Exploring the Therapeutic Potential and Mechanisms of Zizyphus jujuba Miller in Chronic Pancreatitis: A Network Pharmacology and Molecular Docking Approach

Article information

J Korean Med. 2024;45(4):99-115

Publication date (electronic) : 2024 December 1

doi : https://doi.org/10.13048/jkm.24060

Yebin Lim¹, Jin-Young Oh¹, Gyeong-Ran Noh¹, Jihyun Yu¹, Dong-Uk Kim^1,2,, Bitna Kweon^1,2,, Gi-Sang Bae^1,2,3,4,

¹Department of Pharmacology, School of Korean Medicine, Wonkwang University

²Hanbang Cardio-Renal Syndrome Research Center, Wonkwang University

³Research center of Traditional Korean medicine, Wonkwang University

⁴David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States

Correspondence to: Dong-Uk Kim, Bitna Kweon, and Gi-Sang Bae. Department of Pharmacology, School of Korean Medicine, Wonkwang University, 460 Iksandae-ro, Iksan, 54538 Jeonbuk, South Korea, Tel: +82-63-850-6842, E-mail: ckck202@naver.com/kbn306@naver.com/baegs888@wku.ac.kr

Received 2024 October 19; Revised 2024 November 8; Accepted 2024 November 12.

Abstract

Introduction

Chronic pancreatitis (CP) is an irreversible disease characterized by the replacement of pancreatic parenchyma with fibrous connective tissue due to repeated episodes of inflammation¹⁾. This condition is marked by intermittent or continuous severe abdominal pain and, as it progresses, leads to both exocrine and endocrine pancreatic insufficiencies, severely impacting the quality of life²⁾. Additionally, patients with CP have a 13.3-fold increased risk of developing pancreatic cancer³⁾. The highest prevalence and incidence of pancreatitis are observed in East Asia, particularly in Korea, where a 13-year cohort study reported a significant increase in CP prevalence from 90 per 100,000 individuals in 2002 to 560 per 100,000 individuals in 2015^4,5). Although the overall incidence and prevalence of CP are relatively low, it is associated with considerable morbidity and poses a significant financial burden on both individuals and public health systems²⁾. Despite its severity, CP currently lacks effective treatments, with existing medications providing only limited symptom relief^6–10). For instance, paracetamol is frequently administered to manage pain in CP patients, while opioids may be considered in cases of more severe pain¹¹⁾. As a result, patients are encouraged to implement lifestyle modifications, including abstinence from alcohol and smoking, to help manage the disease⁷⁾. Consequently, there is an urgent need for effective therapeutic interventions for CP.

The Zizyphus jujuba Miller var. inermis Rehder (Jujube) belongs to the Rhamnaceae family and has been widely utilized in traditional medicine across Asia, Europe, and the United States for the treatment of various ailments such as gastrointestinal disorders, liver diseases, obesity, skin infections, anemia, diarrhea, insomnia, and cancer¹²⁾. Several studies have reported the anti-inflammatory and anti-fibrotic properties of Jujube and its varieties^13–17). However, research investigating the effects of Jujube on CP has not yet been conducted.

Network pharmacology encompasses various approaches, one of which involves linking active compounds to target genes, and target genes to diseases, thereby constructing a network to explore drug efficacy^18,19). It has garnered attention as a novel approach to studying the efficacy of traditional herbal medicines with multiple components and multiple targets^18,20). Additionally, network pharmacology research methods are being effectively used to uncover new effects of existing drugs^21,22).

In this study, we aimed to explore the potential of jujube in improving CP through network pharmacology. Accordingly, we constructed a network based on the active compounds and key genes of jujube. Furthermore, using network pharmacology and blind molecular docking techniques, we sought to predict the potential ameliorative effects and mechanisms of jujube on CP.

Material and Methods

Results

1. Selection of active compounds among the constituents of Jujube

A total of 74 compounds constituting Jujube were collected from the HERB and OASIS databases. After excluding compounds with poor oral bioavailability and those without valid target gene information in the PubChem database, 55 compounds were predicted to be active compounds (Table 1). A total of 344 genes were associated with these compounds, and a PPI network was constructed using these genes, resulting in 344 nodes and 2237 edges (Figure 1A).

