Introduction

Sjögren’s syndrome (SS) is a chronic autoimmune disease characterized by dysfunction and destruction of salivary and lacrimal glands associated with a wide range of systemic manifestations (Bombardieri et al., 2020). Males are 10 times more affected by this disease, and the reported mean age of onset is between 50 and 60 years. Various glandular manifestations are also observed in 30–50% of patients. The prevalence of primary Sjögren’s syndrome is inconsistent at around 1:1000–1:100 (Qin et al., 2015). In spite of all the efforts made, its pathogenesis remains poorly understood, and no well-acknowledged therapy that halts the progression of Sjögren’s syndrome has been available (Vivino et al., 2016). Therefore, there is an urgent need for research to develop novel pharmacological interventions.

Traditional Chinese medicine (TCM) contains various active ingredients and carries complex mechanisms of action to treat and prevent diseases. In the Chinese Pharmacopoeia 2020 edition, Hippophae rhamnoides has been described as a dried fruit of Hippophae rhamnoides L. that can strengthen the spleen, help digestion, arrest cough, eliminate phlegm, activate blood circulation, and disperse blood stasis. This edible medicinal fruit is of a food and medicine continuum and contains a wide variety of active ingredients, such as flavonoids, tannins, terpenoids, polysaccharides, and vitamins. These -pharmacological ingredients exert a wide range of pharmacological activities, including anti--inflammatory, antioxidant, hepato-protective, anti-obesity, anti--cardiovascular disorder properties as well as anti--bacterial potency (Gao-Shuang et al., 2121; Heinrich et al., 2021). Further, an increasing number of studies have reported that Hippophae rhamnoides may exert desirable therapeutic effects on Sjögren’s syndrome. However, its potential active ingredients and underlying mechanism remain elusive.

Network pharmacology has been considered a new approach to identify the mechanism of herb–-ingredient–target multimolecular synergy at the systemic level (Li and Zhang, 2013). Furthermore, the research strategy of network pharmacology embodies the TCM’s holistic view of the disease, providing new ideas and methods for relative research. Therefore, this study aimed at deciphering the Hippophae rhamnoides targets and potential -signaling pathways in the treatment of Sjögren’s syndrome by performing network pharmacologic and molecular docking study. The flow chart of the docking study is presented in Figure 1.

Figure 1. Flowchart of the study design.

Materials and methods

Hippophae rhamnoides’ chemical composition search and screening

The Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP) (http://tcmspw.com/tcmsp.php) is an online database used to filter the related targets and active ingredients of Hippophae rhamnoides. This database comprises 499 Chinese medicinal species and provides data about the absorption, distribution, metabolism, and excretion (ADME) properties of various ingredients (Ru et al., 2014). Oral bioavailability (OB) and drug-like (DL) properties are the foremost pharmacokinetic indicators (Huang et al., 2017). Oral bioavailability (OB) and drug-like (DL) properties are the foremost pharmacokinetic indicators (Huang et al., 2017). In this study, compounds with OB ndicatorinal speciesfrom Hippophae rhamnoides were selected as candidate components, and their corresponding target proteins were predicted by searching the HERB (High-throughput Experiment- and Reference-guided database of TCM; http://herb.ac.cn) database (Fang et al., 2021).

Construction of target networks of Hippophae rhamnoides’ components

Initially, active ingredients and targets of Hippophae rhamnoides were imported into the network visualization software Cytoscape 3.8.2 (Shannon et al., 2003). A network map of active ingredients and targets was constructed, and the core ingredients of Hippophae rhamnoides were screened.

Screening of Sjögren’s syndrome-related targets

The Online Mendelian Inheritance (OMIM; http://www.omim.org), the Human Gene Database (GeneCards; https://www.genecards.org), and DisGeNET (http://www.disgenet.org/) databases were screened using “Sjögren’s Syndrome” as keywords to identify the disease-related targets. After deduplication, a Venn diagram was drawn using active ingredient targets of Hippophae rhamnoides. Similarly, a Venn diagram was constructed using the disease targets of Sjögren’s syndrome with the microbiology letter online mapping tool (www.bioinformatics.com.cn) to obtain crossover genes as candidate genes.

