Research progress of probiotics and their protective strategy in the field of inflammatory bowel disease treatment: A review

Abstract

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease characterized by recurrent episodes and difficult-to-cure symptoms. Although the pathogenesis of IBD is closely related to host genetic susceptibility, intestinal microbiota, environmental factors, and immune responses, leading to mucosal damage and increased intestinal permeability. Intestinal mucosal injury in IBD patients causes pathogenic bacteria and pathogenic factors to invade the intestine, leading to disturb the structure and metabolic products of intestinal flora. Researchers have found that probiotics, as live microbial agents, can effectively inhibit the growth of pathogenic bacteria, regulate intestinal flora, optimize intestinal microecology, restore intestinal homeostasis, and promote intestinal mucosal repairing. During the oral delivery process, probiotics are susceptible to adverse physiological factors, leading to reduced bioavailability. Additionally, the oxidative stress microenvironment induced by intestinal mucosal damage makes it difficult for probiotics to colonize the intestinal tract of IBD patients, thereby affecting their probiotic effect. This research mainly introduces and reviews the advantages and disadvantages of probiotics and their protective strategies in the treatment of IBD, and prospects the future development trends of probiotics and their protective strategies. Probiotics can effectively inhibit the growth of harmful microorganisms, regulate the structure of the intestinal microbiota, and promote mucosal repairing, thereby reducing immune stress and alleviating intestinal inflammation, providing a new perspective for the treatment of IBD. The development of single-cell encapsulation technology not only effectively maintaining the biological activity of probiotics during oral delivery, but also endowing probiotics with additional biological functions naturally achieved through surface programming, which has multiple benefits for intestinal health.

1. Introduction

Inflammatory bowel disease (IBD) is a chronic and recurrent nonspecific intestinal disorder, clinically characterized by symptoms such as abdominal pain, diarrhea, and rectal bleeding, accompanied by infiltration of large numbers of neutrophils and macrophages, leading to dysbiosis of intestinal microbiota and sustained intestinal mucosal inflammation.^[1,2] Ulcerative colitis and Crohn disease are the 2 main forms of IBD, which often accompanied with intestinal mucosal injured, inflammatory hyperplasia, and persistent ulcers.^[3] Currently, the primary clinical treatment for IBD still relies on antibiotics, steroids, immunosuppressants, etc, to alleviate intestinal immune stress in IBD patients, prevent the occurrence of related complications, and achieve satisfactory therapeutic effects in many IBD patients.^[4,5] However, the efficacy of these drugs varies among individuals, and their short half-life and inevitable side effects from intravenous or subcutaneous administration pose challenges. Moreover, the pathogenesis of IBD remains unclear, and its recurrent nature and inability to be completely cured, which greatly distress patients mentally and physically, earning IBD the moniker “the cancer that never dies.”

To formulate appropriate treatment strategies to improve the therapeutic efficacy of IBD and reduce the risk of recurrence, it is essential to understand the pathogenic factors of IBD. Research has shown that the pathogenesis of IBD is complex and diverse, mainly induced by various factors such as host genetic susceptibility, dysbiosis of intestinal microbiota, complex external environment, and abnormal immune responses in the body, leading to chronic inflammation or apoptosis of intestinal epithelial cells.^[6] Consequently, the integrity of the intestinal mucosa is compromised, causing intestinal barrier damage, increased intestinal mucosal permeability, invasion of pathogens and pathogenic bacteria, effective recruitment, and activation of inflammatory cells to secrete cytokines.^[7] Imbalance of cytokines leads to the establishment of intestinal inflammatory status, thereby promoting the production of pro-inflammatory factors and disrupting intestinal microbiota balance.

Research on intestinal microbiota and fecal microbiota have found significant changes in the composition of intestinal microbiota in IBD patients compared to normal individuals, including decreased levels of lactobacilli, bifidobacteria, and clostridia, and significantly increased levels of gram-negative bacteria such as Escherichia coli, Actinobacteria, Proteobacterias, and Bacteroides.^[8,9] Toll-like receptor 4 is the prime sensor of gram-negative bacteria-derived lipopolysaccharide in the intestine. In IBD process, lipopolysaccharide is considered to be the primary trigger can act toll-like receptor 4 through either the MyD88-dependent or MyD88-independent pathway, ultimately directly triggering the distal nuclear factor kappa B signaling pathway through an auto-regulatory feedback loop mechanism to further amplify the inflammatory response and result in gut tissue destruction.^[10,11] Therefore, intestinal microbiota playing a crucial role in the occurrence and development of IBD.

