Revealing the anti-inflammatory activity of Euphorbia hirta extract: transcriptomic and nitric oxide production analysis in LPS-Induced RAW 264.7 cells

ABSTRACT

Excessive production of nitric oxide (NO) dan pro-inflammatory cytokines can lead to tissue damage and chronic inflammation. Euphorbia hirta presents an interesting opportunity for plant-derived anti-inflammatory agents as it contains a range of compounds which have demonstrated anti-inflammatory effects. This study investigates the anti-inflammatory properties of E. hirta extract on gene expression and NO production in LPS-treated RAW 264.7 cells. The results demonstrate that E. hirta extracts effectively suppress NO production in RAW 264.7 cells. Furthermore, the extract inhibits iNOS protein expression and reduces the expression of pro-inflammatory cytokines TNF-α and IL−12. RNA sequencing revealed significant alterations in gene expression, including 167 upregulated and 56 downregulated genes-related inflammation pathways. The study concludes that E. hirta extract possesses anti-inflammatory activity by modulating gene expression and inhibiting NO production in LPS-treated RAW 264.7 cells. The extract influences genes associated with inflammation signalling pathways, including cytokine signalling, NF-κB, iNOS and JAK-STAT pathways.

KEYWORDS:

• Anti-inflammatory
• Euphorbia hirta
• nitric oxide
• RAW 264.7

Introduction

Inflammation is a fundamental component of the body's immune response but dysregulated can lead to various chronic diseases. Among many immunocytes, the macrophage is vital in bridging innate and adaptive immunity (Paul, 2011). How macrophages are activated also determines the downstream mechanism in adaptive immune response (Arnold et al., 2015). This event is associated with macrophages’ Antigen-Precenting Cells (APCs) capability. Macrophage secretes nitric oxide (NO) in response to pathogen infection (Palmieri et al., 2020). It is produced by the inducible Nitric Oxide Synthase (iNOS) in response to immunogenic substances (Taechowisan et al., 2010). However, excessive production of NO can lead to tissue damage and chronic inflammation (Laskin et al., 2011). Chronic inflammation can lead to chronic inflammatory diseases, which are significant causes of disability and mortality worldwide. Chronic inflammatory diseases, such as cardiovascular disease, cancer, diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, autoimmune disorders, and neurodegenerative conditions (Pahwa et al., 2023). Beyond NO production, macrophage also produces cytokines to orchestrate inflammation. Most of the cytokines produced by macrophages are inflammatory cytokines (Sharma et al., 2014). Thus, maintaining NO and cytokines levels in an adequate state becomes critical to modulate the inflammation for protective activity against chronic inflammation-related diseases.

Plants have been explored extensively to find natural-derived anti-inflammatory agents, particularly those regulating macrophages or their associated molecules. Plants have been the first source of remedies in the history of humanity, and herbal bioactive compounds have fueled drug development. Among the potential plant species, Euphorbia hirta (EH) becomes one of the promising anti-inflammatory agents. This plant has been traditionally used to treat various conditions, including gastrointestinal disorders, respiratory ailments like asthma, and skin inflammatory diseases such as atopic dermatitis (Sharma et al., 2014). EH extract also exhibits significant immunosuppressive effects on T lymphocytes and Th1 cytokines, suggesting its potential as a potent and safe immunosuppressant for further exploration in drug development (Ahmad et al., 2013b). EH contains several bioactive compounds, ranging from phenolic compounds as the major constituents and several other classes of bioactive like organic acids, terpenes, alkaloids, anthraquinone, isoprenoids, and amino acids (Mekam et al., 2019). Previous research demonstrated that the methanol extract of EH aerial parts which is rich in two flavonoids (Quercetrin, Rhamnetin) and two phenolic acids (Ferulic acid, Gallic acid) exhibited notable inhibition of the COX-2 enzyme in vitro and proved to be an effective anti-inflammatory agent in acute and sub-acute inflammation models in rats (Subbiah, 2007). Furthermore, the ethanol extract of EH possesses anti-inflammatory properties primarily due to its terpenoid constituent, β-amyrin. β-amyrin and other terpenoids like 24-methyl encycloartenol and β-sitosterol, isolated from the n-hexane extract of EH aerial parts, demonstrated significant inhibition of TPA-induced inflammation in mice ears (Meda et al., 2023). Additionally, β-amyrin from EH effectively hindered the functions of the iNOS protein and the production of nitric NO in LPS-induced RAW 264.7 cells (Shih et al., 2010). Thus, EH becomes a promising candidate for exploring natural-derived anti-inflammatory agents.