Protein–protein interaction (PPI) network construction

A PPI network was constructed by importing the processed dataset into the Search Tool for the Retrieval of Interacting Genes/Proteins 11.5 (STRING) online database (https://string-db.org) and the Cytoscape 3.8.2 software. The Cytohubba plug-in of Cytoscape was utilized to screen out key genes.

Functional enrichment analysis

Gene ontology (GO) enrichment analysis was performed on target proteins obtained after network merging in the Metascape database (Zhou et al., 2019), and the R language toolkit was used to visualize biological processes, cellular components, molecular functions, and signaling pathways.

Compound–Target–Pathway (C-T-P) network construction

To further investigate the therapeutic mechanism of Hippophae rhamnoides in Sjögren’s syndrome, a C-T-P network was created using Cytoscape 3.8.2.

Molecular docking

Molecular docking was used to assess the binding activity of core compounds to key targets. Using core compounds as ligands and core target genes obtained from PPI network topology analysis as receptors, the AutoDock Vina 1.5.7 software developed by Olson’s research group in Scripps Research Institute (CA, USA) was applied for molecular docking (Trott and Olson, 2010). The top five targets of the PPI network and the top 10 core compounds of Hippophae rhamnoides were selected and downloaded from the RCSB PDB database (http://www.rcsb.org/) (Goodsell et al., 2020), while their chemical structures were downloaded from the TCMSP platform. The candidate compounds were converted to Program Database (PDB) format using AutoDock tools. The protein crystal structures of core target genes were pre--processed by dehydration, hydrogenation, modeling the missing loop regions, calculation of protein ionization, and protonation of protein structures. The compounds and proteins were converted into Protein Data Bank, Partial Charge (Q), & Atom Type (T) (PDBQT) format using AutoDock tool, and the position of each protein or its ligand was determined to figure out the active pocket of the protein. When the binding energy value is zero, the molecular proteins are considered to bind and interact spontaneously. Therefore, the lower the binding energy required for docking, the more stable the molecular conformation, and the best receptor-ligand binding complex is selected for visualization.

Results

Composition prediction of Hippophae rhamnoides

In all, 33 active compounds were screened using the requirements of OB ≥ 30% and DL ≥ 0; 18 and 22 active ingredients were obtained after deleting duplicates by searching the TCMSP database (Table 1). The active ingredients were entered into HERB to acquire drug target names, and 208 gene targets were obtained after removing duplications.

Construction of target networks for Hippophae rhamnoides components

The network comprised 231 nodes (22 nodes for compounds, 208 nodes for targets, and one node for the herb) and 538 edges (Figure 2). Quercetin was connected to 161 targets with the highest degree of network connectivity, followed by kaempferol with 65 targets and β-sitosterol with 48 targets. The properties of this network were suitable for presenting complex compounds with multiple targets and compound–target interactions. The OBs of the three above-mentioned compounds were 46.43%, 41.88%, and 36.91%, respectively, suggesting that Quercetin, kaempferol, and β-sitosterol might be the potential key active compounds. Detailed information on the complex target network is provided in Supplementary Table S1.

Figure 2. Compound–target network. Yellow circles represent Hippophae rhamnoides, purple hexagons represent its active compounds, and red circles represent its related targets (IDs of the components are presented in Table 1).

Collection of Sjögren’s syndrome-related targets

A total of 2,185 Sjögren’s syndrome targets were integrated from multiple databases, including 643 targets from OMIM, 1,061 targets from GeneCards, and 481 targets from DisGeNET. After removing duplicates, a final list of 1,876 Sjögren’s syndrome-related targets was obtained (Table S2). In this study, 87 cross-targets were identified and collected for further mechanistic studies (Figure 3 and Table S3).

