Anti-Allergic and Anti-Inflammatory Signaling Mechanisms of Natural Compounds/Extracts in In Vitro System of RBL-2H3 Cell: A Systematic Review

Various extracts are tested for anti-allergic or anti-inflammatory properties on in vitro models. RBL-2H3 cells are widely used in allergic or immunological studies. FCεRI and its downstream signaling cascades, such as MAPK, NF-κB, and JAK/STAT signaling pathways, are important allergic or inflammatory signaling mechanisms in mast and basophil cells. This systematic review aims to study common signaling pathways of the anti-allergic or anti-inflammatory compounds on RBL-2H3 cells. We selected the relevant research articles published after 2015 from the PubMed, Scopus, Science Direct and Web of Science databases. The risk of bias of the studies was assessed based on the modified CONSORT checklist for in vitro studies. The cell lines, treatments, assay, primary findings, and signaling pathways on RBL-2H3 cells were extracted to synthesize the results. Thirty-eight articles were included, and FCεRI and its downstream pathways, such as Lyn, Sky, PLCγ, and MAPK, were commonly studied. Moreover, the JAK/STAT pathway was a potential signaling mechanism in RBL-2H3 cells. However, the findings based on RBL-2H3 cells needed to be tested along with human mast cells to confirm its relevance to human health. In conclusion, a single plant extract may act as an anti-inflammatory reagent in RBL-2H3 cells via multiple signaling pathways besides the MAPK signaling pathway.

