Muntingia calabura Leaves Extracts to Ameliorate chronic obstruction pulmonary diseases by Inhibiting IL-17a Signaling: In silico and in vivo studies

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is a disease of the lower respiratory system, which can be emphysema and bronchitis, characterized by impaired breathing patterns and limited airflow. The causes of COPD are very complex, involving interactions between genetic factors, cell aging, and continuous exposure to harmful particles, which will disrupt the respiratory system’s homeostatic conditions to make morphological changes and functional organ respiration (Hikichi et al., 2019). Symptoms seen in COPD patients are shortness of breath, wheezing, chest tightness, and coughing, and they can be accompanied by mucus production, loss of appetite, and weight loss (Wang et al., 2020).

COPD ranks as the third leading cause of global death (after ischemia and stroke), with 80% coming from low to middle-income countries. The 2021 Global Initiative for Chronic Obstructive Lung Disease (GOLD) report states that there are at least 3 million deaths each year due to COPD, with a projected 5.4 million in 2,060 (GOLD Committee, 2021). In Indonesia alone, in 2013, there were 9.3 million people with COPD with various causes, including exposure to air pollution due to industrialization and continuous exposure to cigarettes. 33.8% of Indonesia’s population are smokers, including 9.1% of Indonesian teenagers (aged 10–18 years) in 2018.

Current COPD therapy options are still limited, referring to the improvement of symptoms that arise, such as giving bronchodilators (β2 receptor agonists and anti-cholinergic), mucolytics, PDE-4 inhibitors, and glucocorticoids (GOLD Committee, 2021), so there is a need for therapeutic candidates who can provide intervention based on COPD pathogenesis (Wang et al., 2020). Various antioxidants can be given as a therapy in COPD management (Hikichi et al., 2019).

The potential of Indonesia’s biological resources is always interesting to study. One of them, the Muntingia calabura plant, known as Kersen, talok, or cherry, is a plant that grows and is easy to cultivate in Southeast Asia, the United States, India, and Brazil (Ansori et al., 2021). Kersen is a plant with antioxidants (Rahmawati et al., 2018). Biflavans, flavonoids, and dihydrochalcones from the stems of this plant can affect the number of human proinflammatory neutrophils in vitro (Kuo et al., 2014). The fruit can suppress proinflammatory cytokines (Lin et al., 2017). Kersen leaves have cytotoxic activity (Chen et al., 2005) and can improve colorectal cancer conditions, regulation of apoptosis, and inflammation (Jisha et al., 2020). In addition, Kersen leaves are hepatoprotective (Indriawati, 2020; Rofiee et al., 2015; Zakaria et al., 2018), antihypercholesterolemia (Ranti et al., 2021), gastroprotective (Zakaria et al., 2018), antinociceptive (Mohamad et al., 2013), and antiproliferative (Desrini and Purnamasari, 2017) and has antimicrobial activity ( Buhian et al., 2016; Sufian et al., 2013). The main bioactive content that contributes to the pharmacological effects of Kersen is flavonoids (Sari et al., 2020), where the value of flavonoids in Kersen leaves is higher than in the stem.

Kersen leaf ethanol extract 6 mg/20-g weight of mice made a difference in alveolar edema, septal destruction, and lung inflammatory cell infiltration of mice exposed to cigarette smoke. Meanwhile, Kersen fruit extract at 100 mg and 200 mg/kg BW can reduce the number of goblet cells in the rat trachea. However, it is not yet known how the molecular mechanism of Kersen can improve the test animals’ condition. By linking the pathogenesis of COPD and the potential possessed by Kersen leaves, it is necessary to test and analyze the mechanism of this plant as a therapy for improving COPD conditions, which is the aim of this study.