Although much research has been conducted to elucidate the anti-inflammatory activity of EH, the mechanism associated with macrophage activity remains limited and inconclusive. Previous research demonstrated that EH exhibits anti-inflammatory by down regulating iNOS protein with less effect on the Prostaglandin E2 (PGE2) level (Shih et al., 2010) and TNF-α (Ahmad et al., 2013a). Confusingly, other studies reported the inhibitory activity of EH in suppressing PEG2 levels (Upadhyay et al., 2014). However, both studies described the same inhibitory activity of EH against Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin (IL)−6 (Shih et al., 2010; Upadhyay et al., 2014). Fortunately, with the current advancement in Nanopore sequencing technology, whole RNA sequencing could be performed to elucidate a particular mechanism at the transcription level efficiently (Jain et al., 2022). For instance, this approach identifies Caesalpinia sappan's anti-cancer mechanism through direct RNA-sequencing analysis (Widodo et al., 2022). Therefore, this study will discover the potential mechanism of the anti-inflammatory activity of EH through modulation of macrophage activity in vitro by employing direct RNA-sequencing analysis.

Materials and method

E. hirta extract preparation

Dry powder of EH was purchased from UPT Laboratorium Herbal Materia Medica Batu, Indonesia with voucher specimen number 200506.PTK.R.001. The microwave-assisted extraction method was used for extraction by dissolving the powder in ethanol in a 1:10 ratio (herb: solvent; w/v) using a holding temperature of 50°C, warming at 50°C for 5 mins, holding time of 10 mins, cool down of 5 mins, and power of 1500 W. The extract was filtered using filter paper and then evaporated using a rotary vacuum evaporator (Buchi: Buchi R-210 Rotavapor System). The extract was freeze-dried until a dried extract was obtained.

RAW 264.7 Cell Culture

Mouse macrophage cell line RAW 264.7 was obtained from Elabscience® (Catalog No.: CL-0190, Elabscience Biotechnology Inc., USA). This cell was set as the inflammation cell model because RAW 264.7 cells exhibit characteristics similar to primary macrophages. Additionally, RAW 264.7 cells can be polarized into either pro-inflammatory M1 or anti-inflammatory M2-type macrophages, making them a suitable model for studying immune responses and inflammation (Taciak et al., 2018). The RAW 264.7 cell was cultured in Dulbecco’s Modified Eagle’s Medium  – high glucose (DMEM-HG) (Sigma-Aldrich, Co., Merck KGaA) containing 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Co., Merck KGaA) and 1% antibiotics (100U/ml penicillin and 100U/ml streptomycin) (Sigma-Aldrich, Co., Merck KGaA) in a 5% CO₂ incubator 37°C.

Determination of nitric oxide level

RAW 264.7 cells were seeded on a 24-well plate with a density of 1 × 10⁵ cells/well and incubated in a 5% CO2 incubator, 37°C for 24 hours to obtain a stable condition. The control group was only treated with DMEM maintenance medium. To generate the inflammation cell model in RAW 264.7 cells, lipopolysaccharide (LPS) was used (Taechowisan et al., 2009; Taechowisan et al., 2010). LPS group was treated with 1 µg/mL LPS (Sigma-Aldrich, Co., Merck KGaA). In LPS + EH group, DMEM maintenance medium containing EH (25, 50, 100 μg/ml) and LPS (1 µg/mL) were added at the same time. Cells were then incubated in the cell CO₂ incubator for 24 hours. After 24 hours, the concentration of nitrite in the culture medium was measured as an indicator of NO production. 75 µL of the cell culture supernatant was reacted with 75 µL of Griess reagent modified (Sigma-Aldrich, Co., Merck KGaA) containing 1:1 mixture of 0.1% N-(1-naphthyl) ethylene-diamine dihydrochloride in water and 1% sulphanylamide in 5% phosphoric acid, in a 96-well plate. The absorbance was then measured by microplate reader at 571 nm. The concentration of nitrite in the sample was determined from the standard curve of sodium nitrite (NaNO₂). Experiments were carried out in triplicate and repeated five times with different cell passage numbers.