To increase the levels of beneficial bacteria and reduce the composition of harmful bacteria in the intestines of IBD patients, thereby improving the intestinal microbiota of IBD patients, promoting intestinal mucosal repairing, and maintaining intestinal mucosal barrier function. In recent years, methods such as probiotics, prebiotics, or fecal transplantation have been effective in regulating the composition of intestinal microbiota, restoring intestinal homeostasis, and alleviating symptoms of IBD.^[1,12] Among many treatment options, probiotics, as live microbial drugs, not only inhibit the growth of pathogenic bacteria, inhibit and eliminate toxins produced by pathogenic bacteria, but also regulate intestinal metabolites, enhance immune function, and have been widely applied in the treatment of IBD.^[13] Researchers have found that probiotics are called “living microorganisms,” and sufficient amount of probiotics (least at 10⁶ CFU/g) is very beneficial to host health. Therefore, the maintenance of probiotic activity is an important indicator to evaluate the therapeutic effect of probiotics.^[14] However, during the oral delivery process, probiotics are susceptible to adverse physiological and pathological factors in the body, leading to a decrease in their biological activity and subsequently affecting their probiotic properties.^[15] This research mainly introduces and reviews the advantages and disadvantages of probiotics and their protective strategies in the treatment of IBD, and looks forward to the future development trends of probiotics and other biological preparations in the treatment of IBD.

2. Probiotics can effectively improve the imbalance of intestinal microbiota in patients with IBD

IBD, a chronic nonspecific intestinal disease, has been on the rise in China, posing a significant burden on human health and socio-economic development.^[2] The intestinal microbiota, a vast microbial community residing in the intestine, plays a pivotal role in maintaining host homeostasis and immune regulation.^[16] This symbiotic relationship between the intestinal microbiota and the host has been established since embryonic development through maternal–fetal interaction, earning the intestine the moniker “second brain” of the human body.^[17] Researchers have observed that the intestinal immune stress response triggered by IBD can lead to rearrangement of the intestinal epithelial cytoskeleton.^[18] This disruption compromises the tight junctions between intestinal epithelial cells, resulting in increased intestinal mucosal permeability and leakage of intestinal mucosal tissue proteins into the intestinal lumen. Consequently, toxic substances and microorganisms from the intestinal lumen can infiltrate the body, exacerbating intestinal inflammation, and mucosal damage.^[19] The dysbiosis of intestinal microbiota induced by IBD can adversely affect host intestinal health in several ways (Fig. 1A): (1) aberrant immune signal network: the intestine, a vital immune organ, plays a pivotal role in innate and adaptive immune responses. Dysbiosis of intestinal microbiota in IBD can trigger intestinal stress responses, prompting intestinal epithelial cells to upregulate cytokines and chemokines, thereby inducing intestinal inflammation. This inflammatory cascade results in intestinal tissue damage, heightened intestinal mucosal barrier permeability, and the infiltration and translocation of pathogenic bacteria and pathogens. Subsequently, immune cells and inflammatory cells are activated, leading to the release of pro-inflammatory factors and further amplifying the inflammatory response.^[20] (2) Disrupted chemical signal network: IBD-induced disruption of the intestinal environment not only alters the structure and composition of intestinal microbiota but also disrupts their metabolism. Especially, the metabolism of short-chain fatty acids and amino acid are reduced, thus affecting the biological function of the host gastrointestinal tract.^[21] (3) Altered neural signal network: dysbiosis of intestinal microbiota in IBD disrupts intestinal homeostasis, directly influencing the secretion and synthesis of neurotransmitters such as serotonin and dopamine by intestinal epithelial cells and enterochromaffin cells. Consequently, intestinal motility is affected.^[22] Therefore, the increase in intestinal mucosal permeability triggered by IBD not only facilitates the invasion and translocation of pathogenic bacteria and pathogens, leading to pathological changes in the structure and function of intestinal microbiota, but also predisposes to disorders in intestinal microbial metabolites and intestinal homeostasis. These disruptions ultimately affect the biological function of the host intestine.

Figure 1.