IL-4 and IL-13 increase pro-inflammatory gene expression in allergic diseases via JAK/STAT signaling pathways [7].These cytokines bind receptors, which lead to receptor dimerization and the recruitment of Janus kinase (JAK).The activated JAKs activate the receptors and recruit STATs to the receptors.The activated STATs dissociate from the receptors as homo or heterodimers and are translocated into the nucleus, bind into DNA, and regulate the gene expression [7,36] (Figure 3).MAPK can activate NF-kB [28].Inactive NF-kB in cytoplasm is found in trimeric form with an NF-kB inhibitor (IkB).Signals from MEKK1 activate the IkB kinase complex (IKK) [37], which further activates IkB (at Ser 32 and Ser 36 residues), releasing NF-kB into the nucleus where it binds to the kB binding site of promotor regions and activates gene expression and mediators-for example, COX-2, TNF-α, IL-1β, -6, and -8 [38][39][40].The activated NF-kB presents in the heterodimer form of p65 and p50 subunits where the activated IkB is ubiquitinated by the 26S proteasome [39].
Mast cells are vital for allergic reaction, as their activation leads to the release of mast cell mediators such as histamine, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2), causing early reaction.Other mast cell mediators such as IL-3, IL-5, IL-8, and tumor-necrosis factor (TNF) recruit eosinophils, neutrophils, and Th2 cells, and they also interact with other tissue cells to start late-phage allergic reaction [41].Since mast cells regulate allergic reactions in a multi-faceted way, anti-allergic drugs to control the allergic reaction due to mast cells could be used at various levels such as (a) targeting mast cell mediators and their receptors, (b) mast cell activators, their receptors, and signal transduction, (c) mast cell inhibitory receptors, and (d) reducing mast cell numbers [42].In other words, mast cells can be targeted with anti-allergic drugs at three levels: the cell membrane (e.g., Omalizumab, which targets free IgE and reduces the IgE attachment to FCεRI), intracellular (e.g., Syk kinase inhibitors, which block IgE-FCεRI-mediated downstream phosphorylation) or extracellular (e.g., H1-4 receptor antagonists which prevent binding and the effect of histamine on target cells) [43,44].However, since mast-cell-derived mediators are also produced by other cells, targeting those mediators would not provide the answer to the absolute necessity of mast cells for the reaction.On the other hand, mast cell activating receptors are unique to those cells, and blocking them, for example by using anti-IgE antibodies omalizumab and ligelizumab, could provide the definite role of mast cells for the reaction [42].These monoclonal antibodies block the attachment of IgE to FCεRI MAPK can activate NF-kB [28].Inactive NF-kB in cytoplasm is found in trimeric form with an NF-kB inhibitor (IkB).Signals from MEKK1 activate the IkB kinase complex (IKK) [37], which further activates IkB (at Ser 32 and Ser 36 residues), releasing NF-kB into the nucleus where it binds to the kB binding site of promotor regions and activates gene expression and mediators-for example, COX-2, TNF-α, IL-1β, -6, and -8 [38][39][40].The activated NF-kB presents in the heterodimer form of p65 and p50 subunits where the activated IkB is ubiquitinated by the 26S proteasome [39].
Mast cells are vital for allergic reaction, as their activation leads to the release of mast cell mediators such as histamine, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2), causing early reaction.Other mast cell mediators such as IL-3, IL-5, IL-8, and tumor-necrosis factor (TNF) recruit eosinophils, neutrophils, and Th2 cells, and they also interact with other tissue cells to start late-phage allergic reaction [41].Since mast cells regulate allergic reactions in a multi-faceted way, anti-allergic drugs to control the allergic reaction due to mast cells could be used at various levels such as (a) targeting mast cell mediators and their receptors, (b) mast cell activators, their receptors, and signal transduction, (c) mast cell inhibitory receptors, and (d) reducing mast cell numbers [42].In other words, mast cells can be targeted with anti-allergic drugs at three levels: the cell membrane (e.g., Omalizumab, which targets free IgE and reduces the IgE attachment to FCεRI), intracellular (e.g., Syk kinase inhibitors, which block IgE-FCεRI-mediated downstream phosphorylation) or extracellular (e.g., H1-4 receptor antagonists which prevent binding and the effect of histamine on target cells) [43,44].However, since mast-cell-derived mediators are also produced by other cells, targeting those mediators would not provide the answer to the absolute necessity of mast cells for the reaction.On the other hand, mast cell activating receptors are unique to those cells, and blocking them, for example by using anti-IgE antibodies omalizumab and ligelizumab, could provide the definite role of mast cells for the reaction [42].These monoclonal antibodies block the attachment of IgE to FCεRI on mast cells and indirectly reduce the FCεRI expression in the cell, resulting in downstream signaling reduction [43].
The rat basophilic leukemia cell (RBL-2H3) line is commonly used for various allergic and immunological studies, specially to study the IgE-FCεRI-mediated signaling mechanism [8,10,12,17,23,45], as it has similar characteristics (such as the presence of histamine, β-hexosaminidase, and the expression of FCεRI) [46] and response behavior as those of mast cells and basophil cells [2,10,21,22,25,47].However, detailed information on RBL-2H3's potential to test multiple signaling pathways/mechanisms of various anti-allergic or anti-inflammatory compounds or extracts on RBL-2H3 cell lines is scarce.Thus, this systematic review aims to study the common signaling pathways of anti-allergic and anti-inflammatory compounds in RBL-2H3 cells.

Materials and Methods
This systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines [48], and the PRISMA flow chart shows the detail of the articles' selection process and search results (Figure 4).Out of the 657 total articles, 18 were not fully retrieved, 345 were published before 2015 and were not original research articles, 195 were not closely related to the topics, and 61 included both in vitro and in vivo systems, leading to the final 38 articles for the review.
on mast cells and indirectly reduce the FCεRI expression in the cell, resulting in downstream signaling reduction [43].
The rat basophilic leukemia cell (RBL-2H3) line is commonly used for various allergic and immunological studies, specially to study the IgE-FCεRI-mediated signaling mechanism [8,10,12,17,23,45], as it has similar characteristics (such as the presence of histamine, β-hexosaminidase, and the expression of FCεRI) [46] and response behavior as those of mast cells and basophil cells [2,10,21,22,25,47].However, detailed information on RBL-2H3's potential to test multiple signaling pathways/mechanisms of various anti-allergic or anti-inflammatory compounds or extracts on RBL-2H3 cell lines is scarce.Thus, this systematic review aims to study the common signaling pathways of anti-allergic and antiinflammatory compounds in RBL-2H3 cells.