Cell viability assay

The cell culture medium, which had been used for the NO assay in the previous method, was discarded and replaced with 120 µL of media containing 7.5 µL of WST-1 reagent (Roche Diagnostics GmbH, Germany) in each well. Then it was incubated for 30 minutes in a 5% CO₂ incubator, 37°C. After 30 minutes, 100 µL of media was transferred to a 96-well plate and then read by microplate reader at 450 nm.

Flowcytometry Analysis for iNOS, TNF-α, and IL-12 protein

RAW 264.7 cells were seeded on a 24-well plate with a density of 1 × 10⁵ cells/well and incubated in a 5% CO2 incubator, 37°C for 24 hours to obtain a stable condition. The control group was only treated with DMEM maintenance medium. LPS group was treated with 1 µg/mL LPS. In LPS + EH group, DMEM maintenance medium containing IC₅₀ dose of EH extract and LPS (4 µg/mL) were added at the same time. Cells were then incubated in the cell CO₂ incubator for 24 hours. After incubation for 24 h, the treated RAW 264.7 cells were harvested and dyed using iNOS (inducible Nitric Oxide Synthase), TNF-α, IL-12 antibodies for 20 minutes. The cells were then analyzed using the flow cytometer (BD FACS Calibur™, San Jose, CA). FACS data were analyzed using FlowJo™ v. 10 Software (Vancouver, BC). The gating was designed to identify and characterize specific cell populations within the sample, while excluding debris and nonspecific events.

Rna sequencing analysis

Cells were seeded in a 100 mm dish at a density of 2.2 × 10⁶ cells and incubated overnight. Next, they were treated by 1 µg/mL LPS with or without 53.40 µg/ml (IC₅₀) EH leaves extract for 24 hours. Three replicates were done for each treatment. Total RNA was extracted using the Quick RNA MiniPrep Kit. RNA quality and quantity were assessed using agarose gels, Bioanalyzer 2100, and NanoPhotometer® spectrophotometer.

For RNA sample preparation, 1 µg of total RNA was used. Sequencing libraries were created with the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the provided guidelines. mRNA was isolated from total RNA using poly-T oligo-linked magnetic beads. Fragmentation was achieved using divalent cations at high temperatures in NEBNext First Strand Synthesis Reaction Buffer. First-strand cDNA synthesis used a random hexamer primer and M-MuLV Reverse Transcriptase. The second strand of cDNA was produced using DNA Polymerase I and RNase H. Blunt ends were created from remaining overhangs with exonuclease/polymerase activities. NEBNext Adapter with a hairpin loop structure was ligated to DNA fragment ends for hybridization. Library fragments were purified using the AMPure XP system to select cDNA fragments of 150∼200 bp length. USER enzyme was utilized for further processing before Polymerase Chain Reaction (PCR). PCR was performed using Phusion High-Fidelity DNA polymerase with universal PCR primers and Index (X) Primer. The PCR product was purified using the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system.

The qualities of cleaned reads were assessed using FastQC version 0.11.9 (https://github.com/s-andrews/FastQC) and the reports were compiled using MultiQC version 1.1 (https://multiqc.info). The transcript was quantified using the pseudo-alignment method employing kallsito version 0.461 with cDNA sequence downloaded from Ensembl (GRCm39, GCA_000001635.9: contigs; annotation). Differential expression analysis was performed using EdgeR version 3.34.0. GO enrichment was performed using Blast2GO (https://www.blast2go.com/). The data were visualized using ClustVis website 2.0 (https://biit.cs.ut.ee/clustvis).