(A) Intestinal microenvironment in IBD patients. (B) The intestinal microenvironment of IBD patients who had taken probiotics. IBD = inflammatory bowel disease.

The primary clinical approach for treating IBD currently relies on antibiotics, steroids, immunosuppressants, and similar medications.^[4,5] While effective in eliminating pathogenic bacteria and alleviating intestinal inflammation, prolonged use of these drugs can lead to drug resistance and detrimental toxic effects on the body. This extended exposure diminishes the concentration and diversity of intestinal microbiota, resulting in immune imbalances and further disruption of the intestinal immune microenvironment.^[23] Consequently, there is an urgent need to identify novel, safe, and effective treatment modalities for IBD. Research has demonstrated that probiotics offer promising potential in this regard. Probiotics have been shown to eliminate or hinder pathogen colonization within the intestine by directly influencing symbiotic microbiota or producing antibacterial compounds, thereby enhancing the structure and function of the intestinal mucosa and suppressing inflammatory responses.^[24] Additionally, probiotics facilitate mucus production and reinforce the tight connections between intestinal epithelial cells by modulating signaling pathways, such as activating the nuclear factor kappa B and mitogen-activated protein kinase pathways, thereby bolstering the intestinal mucosal barrier function.^[25] Furthermore, probiotics playing a pivotal role in regulating host immune responses, downregulating the expression of inflammatory factors, and upregulating the expression of anti-inflammatory factors, thus preserving local or systemic immune homeostasis (Fig. 1B).^[26] Consequently, supplementing with probiotics to inhibit pathogen colonization and harness their beneficial effects represents an effective strategy for actively managing the balance of intestinal microbiota.

Whether as a pharmaceutical ingredient or an additive in food, probiotics have emerged as a promising avenue for treating IBD. While probiotics can effectively enhance the balance of gut microbiota in IBD patients, thereby mitigating symptoms, their stability during oral administration is inferior to that of other pharmaceutical compounds. They are vulnerable to adverse physiological factors such as gastric acid, digestive enzymes, and bile acids.^[27] Moreover, IBD patients experience immune stress, resulting in reduced probiotic counts and activity as well as the damaged intestinal mucosa impedes probiotics from adhering to its surface, thereby diminishing their efficacy.^[24] In conclusion, ensuring high survival rates and efficient colonization of probiotics in the intestine are crucial prerequisites for them to exert their therapeutic effects in the host.

3. Probiotic microencapsulation is effective in maintaining the biological activity of probiotics

Currently, there are various methods to maintain the biological activity of probiotics during oral delivery and improve their intestinal colonization efficiency, such as engineered probiotics, microencapsulation, and other technologies.^[28,29] Given probiotics’ unique advantages as live drug carriers in the treatment of IBD, researchers have introduced nucleic acid fragments, proteins, or transgenic segments into probiotics to effectively alleviate symptoms of colitis in mice.^[30,31] Furthermore, research by Zhang et al showed that engineered probiotics can effectively reduce the ratio of pro-inflammatory/anti-inflammatory cytokines in the colon tissues and plasma of colitis mice, regulate colonic oxidative stress, modulate the structure of intestinal microbiota, promote intestinal mucosal repair and its metabolites, effectively restore intestinal microbial homeostasis, and alleviate symptoms of IBD.^[32] However, the colonization of engineered probiotics in the intestine is insufficient to maintain long-term efficacy as well as frequent intake is required. Moreover, engineered probiotics are prone to genetic transfer at the gene level, posing a risk of host mutation, indicating obstacles in using engineered probiotics for IBD treatment.^[33,34] Considering the potential threat to human health posed by engineered probiotics in the treatment of IBD, their application is not emphasized in IBD treatment.