Materials and Methods
This systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines [48], and the PRISMA flow chart shows the detail of the articles' selection process and search results (Figure 4).Out of the 657 total articles, 18 were not fully retrieved, 345 were published before 2015 and were not original research articles, 195 were not closely related to the topics, and 61 included both in vitro and in vivo systems, leading to the final 38 articles for the review.We searched literature utilizing four databases: PubMed, Scopus, Science Direct, and Web of Science until 5 March 2024.We used the following keywords, in the same order, in Scopus and Science Direct: ("RBL-2H3") AND (MAPK OR "JAK/STAT" OR "NFkB" OR "FCεRI" OR "signaling pathway") AND ("allergy" OR "peanut allergy") AND We searched literature utilizing four databases: PubMed, Scopus, Science Direct, and Web of Science until 5 March 2024.We used the following keywords, in the same order, in Scopus and Science Direct: ("RBL-2H3") AND (MAPK OR "JAK/STAT" OR "NFkB" OR "FCεRI" OR "signaling pathway") AND ("allergy" OR "peanut allergy") AND (polyphenol).In Web of Science, all keywords mentioned above were identical except that "Polyphenol" was omitted.For PubMed, we used following keywords: (("RBL-2H3") AND (MAPK OR "JAK/STAT" OR "NFkB" OR "FCεRI" OR "signaling pathway") AND ("allergy" OR "peanut allergy")) and the following Medical Subject Headings (MeSH) separately: ((((rbl-2h3) OR ("Mast Cells" [Majr])) AND ("Signal Transduction" [Majr])) AND ("Hypersensitivity" [Majr])).Two authors (T.S.R. and R.B.) screened the articles, and any disagreements were solved through discussion and consultation with a third author (L.L.W.).
The PICO framework was used to determine the evidence in the articles as follows: population: RBL-2H3 cells only (or along with other cell lines); intervention: natural compounds/extracts; comparison: extract/compound-treated cells versus untreated cells; outcome: signaling pathways, cell degranulation, and inflammatory cytokines.
The articles were deduplicated by uploading them in Excel sheets followed by the title and abstract screening for inclusion/exclusion criteria and were further evaluated on full text for their eligibility.Two reviewers (T.S.R. and R.R.B.) independently screened the title and abstract for the inclusion.The original research articles using an in vitro system, published in the English language in peer-reviewed journals from 2015 to 2024 (5 March), were included for the review.Review articles, proceedings, or articles published in languages other than English, articles published before 2015, articles not closely related to the topic (i.e., those that did not include RBL-2H3 cells, pro-inflammatory cytokines, or signaling pathways/molecules), and articles that included both in vitro and in vivo systems were not selected in this review.We excluded studies that used both in vitro and in vivo systems to focus our review on an in vitro system using RBL-2H3 and other cells in order to avoid variability and complexity in our study.Two authors (T.S.R. and R.R.B.) agreed upon the selection criteria and on selected articles.
The following items were manually and independently extracted and agreed by two authors (T.S.R. and R.R.B.) from the selected articles: publication year, objectives, cell lines used, dose and duration of the treatment/control, sequence of sensitization, treatment and stimulation, assay/techniques used, primary findings, and treatment effect on signaling pathways/molecules on RBL-2H3 cells.When information on signaling pathways were not provided in the included articles, cell degranulation signature molecules such as histamine and β-hexosaminidase and cytokines expression data were obtained.Moreover, the compounds' extraction methods were not included from the selected articles.
The risk of bias of selected articles was independently assessed and agreed by two authors (T.S.R. and R.R.B.) using a modified CONSORT checklist for in vitro study [49,50].The comprehensiveness and unbiased reporting on the following items of the articles were assessed: abstract, background and rationale, objectives, hypothesis, intervention, outcome, statistical method, outcome estimation, limitations, and funding.As the articles were diverse in terms of treatment used, objectives, and design, we used a narrative synthesis approach to synthesize the articles.The information were tabulated according to cell line used, i.e., either only RBL-2H3 or RBL-2H3 along with other cell lines.The synthesis steps were conducted by two authors (T.S.R. and R.R.B.) and agreed among all three authors (T.S.R., R.R.B., and L.L.W.).