Statistical analysis

The data NO assay and flowcytometri are representative of five independent experiments from different passage of cell line that yielded similar results. The values are presented as the means ± standard deviation (SD). The differences were analyzed using Student’s t-test. The statistical significance was set at p < 0.05 and p < 0.01.

Result

E. hirta extracts suppress NO production in RAW 264.7 without cytotoxicity

We examined the effect of E. hirta (EH) leaves extract on NO production in LPS-induced RAW 264.7 cell. This study demonstrated that EH dose-dependently suppressed the levels of NO production in LPS-induced RAW 264.7 cell (Figure 1(A)). All dose of EH showed great NO inhibitory activity in dose depend manner (Table 1). Furthermore, the half-maximal inhibitory concentrations (IC₅₀) were determined to know the potency of EH in inhibiting NO production. EH demonstrated NO suppression activity with IC₅₀ values of 53.40 ± 9.48 μg/mL (Table 1).

Figure 1. E. hirta leaves extract suppress nitric oxide (NO) production in LPS-induced RAW 264.7. A. The effects of E. hirta leaves extract on the induction of NO production. B. The cell viability of (A) assesed by WST assay. The RAW 264.7 cells were treated with LPS and/or extracts for 24 h. The NO levels in the medium and WST-1 were measured in triplicate (n = 3) and are shown as a means ± standard deviation (SD). * P < 0.05 and ** P < 0.01 versus LPS alone.

Table 1. The potency of E. hirta leaves extract in inhibiting NO.

Euphorbia hirta	NO Production (µM)	NO Inhibitory Activity (%)	IC50 Value (µg/mL)
0 µg/mL	11.70 ± 0.35	0.00 ± 3.02
25 µg/mL	9.73 ± 0.42**	16.86 ± 3.63**	53.40 ± 9.48
50 µg/mL	5.24 ± 0.12**	55.23 ± 1.01**
100 µg/mL	1.29 ± 0.20**	88.95 ± 1.74**

Note: The RAW 264.7 cells were treated with LPS and/or extracts for 24 h. The NO levels in the medium were measured in triplicate by Griess method. NO inhibitory activity was measured in (%) based on NO level in medium. The half-maximal inhibitory concentration (IC₅₀ value) of NO production in LPS-induced RAW 264.7 cells, and at least three experiments were performed. Data are shown as a means ± SD. ** P < 0.01 versus LPS alone.

To monitor cytotoxicity, the viability of cell by WST-1 assay. As shown in Figure 1(B), the range dose of EH used in this study did not showing any toxicity. These results suggest EH was not toxic to the RAW 264.7 cell lines at the concentration indicated.

E. hirta extract inhibits iNOS protein expression

The production of NO in the cell is catalyzed by inducible nitric oxide synthase (iNOS), which can be induced in several cell types (Jain et al., 2022; Taechowisan et al., 2010), including macrophages. Thus, we investigated the effects of EH leaves extract on iNOS protein expression. As shown in Figures 2 and 3(A), flowcytometri analysis indicated that the IC₅₀ dose of EH decreased the iNOS protein expression in LPS-induced RAW 264.7. These data suggest that EH leaves extract suppress NO production through inhibition of iNOS protein expression.

Figure 2. The effects of EH leaves extract on the induction of iNOS protein and pro-inflammatory cytokines. The RAW 264.7 cells were treated with LPS and/or extracts for 24 h. The iNOS protein level was measured by flowcytometri and then the histogram image was visualized with FlowJo software. The red line separates cells that positively express the target protein. Peaks to the left of the red line are cells that do not express the target protein, while those to the right of the red line are peaks of cells that positively express the target protein.

Figure 3. The extract of EH suppress the expression level of iNOS protein (A) and pro-inflammatory cytokines TNF-α and IL-12 (B). The RAW 264.7 cells were treated with LPS and/or extracts for 24 h. The pro-inflammatory cytokines TNF-α and IL-12 level were measured by flow cytometri in quadruplicate (n = 4). Data are shown as a means ± standard deviation (SD). ^# P < 0.05 and ^## P < 0.01 versus control; * P < 0.05 and ** P < 0.01 versus LPS alone.