Figure 3. Venn diagram of intersection targets of Hippophae rhamnoides and Sjögren’s syndrome.

Potential target PPI network analysis

The STRING database was utilized to obtain the PPI network topology by importing 87 potential targets of Hippophae rhamnoides for improving Sjögren’s syndrome. The first 58 targets of the PPI network are shown in Figure 4. The network had 58 nodes and 269 edges with an average node degree of 9.276 and an average local clustering coefficient of 0.597. The built-in Network Analyzer function in Cytoscape 3.8.2 demonstrated that ALB, epidermal growth factor receptor (EGFR), epidermal growth factor receptor/Caspase-3 (CASP3), peroxisome proliferator-activated receptor gamma (PPARG), and estrogen receptor (ESR1) were the targets with large values and were of 43°, 28°, 25°, 24°, and 22°, respectively. It suggested that the efficacy mechanism of Hippophae rhamnoides in treating Sjögren’s syndrome could be closely related to these proteins.

Figure 4. Protein–protein interaction network of Hippophae rhamnoides for Sjögren’s syndrome.

Potential targets of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analysis

The Metascape database and the R language toolkit were adopted to perform the GO and KEGG pathway-enrichment analysis of Hippophae -rhamnoides in treating Sjögren’s syndrome. The biological processes (BP) involved in the GO-enrichment results were mainly responses to inorganic substances and lipopolysaccharides, negative regulation of cell population proliferation, positive regulation of cell death and cell migration, response to radiation and nutrient levels, and cellular response to organic cyclic compounds (Figure 5A). Relevant cellular components (CC) included were membrane raft, vesicle lumen, transcription regulator complex, side of membrane, extracellular matrix, myelin sheath, and endocytic vesicle (Figure 5B). The molecular functions (MF) involved cytokine receptor binding, protein homodimerization activity, DNA-binding transcription factor binding, protein structure, transcription factor binding, protein domain-specific binding, and oxidoreductase activity (Figure 5C). The KEGG pathway analysis suggested that 171 KEGG pathways were significantly enriched with the potential therapeutic targets of Hippophae rhamnoides. Key pathways with high P-values and literature of relative diseases were extracted, and the 14 pathways closely associated with Sjögren’s syndrome were displayed, with lipid and atherosclerosis, and tumor necrosis factor (TNF) signaling pathway as the top two pathways (Figure 5D).

Figure 5. Gene ontology analysis for (A) BP, (B) CC, and (C) MF and pathway enrichment. (D) KEGG analysis of Hippophae rhamnoides–Sjögren’s syndrome overlapping target genes.

The component–target–pathway network diagram

The network analysis revealed that the top five targets in the highest degree were transcription factor p65 (RELA), AKT1, CASP8, CASP3, and transcription factor Jun (JUN), and the top five active ingredients in the highest degree were quercetin, kaempferol, β-sitosterol, folic acid, and isorhamnetin (Figure 6). It was indicated that these were the important components and targets of Hippophae rhamnoides for its potential therapeutic effect on Sjögren’s syndrome.

Figure 6. Component–target–pathway network. Blue circles represent active ingredients of Hippophae rhamnoides; green -circles represent signaling pathways of Sjögren’s syndrome; and purple circles represent the targets.

Molecular docking

The top 10 active compounds obtained from the analysis of component target network were docked with the five potential core targets, ALB, EGFR, CASP3, PPARG, and ESR1, obtained from the PPT network analysis. Detailed information on compounds and targets is demonstrated in Table 2 and Figure 7. Binding energies are presented in Table 3. When the binding activity was less than 0, the ligand molecule could spontaneously bind to the receptor protein. The ligand molecule had the desired binding affinity if the binding energy was less than –5.0 kJ-mol^–1 (Yang et al., 2021). It was observed that all compound–target pairs had zero binding energy, suggesting that each core compound had good binding affinities for the first five targets. Docking pattern analysis was also performed for strong docking binding activity, as shown in Figure 8. The docking predictions might provide an initial basis for further drug target studies.