Study Characteristics, Risk of Bias, and Reporting Quality
The selected studies varied in their objectives and overall experimentation.Out of the total of 38 chosen studies, 26 studies used only RBL-2H3 cells and 12 studies used RBL-2H3 along with other cells such as RAW264.7 (5 studies), HaCaT (2 studies), HiMC (1 study), BMMC (1 study), Caco2 (1 study), MoLT-4 (1 study), HMC-1 (1 study), KU812 (1 study), human neutrophils (2 studies), human basophils (1 study), and bacteria (Klebestella pneumonia) (2 studies) (Tables 1 and 2).According to the CONSORT checklist used [49,50], although most of the studies did not mention the hypothesis and limitation of their studies clearly, the majority of them clearly explained the experimental details so that the experiment could be replicated and also reported complete results showing quality results with low risk of bias (Table 3 and Figure 5).
For MOLT-4: C1   of their studies clearly, the majority of them clearly explained the experimental details so that the experiment could be replicated and also reported complete results showing quality results with low risk of bias (Table 3 and Figure 5).

Signaling Pathways
Data extracted from the included studies are summarized in Tables 1 and 2. Studies had diverse compounds tested for their anti-allergic or anti-inflammatory properties and possible signaling mechanisms.For example, studies including only RBL-2H3 had extracts from plants, shrimps, fungi, sponges, and chemicals (sodium sulfite) (Table 1).However, the majority of the studies were related to plant extracts.Similarly, studies those used multiple types of cells also had all plant extracts except one study with enzymatically synthesized glycogen (Table 2).Some studies did not explore the signaling mechanisms of the tested compounds in depth.In contrast, most of the studies had shown that the anti-allergic or anti-inflammatory activities of tested compounds on RBL-2H3 were via the regulation of MAPK, FCεRI, NF-kB signaling pathways, or their upstream or downstream regulators such as Syk, Lyn, Akt, PLCγ, PI3K, PKC, and LAT (Tables 1 and 2).In addition to FCεRI and its downstream signaling, some studies have also shown that RBL-2H3 cells demonstrate JAK/STAT and NLRP3 signaling pathways when they were tested for antiallergic mechanisms of fungus extract and sodium sulfite, respectively (Table 1).Moreover, the studies had shown that a single compound can have multiple signaling pathways (Tables 1 and 2).