Suppression of the expression of pro-inflammatory cytokines TNF-α and IL-12 by E. hirta leaves extract

During inflammation, apart from nitric oxide, many pro-inflammatory cytokines are also secreted. The presence of these pro-inflammatory cytokines can worsen inflammatory conditions. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-12 play key roles in inflammation. The levels of these pro-inflammatory cytokine levels were induced by LPS stimulation of RAW 264.7. Therefore, we examined whether EH leaves extract affects these protein levels in RAW 264.7 cell line induced by LPS. When the IC₅₀ dose of EH leaves extract was added to the medium, the protein expression levels of the pro-inflammatory cytokines TNF-α and IL-12 significantly decreased (Figures 2 and 3(B)). These results indicate that EH leaves extract suppresses not only NO but also the pro-inflammatory cytokine expression.

Gene expression profile of RAW cell treated by E. hirta extract

The gene expression patterns in cells subjected to both the control and IC₅₀ treatment were analyzed and presented visually using a heatmap. The heatmap used higher Transcript per million (TPM) values to represent elevated expression and lower values to represent decreased expression. Notable distinctions in gene expression were observed between the control and IC₅₀ treatments, as evident in Figure 4(A). Moreover, alterations in gene expression within RAW cells following treatment with EH leaves extract were depicted through a volcano plot. This plot was constructed based on Log2FoldChange and p-value, with defined criteria for significant differences set at Log2FoldChange < 1/−1 and p-value < 0.05. The volcano plot displayed 167 upregulated genes and 56 downregulated genes in RAW cells treated with EH leaves extract, as illustrated in Figure 4(B).

Figure 4. Gene expression profile of RAW cells due to administration of IC₅₀ dose of extract. (A) Heatmap, (B) volcano plot

RNA-seq results showed that 16 genes related to inflammation experienced significant changes in expression due to the administration of E. hirta extract. These genes are IL11RA, NOSIP, ILF3, IL12RB1, TNFRSF9, TNFRSF1A, IL36A, IL36G, IL6, IL6R, TNFAIP2, TNFAIP3, AKT1S1, NFKB1, IL7R, and STAT1 (Figure 5(B)). From these 16 genes, a PPI network was then compiled to analyze the interactions between them and other proteins related to inflammation. The PPI network shows that these 16 genes interact with other inflammation-related proteins such as IL1, TNF, STATs, JAKs, IL7, and so on (Figure 5(A)). Based on functional annotation, the genes are mainly involved in inflammatory mechanisms. There are 30 genes that play a role in the cytokine-mediated signaling pathway and 28 genes related to the inflammatory response (Figure 5(C)). These genes are predominantly proteins located in the cytosol, the majority being of the protein binding type (Figure 5(D&E)). According to the KEGG pathway, these genes have a close relationship with signaling pathways related to inflammation, such as Th17 cell differentiation, JAK-STAT signaling pathway, Cytokine-cytokine receptor interaction, TNF signaling pathway, and so on (Figure 5(F)).

Figure 5. PPI network based on proteins that experience changes in expression due to extract administration and functional annotation analysis. (A) PPI networks, red color indicates the proteins that experienced changes in expression due to the administration of EH extract. (B) Changes in the expression of inflammation-related proteins. (C-F) Functional annotation based on PPI network.