Probiotic microencapsulation technology is considered one of the key high-tech research and development areas of the 21st century. It can effectively enhance the resistance of probiotics to adverse gastrointestinal environments, promote their colonization on damaged intestinal mucosa, maintain the biological activity of probiotics effectively, and subsequently promote intestinal mucosal repair (Fig. 2).^[35] Therefore, it has been widely used in the treatment of IBD. Currently, probiotic microencapsulation technologies mainly including compression method, emulsion cross-linking method, spray drying method, and composite gel method.^[36] In addition, the selected wall material for probiotic microencapsulation should have good biocompatibility with probiotics and their metabolites and effectively degrade according to specific physiological and pathological conditions to release the encapsulated probiotics.^[36] The available wall materials can be classified into natural polymer materials, semisynthetic polymer materials, and synthetic polymer materials based on their sources. Among them, natural polymer materials such as gelatin, arabic gum, sodium alginate, chitosan, proteins, and starch are the most commonly used wall materials in probiotic microencapsulation technology due to their stable performance, nontoxicity, biodegradability, good biocompatibility, and excellent film-forming or spherical properties.^[37] Sodium alginate is a natural polysaccharide present in brown algae cell walls, due to their high biocompatibility, easily forms gels with cations, and the formed ion-type colloid is stable at low pH and can dissolve in neutral or slightly alkaline environments, making it widely used as a wall material for probiotic microencapsulation.^[38] However, the gel stability of sodium alginate colloid decreases in the presence of high-affinity ions or high concentrations of nonionic gel inducers. Therefore, depending on the biological characteristics of the encapsulated core material, sodium alginate is often used alone or in combination with chitosan, porous starch, gelatin, or whey protein and other traditional natural polymer wall materials in single or multiple combinations. Nonetheless, the existing research on the functional evaluation of microencapsulated probiotics tends to focus more on “in vitro” research, and a large number of research on the functionality of microencapsulated probiotics remain in the “in vitro” stage, such as simulating gastrointestinal digestion, in vitro antibacterial effects, and simulating adhesion, lacking further validation through in vivo experiments in animals and clinical trials in humans. Therefore, Fang et al successfully prepared alginate/barium sulfate microcapsules with uniform particle size and real-time tracking imaging function for encapsulating Bifidobacterium by combining alginate, sodium sulfate, and Bifidobacterium as the spraying solution, and barium chloride as the receiving solution through electrostatic spraying method, followed by coating the microcapsules with a layer of chitosan outer shell, preparing CA/BaSO₄ microcapsules.^[39] Upon entering the stomach, the electrostatic repulsion prevents hydrogen ions from entering the microcapsule interior, thereby enhancing the biological activity of Bifidobacterium. Upon entering intestinal tissues, CT imaging results showed that CA/BaSO₄ microcapsules can effectively colonize the intestines of IBD. Additionally, fecal microbiota analysis in mice showed that CA/BaSO₄ microcapsules can effectively increase the richness of intestinal microbiota and optimize the composition of intestinal microbiota, promote intestinal mucosal repair, and alleviate symptoms of colitis in mice.

Figure 2.

Representation of probiotic cells without protection and bulk encapsulation of probiotic via the gastrointestinal tract and the importance of probiotic encapsulation.

Although the above methods maintain the biological activity of probiotics to a certain extent during oral delivery, they inevitably suffer from complex operation, low yield, mechanical damage to probiotics during preparation, and increased local oxidative stress in the intestine induced by IBD, making it difficult for probiotics to directly contact the intestinal mucosa, thereby affecting the bioavailability of probiotics. Therefore, there is an urgent need to develop a simple and effective probiotic protection strategy to enhance the resistance of probiotics to gastrointestinal stress environments, prolong the residence time of probiotics in the intestine, and effectively reverse intestinal inflammation. Hence, improving the colonization of probiotics in extreme conditions and ensuring their directly contact with intestinal mucosa are key to ensuring the optimal therapeutic effect of probiotics.