To predict the possible mechanism of a substance against certain diseases, a bioinformatics approach can be used through network pharmacology and molecular docking, which is then confirmed by in vitro or in vivo studies (Cao et al., 2021). Network pharmacology is a combination of genomic technology and biological systems which, through computational applications, can describe a complex relationship between drug compounds and disease targets by clarifying these relationships through possible mechanisms based on available databases [Sakle et al., 2020]. The disease targets in the form of genes or proteins (macromolecules) are then tethered with the ligands of the test compounds through molecular docking to determine the affinity and interactions that occur. The conformational change, stability, and interaction mechanism of the tethered protein-ligand complex were then analyzed using molecular dynamics simulations, and in vivo analysis using the COPD mice model will be evaluated in this paper.

METHODS

RESULTS

Flavonoid content

Determination levels of flavonoids are using quercetin as standard. The flavonoid content of ethanolic Kersen extract is 46.05 mg quercetin equivalent/gram or 0.0465% w/b. Other researchers found that the total flavonoid content is 0.06% w/b, equivalent to quercetin/g (Pertiwi et al., 2020). The previous study has a few differences in total flavonoid yield, although extraction is done on type plants and the same solvent. The difference in the content metabolites secondary Kersen could be because of different cultivation areas plant as altitude, carbon dioxide level, presence of pests, and species and population of bacteria in the region.

Table 1. Cigarette smoke content by GC/MS. [Click here to view]

The main bioactive content that contributes to the pharmacological effects of cherries is flavonoids (Sari et al., 2020), where the value of flavonoids in Kersen leaves is more than in the stem (Buhian et al., 2016).

Analysis of in silico bioavailability of Kersen compound

Study-related content extract of 50% ethanol leaves Kersen (Pertiwi et al., 2020) shows 42 compounds. Each compound then downloads from canonical SMILES, structure two and three dimensions in SDF format using facilities available at PubChem (https://pubchem.ncbi.nlm.nih.gov). The important step in choosing the most suitable structure is matching heavy and formula molecules following LC/GC-MS information to extract 50% ethanol. Each compound is then selected to score its bioavailability through the SwissADME (http://www.swissadme.ch/index.php) and SCFBio (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp. There are 34 compounds with a bioavailability score of 0.17–0.55, as shown in the table below.

Some of the compounds in Table 2 have been studied for their potential for pathological conditions of the respiratory system, for example, quercetin which can improve lung damage due to cigarette smoke and has entered phase 1 clinical trials (https://ichgcp net/), quercitrin which in vitro can inhibit apoptosis and oxidative stress of human bronchial epithelial cells induced by cigarette extracts, arbutin affects mice models of sepsis pneumonia, and LPS-induced, catechin which can improve the symptoms of COPD patients, and epigallocatechin can reduce neutrophilic inflammation and mucus hypersecretion in rats exposed to cigarettes and improve acute lung damage due to Pseudomonas aeruginosa (Liang et al., 2018).

Table 2. Compound putative extract of Kersen with 50% ethanol with a score bioavailability ≥0.17. [Click here to view]

Gene ontology and pathway analysis

The gene is then analyzed in terms of its enrichment and gene ontology to see the most likely pathways and carried out with the STRING platform https://string-db.org/, DAVID: Functional Annotation Tools (ncifcrf .gov) and metascape.org which is linked to other gene ontology servers such as the genetic association disease database Disease Genetic Association Disease Database. [https://geneticassociationdb.nih.gov/] and the KEGG Pathways database https://-www.genome.jp/ and Human References Interactome http://www.interactome-atlas.org/ as shown in Figure 3.

Figure 1. Venn diagram of COPD and Kersen extract target genes. [Click here to view]

Figure 2. Custer COPD - Kersen target gene. [Click here to view]

Figure 4 shows that these genes are genes involved in response to stimuli, in line with the supporting theory that COPD is a disease involving the immune defense in the respiratory system. With consistent exposure to inhaled particles, reactive oxygen species (ROS) and incoming pathogens will be recognized as pattern recognition receptors on the plasma membrane of lung epithelial cells, activating an inflammatory response cascade. If macrophages are activated, these cells will secrete chemokines and cytokines such as tumor necrosis factor-α (TNF-α), which will then induce the expression of adhesion molecules on endothelial cells so that inflammatory cells can migrate. Macrophages also produce ROS, metalloproteinases (MMPs), and cathepsins, which damage alveolar structures (Hikichi et al., 2019).