Table 1

List of 55 active compounds of Jujube

Fig. 1

(A) Network of Zizyphus jujuba Miller var. inermis Rehder (Jujube) with 344 nodes and 2237 edges. (B) Venn-diagram showing the relationship between Jujube and chronic pancreatitis (CP).

2. Exploring Key genes and Network analysis between Jujube and CP

To determine the association between CP-related genes and the target genes of Jujube, we identified common genes between the 1,241 CP-related genes collected from the GeneCards database and the target genes of Jujube. The analysis revealed a total of 156 overlapping genes (Figure 1B, Table 2). A PPI network of 9 nodes and 28 edges was constructed from 156 overlapping genes through topological analysis in Cytoscape 3.10.2 (Figure 2). The 9 nodes identified as key genes include TP53, IL6, AKT1, TNF, IL1B, INS, STAT3, BCL2, and TLR4. The respective DC, BC and CC values are presented in Table 3.

Table 2

List of overlapping genes of Jujube and CP

Fig. 2

Process of topological screening: (A) Network illustrating the intersection of target genes from Jujube and gene sets associated with CP. (B) and (C) show the networks of the target genes during the initial and final screening, respectively.

Table 3

The DC, BC and CC values of key genes

To investigate the potential mechanisms and effects of Jujube on CP, 9 key genes were analyzed through GO enrichment analysis and KEGG pathway analysis. The GO enrichment analysis identified 65 items in the Molecular Function (MF) category, 26 items in the Cellular Component (CC) category, and 822 items in the Biological Process (BP) category. From each category, the top 10 items with the lowest p-values were selected. The MF category was associated with ‘Cytokine Activity’ and ‘Cytokine Receptor binding’, the CC category with ‘Endoplasmic Reticulum Lumen’ and ‘Mitochondrial Outer Membrane’, and the BP category with ‘Positive Regulation Of NF-kappaB Transcription Factor Activity’, ‘Positive Regulation Of Cytokine Production Involved In Inflammatory Response’, and ‘Positive Regulation Of Interleukin-6 Production’ (Figure 3).

Fig. 3

GO enrichment analysis of the key genes. Top 10 biological processes, cellular components, and molecular functions were identified, ranked by the lowest p-values.

Additionally, KEGG pathway analysis identified 153 items associated with the 9 key genes. Among these, the top 30 items were selected based on p-value for further analysis. Ultimately, 4 top items related to the mechanism were identified: ‘HIF-1 signaling pathway’, ‘Toll-like receptor signaling pathway’, ‘PI3K-Akt signaling pathway’, and ‘NOD-like receptor signaling pathway’ (Figure 4, Table 4).

Fig. 4

(A) KEGG pathway analysis of the key genes. Pathways were listed in order of significance based on their p-values. (B) SRPLOT was used to illustrate the association between the key genes and KEGG pathways.

Table 4

Top 4 items of KEGG pathway related to CP mechanism

4. Exploring the Mechanism of Jujube’s Therapeutic Effects on CP through JAKP network analysis

DC in network pharmacology measures a node's influence based on its number of connections and is crucial for identifying potential drug targets^23,24). In the JAKP network analysis of Jujube, compounds closely associated with CP were ranked by their DC. The top 5 active compounds were Apigenin, Caffeic acid, Ferulic acid, Quercetin, and Ursolic acid. They were found to be connected to all 9 key genes: IL6, TNF, INS, AKT1, BCL2, TP53, TLR4, STAT3, and IL1B. Among the 4 pathways identified through KEGG pathway analysis, ‘HIF-1 signaling pathway’ and ‘PI3K-Akt signaling pathway’ had a DC value of 6, while ‘Toll-like receptor signaling pathway’ and ‘NOD-like receptor signaling pathway’ had a DC value of 5 (Figure 5).

Fig. 5

The compound, target, and pathway network of Jujube in the treatment of CP. The blue represents Jujube, the sky blue represents active compounds, the green represents key genes and the yellow represents pathways.