Discussion

This research explores the anti-inflammatory effects of EH extract on gene expression and NO production within LPS-induced RAW 264.7 cells. EH leaves extract inhibited the NO production and less toxic to RAW 264.7 cell line. The extract also decreased the expression of iNOS, TNF-a, and IL-12 based on flowcytometry analysis. Interestingly, this study revealed substantial changes in gene expression, with 167 genes upregulated and 56 genes downregulated within the inflammation pathway through the RNA sequencing analysis. Notably, 16 inflammation-related genes, including IL11RA (Interleukin 11 receptor alpha subunit), NOSIP (Nitric Oxide Synthase Interacting Protein), ILF3 (interleukin enhancer binding factor 3), IL12RB1 (interleukin 12 receptor subunit beta 1), TNFRSF9 (TNF Receptor Superfamily Member 9), TNFRSF1A (TNF Receptor Superfamily Member 1A), IL36A (interleukin 36 alpha), IL36G (Interleukin 36 Gamma), IL6 (interleukin 6), IL6R (interleukin 6 receptor), TNFAIP2 (TNFα-induced protein 2), TNFAIP3 (TNFα-induced protein 3), AKT1S1 (AKT1 Substrate 1), NFKB1 (Nuclear Factor Kappa B Subunit 1), IL7R (interleukin 7 receptor), and STAT1 (Signal transducer and activator of transcription 1), experienced significant expression changes upon treatment with EH extract. Previous study notes that ethanolic extract of EH decreases the NO production of RAW 264.7 cell induced by LPS and demonstrate low toxicity (Upadhyay et al., 2014). Another study showed that this plant had an anti-inflammatory effect on Sprague–Dawley neonatal rats with asthma by reducing the expression of pro-inflammatory proteins such as TNF  – α (tumor necrosis factor alpha), IL-6 (interleukin 6), iNOS (inducible Nitric Oxide Synthase), and COX-2 (Cyclooxygenase-2) (Xia et al., 2018). EH extract also showed significant inhibition of IL-1β (interleukin 1 beta), TNF-α, IL-2 and IFN-γ (interferon gamma) cytokines in rat (Ahmad et al., 2013b) and mice (Ahmad et al., 2014) adjuvant-induced arthritis. The most current study reported that leaf ethanol extract of EH has anti-inflammatory activity through inhibiting albumin, proteinase and lipoxygenase activities (Das et al., 2022).

Nitric oxide synthases (NOSs) constitute a distinct group of enzymes that utilize oxygen and nitrogen, derived from the amino acid Arginine, to catalyze the generation of Nitric Oxide (NO). NO is characterized as a free radical possessing an unpaired electron and exhibiting signaling properties. NO accumulation causes chronic inflammation (Anavi & Tirosh, 2020). TNF-α is a cytokine generated in response to inflammation and plays a significant role in stimulating acute-phase proteins and various indicators of persistent inflammation (Chen et al., 2010; Francés et al., 2013). TNF-α can activate MAP kinase and NF-κB which are important proteins in cell inflammatory responses (Sabio & Davis, 2014). IL-12 has a role in activating various signaling pathways related to inflammatory responses. IL-12 activates non-receptor Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2), resulting in the phosphorylation of members of the signal transducer and activator of transcription (STAT) family – specifically, STAT1, STAT3, STAT5, and, notably, STAT4 homodimers (Teng et al., 2015). Inhibition of iNOS, TNF-α, and IL-12 expression by EH will be very effective in suppressing inflammation. However, EH may also inhibit the expression of other genes related to inflammation. Therefore, RNA-sequencing was carried out to analyze the effect of EH leaves extract on the expression profile of inflammation-related genes in RAW 264.7 cells.

Based on the RNA-sequencing results, there were changes in the expression of genes related to inflammation. Most of the genes affected by administration of the extract are pro-inflammatory cytokines and their receptors. Several cytokines such as ILF3, IL36A, IL36G, IL6, TNFAIP2, and TNFAIP3 experienced a significant decrease in expression. Interleukin enhancer binding factor 3 (ILF3), a RNA-binding protein, is most recognized for its involvement in the innate immune response. Previous study notes that ILF3 have a role in induction of inflammation injury in septic H9C2 (Yao et al., 2020). IL36A and IL36G are pro-inflammatory proteins that bind to the heterodimeric receptor composed of IL-36R and IL-1R accessory protein (IL-1RAcP) which activates inflammation-related signaling pathways (Sachen et al., 2022). TNFAIP2 and TPFAIP3 are markedly expressed in immune cells, and their expression is regulated by multiple transcription factors and signaling pathways, including NF-κB, KLF5, and retinoic acid. Physiologically, TNFAIP2 and TNFAIP3 serves as a significant mediator of inflammation (He et al., 2018; Jia et al., 2018). Interleukin 6 (IL6) is a proinflammatory cytokine. IL-6 will bind to IL-6R and will activate the JAK-STAT signaling pathway which results in the transcription of pro-inflammatory proteins (Tanaka et al., 2014).

