in the liver (score: 4.64). SRC kinase
   families are non-receptor tyrosine kinases observed in all cell types.
   However, SRC is demonstrated to be active in renal and pulmonary
   fibrosis [[192]103,[193]104]. SRC expression varies dramatically
   depending on the stage of liver cirrhosis [[194]105]. According to
   Western blot and immune-histochemical analyses, src-related tyrosine
   kinases were discovered in large numbers in patients with liver
   cirrhosis and hepatocellular carcinoma, suggesting that SRC-RTK may
   have a role in the formation and progression of both diseases
   [[195]106]. Other research suggests that liver cirrhosis might be
   attenuated by using SRC inhibitors [[196]107]. In cirrhosis patients
   and thioacetamide (TAA)-induced fibrotic mice livers, SRC expression
   was shown to be increased, and phospho-SRC was elevated after the
   activation of hepatic stellate cells. Src inhibition also decreased
   TGF-β and lowered the expression of α-SMA in primary hepatic stellate
   cells. The downregulation of smad3 was linked to the anti-fibrotic
   impact of SRC inhibitors. Furthermore, inhibiting SRC enhanced
   autophagy flux and protected the liver against fibrosis [[197]103]. An
   in vitro kinase investigation indicated that curcumin directly inhibits
   SRC activity [[198]108]. In a study by Yang et al., SRC is an upstream
   signaling transducer of several genes including JNK and Smad3, and has
   a close relation with TGFβ1. They also commented that curcumin
   significantly abolished the TGFβ1-induced CCN2 by hindering the
   phosphorylations of SRC, Smad3, and JNK, and it also interfered with
   α-SMA and TGFβ1 expression [[199]109]. Others have reported that
   curcumin affects the SRC by promoting hypomethylation of the mir-203
   promoter and increasing the expression of this microRNA; mir-203 is the
   target of SRC gene inhibition [[200]110].

   AKT is a serine/threonine-protein kinase that has a primary function in
   the development of liver fibrosis. Akt is composed of three specific
   isoforms including AKT-1, AKT-2, and AKT-3 [[201]111]. The induction of
   fibrosis in the liver of mice reported that all AKT forms were
   triggered with a concomitant rise in the activated forms of mammalian
   target-of-rapamycin complex 2 (mTORC2) phosphoinositide-dependent
   kinase-1 (PDK1) and PI3K, leading to the increasing level of
   inflammatory, fibrogenic, and proliferative genes [[202]112]. An RNA
   sequencing study on patients with hepatitis B cirrhosis indicated that
   AKT is associated with liver cirrhosis [[203]58]. The PI3K/Akt pathway
   plays a key part in apoptosis, motility, and cell growth, and the
   overexpression and activation of Akt1 and Akt2 boost the K8/18 protein
   levels [[204]113]. Another investigation demonstrated the abolishing of
   AKT-mediated phosphorylation of glycogen synthase kinase 3β (GSK3β) on
   Ser9 in cirrhosis compared to the normal cells [[205]114]. In the
   cirrhosis liver of rats, phosphorylation of AKT and eNOS were obviously
   impaired [[206]115]. In several lines of research, curcumin was
   introduced as a regulator of AKT1. In an in vitro study, the incubation
   of curcumin in MDA-MB231 cells reduces the level of AKT1 [[207]116]. A
   recent study showed that when curcumin was combined with bile, it
   inhibited Akt1 much more effectively [[208]117]. Others reported that
   the constitutive activation of AKT1 led to the inhibitory effect of
   curcumin on cell proliferation and migration in human bladder cancer
   [[209]118]. Using molecular docking, a study to find possible
   applicable curcumin targets for treating colorectal cancer showed that
   curcumin could stably combine with EGFR, STAT3, and AKT1. AKT1, the
   strongest binding to curcumin, was shown in their study [[210]119]. In
   accordance with our study, EGFR with binding affinity energy: −8.9
   kcal/mol, STAT3: −7.4 kcal/mol, and AKT1: −6.9 kcal/mol interacted with
   curcumin. Here, we showed AKT1 with a Degree of 156, Closeness of
   0.4619, and Betweenness of 0.0403, a crucial protein target in liver
   cirrhosis. Furthermore, it displayed a high specific presentation in
   the liver tissue (score: 4.88).

   Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor
   triggered by a variety of ligands, resulting in the activation of a
   number of signaling pathways that affect differentiation,
   proliferation, and survival. The EGFR signaling axis has been
   demonstrated to be important in developing liver diseases such as
   cirrhosis and liver cancer [[211]120]. Inhibiting EGFR reduces liver
   fibrosis and HSC activation in NAFLD based on recent research,
   indicating the dysregulation of EGFR in animal models of liver damage
   [[212]121,[213]122]. EGFR is phosphorylated in the liver tissues of
   high-fat diet (HFD) mice with NAFLD. Diet-induced oxidative stress,
   lipid accumulation, HSC activation, and matrix precipitate were all
   reduced by inhibiting EGFR. HSC activation was mediated by EGFR, which
   led to a pro-fibrogenic phenotype [[214]123]. A previous clinical study
   reported that the positive rates of EGFR expression were 66.22% in
   liver cancer, 44.00% in liver cirrhosis, and 10.00% in normal liver
   tissues. The expression of EGFR was remarkably higher in liver
   cirrhosis than in normal liver tissue [[215]124]. Several molecular
   dynamics and docking studies showed that curcumin has great potential
   for anti-EGFR activity [[216]125,[217]126,[218]127,[219]128]. Curcumin
   exerts its anticancer property by inhibiting proliferation, EGFR
   phosphorylation, and promoting the degradation of EGFR by
   ubiquitination and apoptosis [[220]129]. It has also been confirmed
   that the induction of apoptosis is mediated through the EGFR signaling
   pathway [[221]130]. A study has shown that curcumin could control EGFR
   and EGFR downstream signaling molecules containing Akt, STAT3, and
   ERK1/2 [[222]131]; curcumin inhibited EGFR via efficiently modulating
   gefitinib-insensitive EGFR degradation [[223]132]. The hydroxyl groups
   in curcumin and related analogs were important for their biological
   actions in affecting EGFR expression [[224]133]. In our analysis, EGFR
   showed great impact in the PPI network-liver cirrhosis with a Degree of
   149, Betweenness of 0.0245, and Closeness of 0.4498, which curcumin
   strongly interacts with and blocks its function with a low binding
   affinity energy of −8.9 kcal/mol. Furthermore, our acquired data
   indicates that an EGFR score of 4.75 has high liver specificity.

   The signal transducer and activator of transcription-3 (STAT3) perform
   a critical role in the pathophysiology of liver disorders [[225]4].
   STAT3 is a cytoplasmic signal transcription factor that is a member of
   the Janus kinase (JAK)/STAT pathway and is thought to have an essential
   role in mediating liver damage [[226]134]. STAT3 activation is an
   essential signal for liver renewal in cirrhotic livers from the
   hepatitis C virus (HCV) infection and alcoholics, and suppressing liver
   renewal in liver cirrhosis might be due to reduced STAT3 activation
   [[227]135]. Another study reported that in liver cirrhosis, STAT3
   DNA-binding is impaired by raising the expression of Pias3 [[228]136].
   STAT3 proteins were found in abundance in the nuclei of proliferating
   biliary epithelial cells and hepatocytes in cirrhosis livers. Although
   Stat3 proteins are upregulated in cirrhosis, this transcription factor
   appears to be functionally inactive. Impaired STAT3 activation and
   restoration may thus contribute to the development of cell damage,
   which leads to liver cirrhosis [[229]137]. Moreover, others have
   reported that STX-0119, an inhibitor of STAT3 dimerization, could
   suppress the progress of liver cirrhosis by obstructing hepatic
   stellate cell activation [[230]138]. Differing research indicates that
   curcumin could regulate the STAT3 signaling pathway, with most research
   focusing on the role of STAT3 in cancers and its inhibition by
   curcumin. A study reported that curcumin decreased STAT3
   phosphorylation and prevented STAT3-mediated signaling [[231]139].
   Previously, research in a colitis animal model reported that the
   phospho-STAT3 activity and DNA-binding activity of STAT3 dimers were
   significantly decreased following therapy with curcumin [[232]140]. A
   study showed that expression levels of STAT3 declined after curcumin
   administration in MDA-MB-231 cells [[233]141]. Research by Hahn et al.
   in 2018 determined that the cysteine residue 259 of STAT3 is an assumed
   site for the binding of curcumin. The authors also reported that the
   α,β-unsaturated carbonyl moiety in curcumin appears critical for
   binding to STAT3 [[234]142]. Furthermore, several studies have proposed
   that STAT3 could play a crucial function in inflammation-associated
   tumorigenesis [[235]143,[236]144]. Curcumin can abrogate unusual STAT3
   activation via direct interaction and interfere with STAT3-mediated
   carcinogenesis [[237]142]. Other research also showed that curcumin
   could inhibit PTC cell viability in a dose-dependent manner by
   stimulating caspase-mediated apoptosis and inhibiting constitutively
   active STAT3 by dephosphorylating Tyrosine 705 of STAT3 without
   altering STAT3 [[238]145]. Our study illustrated that curcumin may
   strongly modulate STAT3, which had a binding affinity energy of −7.4
   kcal/mol. Based on the PPI network analysis, STAT3 was recognized as a
   key target that could be harnessed for therapeutic purposes against
   liver cirrhosis.

5. Conclusions

   This study looked at the possible effect of curcumin on the genes,
   proteins, and molecular pathways involved in the progression of liver
   cirrhosis and showed that curcumin may dramatically affect critical
   biological pathways associated with proteoglycans, AGE-RAGE, focal
   adhesion, and the HIF-1 signaling pathways in liver cirrhosis. Curcumin
   was shown to be likely to affect nine crucial genes/proteins concerned
   in this disorder, mostly expressed in the liver. Curcumin is proposed
   to have therapeutic benefits in different liver diseases from the
   literature, but more comprehensive molecular studies with robust
   patient cohorts in clinical trials are needed to confirm this efficacy.

Author Contributions

   A.M.; conceptualization, methodology, software, writing-original draft
   preparation, S.L.A.; methodology, writing-review and editing, T.J.;
   methodology, writing-review and editing, M.B.; methodology,
   writing-review and editing, A.S.; conceptualization, methodology,
   supervision, writing-review and editing. All authors have read and
   agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   Not applicable.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This research received no external funding.

Footnotes

   Publisher’s Note: MDPI stays neutral with regard to jurisdictional
   claims in published maps and institutional affiliations.

References