Discussion
This review has the following limitations.(i) We used peer-reviewed articles published in the English language, which may have excluded important results published in other languages and in preliminary result reports.(ii) We only included in vitro studies and articles published after 2015.(iii) We did not manually search and include the literature from the included articles' reference lists.(iv) There are no standard methods on quality evaluation of the in vitro studies, so some modified methods recommended for the clinical setting were used.These shortcomings prevent the comparison of in vivo and in vitro results and the relevance of in vitro studies' findings for further clinical trials.Moreover, exclusion criteria may lead to missing valuable sources regarding the use of RBl-2H3 for novel signaling mechanisms for allergic or immunological research.Furthermore, the lack of a standard protocol for in vitro study quality may also result in variations in the articles' quality.However, this review provides a trend on the use of RBL-2H3 cells and their potential signaling mechanisms for allergic and inflammatory studies over a decade.
The studies had diverse compounds tested, such as a variety of extracts from plants (on the majority of included articles), fungi [9,21], shrimp [56], sponges [20], agro-industrial waste [6,53,54], and chemical compounds [55].Thus, there is also a need for multiple studies using the same or similar compounds, which may be met by broadening the literature inclusion criteria.Moreover, the included studies primarily studied MAPK signaling pathways, and other critical pathways such as JAK/STAT pathways were barely reported.This might be related to the fact that RBL-2H3 cells are not exactly the same as mast cells or basophil cells.For example, studies have reported that RBL-2H3 cells lack some properties of mast cells, such as the absence of essential elements such as CD14 and MyD88.However, they have Toll-like receptor 4 (TLR4) on their surface [64], which might have limited the cells from being used in TLR-related signaling pathways such as JAK/STAT pathways.However, some studies suggested that the presence of CD14 does not guarantee that the TLR-acting stimulus, such as LPS, directly activates the cells [65].However, some of the included studies had reported STAT6 signaling pathways [9] and NLRP3 and caspase pathways [55] in RBL-2H3 cells, indicating their potential use for diverse signaling mechanisms.Moreover, studies related to new applications of tested compounds/extracts, such as using them as a nutraceutical or in industry-standard applications, still need to be included.
From this study, it is also evident that the dose and duration of the tested compound can impact the level of expression or presence and absence of the tested signaling pathways.The literature also reported the variations on the RBL-2H3 phenotype based on various factors such as cultural condition [47,64], age, and biological sex from which the cell line was isolated [46].Thus, it is important to provide details of the used cell's properties (e.g., cell passage, catalog number) in the studies for their reproducibility.
Despite the mast cells and basophil cells being similar to RBL-2H3 in many properties, we excluded them in this review because RBL-2H3 cells still differ from those cells in various characteristics such as the amount of histamine in the cells, the absence of CD14, TLR-2, the presence of c-kit, which is a receptor for stem cell factor (SCF) [65], the absence of CD123, which is a signature of primary basophil, and not expressing rat tryptase genes and release tryptase, which are found in primary mast cells [46].
It is also essential to critically consider the relevance or implication of RBL-2H3 studies' results to human mast cells.There are various limitations on the use of human mast cells or basophil cells for studies.For example, the isolation and purification of human mast cells and basophil cells is expensive, tedious, impractical, and results in issues in the isolated cells' purity and viability [41,66].Moreover, various human mast cell lines have been developed but suffered from vital issues over time.For instance, HMC-1 and HMC-α failed to degranulate following immunological activation; LAD-2 has a high cost and slow growth rate, so it is not the first choice for routine experimental studies; ROSA KIT D816V does not have histamine and β-hexosaminidase expression; and ROS KIT WT needs stem cell factor (SCF) to survive, which is very expensive and not affordable for routine studies [41,66].Due to these limitations for the use of actual human mast cells, the use of RBL-2H3 cells becomes prominent, as these cells are easy to culture, have homogeneity with a rapid growth rate, have functional FCεRI, and have similar degranulation dynamics to human mast cell and basophil cells [41,66].However, despite some similarities between RBL-2H3 cells and the mast cell (e.g., both have c-kit receptors for SCF), there are differences, too.For example, unlike in mast cells, the TLR4 or its co-receptor CD14 and TLR2 are not expressed in RBL-2H3 [47,65].Moreover, since RBL-2H3 cells are immortalized cells, their physiology might not be the same as that of primary mast cell physiology, and RBL-2H3 cells also show intra-laboratory reproducibility issues.Thus, the results obtained with these cells should be validated along with the human mast cell lines [41,46,47,66].
In the included studies, other types of cells (not RBL-2H3) were used to study the antioxidant properties, pro-inflammatory cytokines, or gene-level expression of some allergic markers.Moreover, the crude extract versus purified compound may have affected the expression of signaling pathways on RBL-2H3 cells.The variation in the dose and time of the tested compounds and sensitizing/stimulating agents such as anti-DNP-IgE (33.3 ng/mL for 2 h-1 µg/mL for overnight), ionomycin (1 µM for 30 min-250 µM for 30 min), DNP-BSA (50 ng/mL for 1 h-10 µg/mL for 30 min), calcium ionophore A23187

Figure 4 .
Figure 4. PRISMA flow diagram showing literature search and selection process.

Figure 4 .
Figure 4. PRISMA flow diagram showing literature search and selection process.

Figure 5 .
Figure 5. Percentages of the reviewed articles that met the modified CONSORT checklist.Figure 5. Percentages of the reviewed articles that met the modified CONSORT checklist.

Figure 5 .
Figure 5. Percentages of the reviewed articles that met the modified CONSORT checklist.Figure 5. Percentages of the reviewed articles that met the modified CONSORT checklist.

Table 1 .
Anti-allergic and anti-inflammatory properties and signaling pathways of various compounds using only RBL-2H3 cells.

Table 2 .
Anti-allergic and anti-inflammatory properties and signaling pathways of various compounds using RBL-2H3 cell and other cell lines.