Cytokine receptors such as IL11RA, IL12RB1, TNFRSF9, TNFRSF1A, and IL7R experienced changes in expression due to administration of EH extract. Interleukin-11 (IL11), belonging to the IL6 cytokine family, is associated with various fibro-inflammatory conditions. IL11 interacts with its specific alpha receptor, IL11RA, and initiates signaling through the gp130 receptor, thereby activating the ERK and STAT3 pathway (Ng et al., 2022). The IL12RB1 receptor affects various aspects of human immunity, impacting T and NK cell differentiation, T cell responses, B cell Ig production, and dendritic cell activity, through interaction with IL-12 and IL-23 cytokines (Ford et al., 2012). TNFRSF9 and TNFRSF1 serve as receptors for tumor necrosis factor-alpha (TNF-alpha), also referred to as cachectin. This potent pro-inflammatory cytokine, TNF-alpha, holds significant importance in the immune system's response to inflammation (Zelová & Hošek, 2013). IL7R, found on immune cell surfaces, forms a heterodimer crucial for lymphocyte development. Sarcoidosis, a persistent granulomatous disorder, involves a significant infiltration of Th1 lymphocytes. Both naïve and memory T cells exhibit heightened expression of the interleukin 7 receptor (Heron et al., 2009). Downregulating the genes encoding cytokine receptors has the effect of suppressing inflammation in RAW cells.

Apart from cytokines and cytokine receptors, several important proteins related to inflammation also experience significant changes in expression. In this research, Nitric Oxide Synthase Interacting Protein (NOSIP) was up regulated. NOSIP is a negative regulator of Nitric Oxide Synthase (NOS). Upregulated NOSIP causes decreased NO production and suppresses inflammation (Zhou et al., 2017). NFKB1 and STAT1 were also downregulated due to administration of EH leaves extract. NFKB1 is a transcription factor that regulates the expression of inflammatory genes such as IL1B, IL6, IL17A, and TNFA (Bank et al., 2014). STAT1 is the main protein in the JAK-STAT signaling pathway. Activated STAT1 translocate to the nucleus and becomes a transcription factor for proinflammatory genes such as IL-6, CCL5 (Chemokine (C–C motif) ligand 5), IRF7 (Interferon regulatory factor 7), iNOS, and SOCS1 (Suppressor of cytokine signaling 1) (Shin et al., 2020). Taken together, the EH extract is a promising anti-inflammatory agent as it has been shown not only to inhibit molecular signaling pathways in the inflammation process but also has been extensively demonstrated at the transcriptomic level in this study that EH affects several genes involved in the inflammatory pathway.

Conclusion

The study's results show that EH leaf extract possesses anti-inflammatory properties, as evidenced by its ability to decrease the production of NO, iNOS, and pro-inflammatory cytokines IL-12 and TNF-α in LPS-induced RAW 264.7 cells. This effect is attributed to its capacity to regulate genes associated with inflammation. Specifically, the extract influences the expression of genes involved in key inflammatory signaling pathways such as the cytokine signaling pathway, NF-κB signaling pathway, iNOS signaling pathway, and JAK-STAT signaling pathway. These genes include IL11RA, NOSIP, ILF3, IL12RB1, TNFRSF9, TNFRSF1A, IL36A, IL36G, IL6, IL6R, TNFAIP2, TNFAIP3, AKT1S1, NFKB1, IL7R, and STAT1. However, it's important to note that this study was limited to cell-based analysis (in vitro), and further research using animal models is necessary to confirm and validate these findings.

Acknowledgment

The authors thank to Brawijaya University for supporting this research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author, [NW], upon reasonable request.

Additional information

Funding

This work was supported by Universitas Brawijaya [Grant Number 3084.17/UN10.F09/PN/2022].