4. Single-cell encapsulation is an effective approach to enhance the biological functionality of probiotics

Inspired by bacteria’s ability to form self-protective membranes in complex environments, generating or covering a protective membrane on the surface of probiotics can significantly enhance their colonization on intestinal mucosa, thereby increasing intestinal mucosal barrier function.^[40,41] In recent years, researchers have explored surface modification of individual probiotics as an alternative strategy.^[42,43] This approach not only enables probiotics to resist adverse physiological conditions and enhances their adhesion to intestinal mucosal epithelial tissues, preventing rapid clearance by intestinal peristalsis, but also imparts probiotics with exogenous characteristics achieved naturally through surface programming, rather than inherent to the probiotics themselves. In contrast to previous encapsulation methods that provided temporary protection, the development of single-cell encapsulation technology allows individual probiotics to be encapsulated in three-dimensional space. This enables selective passage of nutrients, oxygen, and probiotic metabolites through the nano-shell, providing essential nutrients for probiotic growth, maintaining their vitality and functionality both intra- and extracellularly, and facilitating the research of probiotic biological performance at the individual cell level.^[44,45] The most commonly used single-cell encapsulation technique is based on the negatively charged nature of probiotic surfaces. Electrostatic adsorption is utilized to alternately deposit cationic and anionic compounds until the prepared nano-shell achieves the desired thickness, strength, and functionality (Fig. 3A).^[46,47] Although this method effectively enhances probiotics’ resistance to adverse physiological factors, promotes their colonization in the intestine, repairs the intestinal mucosal barrier, restores intestinal microecology, and alleviates IBD, the fragility of probiotic cell membranes presents a challenge when directly exposed to polyoxonium ions, causing membrane perforation.^[48,49] Therefore, the biocompatibility of cationic compounds remains a core issue hindering this assembly method. Additionally, each step of multi-layer nano-shell encapsulation on individual probiotic surfaces requires centrifugation and washing to remove unbound polyelectrolytes, making the process cumbersome and unsuitable for large-scale production. Moreover, the template removal process can compromise the integrity of the nano-shell. Hence, there is an urgent need to develop convenient and efficient probiotic protection strategies to enhance their resistance to harmful or lethal external environments, thereby improving probiotic bioavailability.

Figure 3.

(A) Structures of the phenolic precursors used in this study. (B) Structures of the typical phenolic precursors. (C) Process overview of probiotics-mediated oxidation of dopamine and in situ nanoencapsulation.

Researchers have found that natural biopolymers exhibit superior biocompatibility, effectively overcoming the cytotoxicity of cationic compounds on probiotic biofilms and are considered biologically friendly materials for protecting and maintaining probiotic vitality.^[50] Among these, polyphenolic compounds such as dopamine, caffeic acid, and pyrocatechol, containing pyrogallol and catechol groups in their structures, can be oxidized to quinones under alkaline conditions, undergo a series of cross-coupling reactions and polymerization assembly, and then form phenolic protective nano-shells on individual probiotic surfaces through self-assembly (Fig. 3B and C).^[51,52] Based on the oxidative stress state of IBD intestinal tissues, Li et al utilized dopamine’s anti-inflammatory, antioxidant properties, and its propensity to undergo oxidative polymerization to form polydopamine (PDNI).^[53] They encapsulated Escherichia coli Nissle 1917 (EcN) with PDNI, creating the EcN@PDNI system. After oral delivery, the PDNI shell not only protected EcN from harsh physiological environments to maintain its biological performance but also acted as a dopamine analogue, stimulating regulatory T cells and suppressing initial T cell differentiation into helper T cells to regulate intestinal inflammation, inhibit dendritic cell activation, restore intestinal immune homeostasis, and reverse intestinal inflammation. Furthermore, utilizing polydopamine’s strong adhesive properties on the surface, it effectively adheres to damaged intestinal mucosa, promoting the repair of the intestinal mucosal barrier and restoring normal metabolic pathways of intestinal microecology. Besides, Xie and his coworkers exploited a simple and effective modified prebiotic-based “shield” (Fe–TA@mGN), which was composed of a carboxymethylated β-glucan and a Fe³⁺–tannic acid cross-linking network to arm EcN (EcN@Fe–TA@mGN). After oral administration of EcN@Fe–TA@mGN, Fe–TA@mGN “shield” acts as a dynamic barrier to improve the gastrointestinal stress resistance ability of EcN. Once arrived at intestinal tract, the mGN layer can be degraded by the gut microbiota secreted enzymes to expose the underlying Fe–TA layer, which can aid the colonization of EcN for better combat of IBD.^[2]

In recent years, with the rapid development of single-cell nanocapsulation technology, forming ultra-thin, tough nano-shells on individual probiotic surfaces has evolved from merely restricting external adverse physiological and pathological environmental impacts on probiotic biological activity to providing exogenous catalytic capabilities, enabling probiotics to actively adapt to environmental fluctuations and actively modify their surroundings.^[54,55] Therefore, single-cell encapsulation technology has transitioned from a passive role as an inert physical and chemical barrier to an active participant as an additional component in biological interactions, offering entirely new and even nonbiological functions. This presents enormous prospects for individual probiotics in the treatment of IBD.