5. Molecular docking

In the JAKP network, the top 5 active compounds with the highest DC scores were analyzed for their binding energy with proteins coded by key genes. On average, these compounds exhibited a binding affinity of -6.91 Vina score. Notably, Ursolic acid showed a particularly strong binding affinity with the protein Akt1, coded by the AKT1 gene, with a Vina score of −9.0. Additionally, Ursolic acid also demonstrated strong binding with proteins Stat3 and Ins, coded by the STAT3 and INS genes, with Vina scores of −8.9 and −8.6, respectively (Figure 6).

Fig. 6

(A) Results from molecular docking studies between the proteins encoded by the 9 key genes and the top 5 active compounds. (B)–(D) show the results of the top 3 structure-based blind docking poses with the lowest binding energies; (B) Ursolic acid-Akt1, (C) Ursolic acid-Stat3 and (D) Ursolic acid-Ins.

Discussion

CP is a disease characterized by the progressive loss of the endocrine and exocrine compartments of the pancreas due to long-standing inflammation that results in atrophic, fibrotic, or a combination of both types of tissues^25,26). In particular, the activation of pancreatic stellate cells (PSCs) plays a crucial role in pancreatic fibrosis, which is primarily triggered by exposure to various cytokines, growth factors, and reactive oxygen species (ROS) during tissue injury, repair, and inflammation²⁷⁾. Once activated, PSCs migrate to the site of injury, proliferate, and secrete excessive amounts of extracellular matrix (ECM), leading to fibrosis, while establishing a vicious cycle through autocrine stimulation^28,29). These pathological changes eventually lead to structural alterations of the pancreas, contributing to clinical manifestations of CP, such as reduced secretion of digestive enzymes and endocrine dysfunction^29,30). Although the pathophysiology of CP is highly complex, smoking and excessive alcohol consumption have been studied as major etiological factors for chronic pancreatitis, and particularly in cases of recurrent acute pancreatitis, it may progress to CP through a necrosis-fibrosis sequence^6,31–33).

In traditional Korean medicine, CP is categorized under the concepts of pain, epigastric pain, and the formation of abdominal masses(癥瘕)³⁴⁾. It can be classified into several patterns, including spleen-stomach weakness(脾胃虛弱), heat-dampness stagnation(濕熱鬱蒸), and excess heat with obstruction(實熱結滯)³⁴⁾. Due to the prolonged nature of the condition and the predominance of symptoms such as indigestion and abdominal pain, some perspectives consider spleen-stomach weakness (脾胃虛弱) as a primary pathophysiological mechanism³⁵⁾.

The fruit of Jujube has been used to alleviate symptoms related to weakened digestive function and insufficiency of middle qi (中氣不足), which weakens the spleen and stomach, leading to symptoms such as poor appetite, fatigue, weakness, unexplained palpitations, anxiety, and the passage of unformed stools^36,37). Given these properties, Jujube may offer therapeutic potential for treating chronic pancreatitis, particularly in addressing spleen-stomach weakness. While experimental studies suggest that Ziziphus jujuba may have a fibrosis-preventing effect on cavernosal tissue, Jujube’s protective effects against CP have not yet been elucidated¹⁶⁾.

A total of 55 active compounds from the collected Jujube were found to interact with 344 target genes. Among these, 156 genes overlapped with CP, showing a significant relevance of 45.35%. This overlap is relatively higher compared to previous network pharmacology studies, suggesting that Jujube may have a meaningful effect in improving CP^38,39).

Functional enrichment analysis was conducted to investigate the biological functions associated with Jujube in CP. The results revealed that Jujube may play an important role in inflammatory responses, as indicated by functions such as ‘Cytokine Activity’, ‘Cytokine Receptor Binding’, and ‘Positive Regulation Of Cytokine Production Involved In Inflammatory Response’. Additionally, the KEGG 2021 Human database indicates that Jujube is related to CP through pathways such as the ‘HIF-1 signaling pathway’, ‘Toll-like receptor signaling pathway’, ‘PI3K-Akt signaling pathway’, and ‘NOD-like receptor signaling pathway’. Although all 4 pathways are involved in the regulation of inflammation, the PI3K/Akt signaling pathway, in particular, is closely associated with fibrosis, as several studies have highlighted its potential as a therapeutic target^40–42). For instance, in pancreatic fibrosis, a treatment has been shown to inhibit PSCs autophagy and reduce ECM formation and pancreatic damage through the PI3K/Akt pathway⁴³⁾. Therefore, regulation of the PI3K/Akt pathway could potentially prevent or alleviate fibrosis, a major pathological feature of CP.