Figure 7. Three-dimensional (3D) structural diagram of the top five target proteins.

Figure 8. Molecular docking patterns of active components with ALB, CASP3, and PPARG.

Discussion

Owing to complexity, heterogeneity, and poorly defined active parameters of Sjögren’s syndrome, only a few treatment options are available, although it is one of the most common collagen diseases. TCM has demonstrated a desirable therapeutical potential for Sjögren’s syndrome, and the edible medicinal Hippophae rhamnoides has a variety of active ingredients with substantial anti--inflammatory, antioxidant, and immunity--enhancing properties. However, the composition of TCM is complicated, and its active ingredients are unclear in pharmacology and clinical practice, as specific target genes or proteins for drug action have not been identified completely. Network pharmacology combines system network analysis and pharmacology and may facilitate the systematic molecular study of effective components, targets, and pathways of herbs.

In the present study, the TCM active component–-target network analysis revealed that quercetin, kaempferol, β-sitosterol, FA, and isorhamnetin could act on multiple targets in the network, indicating that these components of Hippophae rhamnoides may exert therapeutic effects on Sjögren’s syndrome. Quercetin has been reported to exert significant improving effects on the loss of saliva flow, salivary gland damage, and inflammatory response in non-obese diabetic (NOD)/Ltj mice. Furthermore, it blocked the increased expression of obesity receptors and its downstream Janus kinase 2/signal transducer and activator of transcription 3 signaling in the salivary glands (Chang et al., 2022). Kaempferol inhibits the nuclear factor kappa B (NF-κB) binding activity of DNA and myeloid differentiation factor 88 and suppresses the release of interleukin 6 (IL-6), IL-1β, IL-18, and TNF-α, enhancing messenger RNA (mRNA) and protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated genes, and restraining the toll-like receptor 4 (Nam et al., 2017; Saw et al., 2014; Tang et al., 2015; Zhang et al., 2017). β-sitosterol may treat Sjogren’s syndrome with an improved prognosis by enhancing the expression of cholinergic receptor muscarinic 3 (CHRM3) and stimulating salivary secretion (Wu et al., 2022). Isorhamnetin can significantly improve innate immunity (Wang et al., 2012) and effectively down-regulate TNF-α, IL-6, and IL-1β and up-regulate IL-10 to reduce inflammatory responses (Zhang et al., 2017).

In the PPI network, 58 targets of Hippophae rhamnoides were identified to act in Sjögren’s syndrome, and the top 10 targets based on node degree were ALB, EGFR, CASP3, PPARG, ESR1, HSP90AA1, PLG, MAPK14, MAPK8, and MAPK1. These core proteins have potent therapeutic effects in Sjögren’s syndrome. Among them, increasing evidence has suggested that the EGFR pathway could have a significant impact on the inflammatory/immune reactions of epithelial cells. The EGFR-extracellular-signal-regulated kinase pathway plays a role in the pro-inflammatory responses mounted by Sjögren’s syndrome salivary gland epithelial cells (Sisto et al., 2015). While CASP3 is closely linked with autoimmunity (Lin et al., 2011), various studies have suggested that PPAR-γ agonists might exert anti-inflammatory activities by inhibiting production of cytokines in a variety of mouse models in chronic inflammatory and autoimmune diseases. Beauregard and Brandt (2003) found that PPAR-α and PPAR-γ can inhibit IL-1β-induced production of nitric oxide (NO) in cultured lacrimal gland acinar cells, suggesting PPAR agonists as useful therapeutic agents for preventing dry eye disease because of NO-mediated lacrimal gland damage. Results of the above research highlight that Hippophae rhamnoides may exert anti-Sjögren’s syndrome activity by modulating such core targets as EGFR, CASP3, and PPARG.