5. Discussion

The application of probiotics in the treatment of IBD integrates the basic principles and technologies of immunology, microbiology, materials science, and gastroenterology. It combines probiotic therapy with comprehensive treatment strategies such as dietary management, lifestyle changes, and psychosocial support, providing patients with more comprehensive treatment options to improve treatment outcomes and quality of life. Particularly, with the deepening of microbiome research, comprehensive understanding of the complexity and dynamic changes of individual gut microbiota has been achieved through high-throughput sequencing techniques and metabolomics analysis.^[56,57] This will enable the application of probiotics to more accurately target individual microbiome imbalances, thus achieving personalized medicine.

In addition, in recent years, the development of targeted delivery systems and novel biosynthetic platforms based on probiotic protection strategies, such as engineered probiotics, probiotic encapsulation, and single-cell encapsulation technology, has been remarkable. These advancements not only improve the survival rate and colonization efficiency of probiotics in the intestine, optimize the releasing and action time of probiotics but also impart probiotics with exogenous characteristics achieved naturally through surface programming, thus enhancing the probiotic efficacy. Therefore, the application of probiotics in the field of IBD treatment is in a dynamic stage of research and development. Although probiotics have many remarkable effects, these different effects depend heavily on “strain specificity.” That is to say, although a specific strain of probiotics has been confirmed through extensive and long-term clinical research to possess a certain specific function, it does not mean that different strains of the same probiotic species will necessarily have the same or similar functions.

To further promote the application of probiotics in the treatment of IBD, we need to address the following issues: (1) explore the influence of probiotics on the intestinal microenvironment, particularly how they act by modulating host immune responses, improving intestinal mucosal barrier function, and affecting the composition and function of the intestinal microbiota. (2) In-depth understanding of the specific mechanisms of different types and stages of enteritis, such as acute and chronic enteritis, Crohn disease, and ulcerative colitis, to provide scientific basis for targeted therapy with probiotics. (3) Further research is needed on the purification, characterization, and functional validation of active ingredients in probiotic preparations to improve the specificity and safety of treatment. (4) Analyze the mechanisms of interaction between probiotics and host cells, and how these mechanisms affect the pathogenesis and therapeutic response of enteritis. (5) Establish a more solid evidence base through rigorous clinical trials to validate the therapeutic effects and safety of probiotics for various types of enteritis, including determining the most effective strain combinations, doses, treatment regimens, and applicable patient populations. At the same time, this research should evaluate the long-term effects and potential risks of using probiotics, as well as their synergistic effects with traditional enteritis treatment methods.

In conclusion, the future prospects of probiotics in the treatment of enteritis are broad, but further scientific research and multidisciplinary cooperation are needed to unlock their full potential and ensure their safe and effective application in clinical practice.

6. Conclusion

In conclusion, probiotics, as a beneficial class of microorganisms, playing a significant role in the treatment of IBD. They effectively inhibit the growth of harmful microorganisms, regulate the structure of the intestinal microbiota, and promote mucosal repairing, thereby reducing immune stress and alleviating intestinal inflammation, providing a new perspective for the treatment of IBD. Various probiotic protection strategies have been widely developed and utilized to avoid the influence of adverse physiological and pathological factors on probiotics during oral delivery. Particularly, the development of single-cell encapsulation technology in recent years not only effectively maintaining the biological activity of probiotics during oral delivery and promoting their colonization in the intestine but also endowing probiotics with additional biological functions naturally achieved through surface programming, which has multiple benefits for intestinal health. Although the application of probiotics brings good news to the vast number of IBD patients, their efficacy is considered strain-specific and may vary due to individual differences. Therefore, the use of probiotics should be based on the specific forms and pathways of action of different probiotic strains on IBD, ensuring their safety and effectiveness based on specific clinical evidence, and under the guidance of medical professionals to optimize treatment plans and improve patients’ quality of life.

Author contributions

Conceptualization: Ming Xiong.

Data curation: Ming Xiong.

Formal analysis: Ming Xiong.

Investigation: Ming Xiong.

Methodology: Ming Xiong.

Project administration: Ming Xiong.

Resources: Ming Xiong, Wanlei Sun.

Software: Wanlei Sun.

Supervision: Wanlei Sun.

Validation: Wanlei Sun.

Visualization: Wanlei Sun.

Writing – original draft: Wanlei Sun.

Writing – review & editing: Wanlei Sun.