The abovementioned 16 genes are involved in several signaling pathways, with the result that the IL-17 signaling pathway is ranked first, indicating that this pathway can be used as a referral pathway for analysis of the target mechanism of Kersen extract on the pathogenesis of COPD. In addition to the IL-17 signaling pathway, interleukin signaling, IL-10 signaling, and others are more suggestive of the inflammatory process (Rodrigues et al., 2021).

Furthermore, in the picture above, the IL-17 signaling pathway initiates the formation of chemokines and cytokines such as IL-6, CXCL-8, TNF-α, and IL-1β. IL-17a originates from Th17 cells, which are part of effector T cells. Besides producing IL-17a, these cells also produce IL-17f and IL-22. When an infection occurs, these interleukins are needed to eliminate external stimulants, such as bacteria and fungi, by inducing antimicrobial peptides, such as defensins. These cytokines also play an important role in autoimmune and allergic pathogenesis, such as rheumatoid arthritis (RA) (Rodrigues et al., 2021).

The receptor that plays a role in the IL-17a signaling pathway in the picture above is IL-17 receptor A (IL-17RA, while the IL-17F receptor is IL-17RC). Currently, four other receptors have been found, namely IL-17RB, IL-17RC, IL-17RD, and IL-17RE (specific receptors for IL-17E, IL-17C, and IL-17F), which have similarities in sequence homology and have a fibronectin III-like domain in the extracellular region and the SEF/IL-17R (SEFIR) domain in the intracellular region. Recent studies have proven that IL-17A and IL-17F are widely expressed in TH17 cells through a heteromeric receptor complex consisting of IL-17RA and IL-17RC, a single transmembrane protein, and are expressed in many places covering various types of cells such as epithelial cells, fibroblasts, and astrocytes (Rodrigues et al., 2021).

IL-17 upregulates proinflammatories such as TNF-α, IL-6, G-CSF, IL-1, CXCL1, CCL20, and matrix metalloproteinase through activating the NF-κB, mitogen-activated protein kinases (MAPK), and C/EBP cascades. After IL-17R is activated, the adapter protein Act1 and tumor necrosis factor receptor-associated factor (TRAF) will activate NF-κB. The IL-17R complex recruits act1 through homotypic interactions from the SEFIR domain due to IL-17 stimulation. Furthermore, TRAF6 will be recruited when there is an interaction between Act1 and TRAF so that the TRAF6 task activates downstream TRAF6-dependent TAK1 to activate NF-κB. Act1 mediates in the TRAF6-dependent and TRAF6-independent pathways. In the TRAF6-independent pathway, TRAF2 and TRAF5 are required to induce IL-17 as a stabilizer for CXCL-1 mRNA. The formation of the Act1-TRAF5-TRAF2-SF complex by IL-17 prevents the degradation process if splicing factor binds to CXCL1 mRNA. IKKε directly phosphorylates act1. This phosphorylation is critical for the stabilization of the mRNA cascade. Thus, if candidate compounds can inhibit this pathway, it will prevent the formation of IL-17-mediated disease (Rodrigues et al., 2021).

Table 3. Kersen-COPD network topology results. [Click here to view]

Figure 3. Gene ontology analysis of biological processes between COPD and Kersen target gene. [Click here to view]

Figure 4. COPD- Kersen signaling pathway. (a) Molecular process pathways. (b) iL-17 signaling pathway. [Click here to view]

IL-17a originates from Th17 cells, which are part of effector other T cells producing IL-17A cells; it also produces IL-17F and IL-22. Exposure to cigarette smoke could increase work from Th17 to increase mechanism proliferation and apoptosis in cell epithelium channel breath (Montalbano et al., 2015). In addition, smoking is also an adjuvant for Th17 (Chen et al., 2005), signaling IL-17RA will induce MMP12 to support emphysema in tissue. The IL-17A/IL-17 RA axis pathway also plays a role in duct fibrosis breath (Yanagisawa et al., 2017).