Gene ontology and KEGG-enrichment analysis of the 87 potential targets were performed to predict the mechanism underlying the therapeutic effect of Hippophae rhamnoides in Sjögren’s syndrome. The results showed that the 20 most significantly enriched BP terms were mainly responses to inorganic substances, lipopolysaccharides, and reactive oxygen species. Numerous studies have proposed that the onset and development of Sjögren’s syndrome are related to the activated immune system and the epithelial cells, targets, and players of autoimmune responses (Bombardieri et al., 2020). The enriched MFs included cytokine receptor binding, transcription factor, mitogen-activated protein kinase (MAPK), and integrin; heme-binding cytokine receptor binding, DNA-binding transcription factor binding, endo-peptidase activity, protein homo-dimerization activity, and protein structure. Further, the enriched CCs covered membrane raft, vesicle lumen, transcription regulator complex, side of membrane, extracellular matrix, and myelin sheath. These findings also illustrate the complexity of the pathological mechanism of Sjögren’s syndrome. To decode the potential mechanism of Hippophae rhamnoides in treating Sjögren’s syndrome, KEGG analysis of the 87 potential targets of Hippophae rhamnoides was conducted. The pathways related to Sjögren’s syndrome involved lipid and atherosclerosis, TNF-signaling pathway, and pancreatic cancer. Atherosclerosis is a major cause of cardiovascular disease involving autoimmune reactions and inflammation in its pathogenesis. During the early onset of atherosclerosis, inflammatory cells (monocytes, macrophages, dendritic cells, and T- and B-cells), and cytokines can be identified in the lesion area, and these cells may provoke cell-mediated immune reactions that (i) modulate the development of atherosclerosis and may (ii) predetermine its progression (Frostegård, 2002; Jonasson et al., 1997). TNF-α is mainly activated by macrophages and lymphocytes, and generated in salivary epithelial cells (Sisto et al., 2009). Several studies indicated that TNF-α plays an important role in the pathogenesis of Sjögren’s syndrome, as it is up-regulated in both plasma and salivary glands of patients with Sjögren’s syndrome as well as major salivary glands of animal models of Sjögren’s syndrome (Humphreys-Beher et al., 1994, 1998). These studies also propose that Hippophae rhamnoides can act on Sjögren’s syndrome at multiple levels through signaling pathway networks that are mediated by direct or indirect targets (Figure 9).

Figure 9. Potential target proteins of Hippophae rhamnoides regulating Sjögren’s syndrome on the predicted pathways (pink nodes are potential target proteins of Hippophae rhamnoides, and green nodes are relevant targets in the pathway).

According to the herb-active ingredient-target network and PPI network results, we selected five active ingredients and five key target genes for docking. The docking results showed that these compounds could bind stably to the active pocket of key protein, suggesting that Hippophae rhamnoides may treat Sjögren’s syndrome by inhibiting ALB, EGFR, CASP3, PPARG, and ESR1 (Figure 9).

Conclusion

This study deciphered the potential mechanism of Hippophae rhamnoides in treating Sjögren’s syndrome based on network pharmacology and molecular docking approach. Compounds such as quercetin, kaempferol, and β-sitosterol may play a critical role in treating Sjögren’s syndrome by affecting ALB, EGFR, CASP3, PPARG, and ESR1. Furthermore, the molecular docking study also demonstrated that the active components of Hippophae rhamnoides might dock well with ALB, EGFR, CASP3, PPARG, and ESR1. However, owing to the limitations of network pharmacology, further experimental investigations and verifications are warranted.

Author Contributions

TEZ, YHC, and LUX: conceptualization. ZL,YHC, and HPX: methodology and visualization. WYH, LL, XJ, and QZY: software. YHC , XLY and ZL: writing-original draft. YHC and TEZ: writing-review and editing. TEZ and QZY: supervision and project administration. All authors have read and agreed to the published version of the manuscript.