The PI3K/Akt signaling pathway is involved in the migration and proliferation of PSCs⁴⁴⁾. The migration of PSCs is induced by platelet-derived growth factor (PDGF), which is upregulated during pancreatic injury and mediated through the downstream pathways of PI3K^45,46). Additionally, PI3K interacts with the ERK pathway, which is involved in the proliferation of PSCs⁴⁵⁾. Indeed, it has been reported that the administration of the PI3K inhibitor wortmannin not only inhibits PSCs migration but also reduces ERK activation⁴⁷⁾. Since activated PSCs migrate to the site of injury, proliferate, and secrete extracellular matrix, thereby contributing to pancreatic fibrosis, targeting the PI3K/Akt pathway to regulate PSC activation could potentially reduce tissue damage and serve as a promising therapeutic approach for CP^9,47,48). Several experimental studies have shown that PI3K/Akt signaling is elevated in CP, while inhibitors that reduce symptoms inhibit the phosphorylation of PI3K and Akt^49,50). There is evidence that mixtures containing Ziziphus jujuba Mill. modulate the PI3K/Akt pathway, but no studies have yet demonstrated that Jujube regulates CP⁵¹⁾. Given the critical role of the PI3K/Akt pathway in CP, Jujube may have the potential to alleviate the condition through the same mechanism.

Subsequently, using blind molecular docking, the binding affinities between the top 5 active compounds with the highest DC in the JAKP pathway and key genes were assessed. The analysis revealed that the binding energy between Akt1, a critical component of the PI3K/Akt pathway, and Ursolic acid was −9.0 vina score, indicating a notably strong interaction. In addition, compounds involved in the JAKP network have shown relevance in this context. In the JAKP network, Apigenin, Caffeic acid, Ferulic acid, Quercetin, and Ursolic acid exhibit high DC, suggesting their significant involvement in various biological processes related to CP. Notably, Ursolic acid, Quercetin, and Apigenin demonstrated strong binding affinities with key genes-coded proteins, with average binding energies below −7 vina score (Figure 6A). These active compounds have been reported to modulate various diseases through the PI3K/Akt signaling pathway^52–59). Particularly Ursolic acid, which stands out with its strong binding affinities to Akt1, has meaningful relevance to fibrosis. For instance, Ursolic acid ameliorates hepatic fibrosis in rats through the ERK, PI3K/Akt, and p38 MAPK pathways⁵⁹⁾. Furthermore, It has been demonstrated that Ursolic acid significantly suppresses the growth and induces apoptosis of resistant pancreatic cancer through activation of the c-Jun-terminal kinase pathway and inhibition of PI3K/Akt/NF-κ B pathway⁶⁰⁾. Such evidence suggests that Jujube has the potential to modulate CP through the PI3K/Akt signaling pathway, with Ursolic acid, in particular, playing a prominent role, alongside Quercetin and Apigenin.

However, this study is based on in silico analysis, and it remains unclear whether Jujube exhibits beneficial effects on CP in vivo. While the potential antifibrotic effects of the aforementioned compounds have been suggested, the precise mechanisms through which Jujube and its active compounds act on pancreatic tissue remain unclear. Additionally, research on the dosage of Jujube that can exhibit pharmacological efficacy is also needed.

Conclusion

In this study, we constructed a network based on the active compounds of Jujube and the associated key genes. By utilizing network pharmacology and molecular docking methods, we explored the potential of Jujube in improving CP and its underlying mechanisms of action. It is anticipated that the findings of this study could provide foundational data for future research on the use of Jujube in improving CP.

Article information Continued