Molecular docking method validation with AutoDock 4.0

The selected IL-17a macromolecule was 8DYI, with the ligand (5P)-5-[5-(benzylamino)pyridine-3-yl]-N-[2-(morpholin-4-yl)ethyl]-1H-indozole-3- amine (C25H28N6 O) (accessed from: https://www.rcsb.org/). As for TNF-α, the macromolecule used is 2AZ5 which has the ligand 6,7-dimethyl-3-[(Methyl{2-[Methyl({1-[3-(Trifluoromethyl))phenyl]-1H-indole-3-yl }Methyl) amino] ethyl} amino) methyl]-4H-chrome-4-ONE. The grid box parameters used after several iterations are listed in Table 4.

Table 4. The grid box parameters. [Click here to view]

Table 5. Conformation best ligand with IL-17a and TNF-α macromolecules. [Click here to view]

Virtual screening with AutoDock Vina

After obtaining the optimal grid size, the parameters obtained are used for the virtual screening process using AutoDock Vina. The results show that quercitrin has the smallest affinity energy for both macromolecules, as shown in Table 5, Figures 5 and 6.

Based on the results of this bioinformatics study, a very potent compound, quercitrin, or another name, quercetin 3-rhamnoside, is a bioflavonoid found in many plants and can be consumed. Quercitrin has a bitter taste with pale yellow crystals. When hydrolyzed, it breaks down into quercetin and rhamnose (Septembre-Malaterre et al., 2022).

An in vitro study conducted by Yu et al. proved that quercitrin could protect human bronchial epithelial cells from apoptosis and oxidative damage in the Nrf2/HO-1/NQO1 pathway and the MAPK/ERK pathway (Sharma et al., 2020).

Quercitrin significantly affects platelet aggregation, granule secretion, ROS production, and intracellular calcium mobilization, inhibiting platelet activation in arterial thrombosis. During cell activation, quercitrin inhibits platelet signal transduction mediated by glycoprotein VI, reduces phosphorylation of TNF receptor components, and inhibits FeCl3 induction, which plays a role in thrombus formation (Oh et al., 2021).

The molecular docking results above show that quercitrin can bind well with IL-17a and TNF-α with a bond energy affinity of −7.3 and −6.7 kcal/mol, respectively. The type of bond between IL-17a and quercitrin is noncovalent, namely van der Waals bonds on residues Arg 69, Ser 70, Ser 72, Ile 115, Gly 143, Thr 145, and Cys 146 and hydrogen bonds which are located on acidic residues. There are amino Tyr 66, Thr 71, and Cys 144, as well as two bonds on other residues, namely Val 142 and Trp 74. In the TNF-α macromolecule, the noncovalent bonds are van der Waals bonds at residues Gln 61, Gly 121, Gly 122, and Ile 155. Hydrogen bonding in Ile 58, Leu 120, and Tyr 151. Hydrogen bonding plays a role in stabilizing molecular interactions between ligands and proteins, such as enzyme catalysis, substrate-inhibitor complexes, and the stability of protein structures. Biological molecules are affected by this bond (Kelepouri et al., 2018).

Figure 5. Visualization of binding and attachment sites of compounds with macromolecule IL-17a. (a) Quercitrin. (b) Myrtillin. (c) Quercetin. [Click here to view]

Dynamic molecular simulation of IL-17a

Molecular dynamic simulation is a method to describe the stability of a ligand to a macromolecule in its binding pocket by taking into account the side chains, the backbone flexibility of the protein, and the influence of the solvent. Dynamic simulations support optimizing research designs with characteristics relevant to the biomolecular system (Durrant and McCammon, 2011).

To see the prediction of the stability of the bond between quercetin and quercitrin with the IL-17a macromolecule, a dynamic simulation analysis was carried out using Gromacs at a period of 10 ns.

The two RMSD images in Figures 7 and 8 showed that both ligands, quercetin and quercitrin, can bind to the macromolecule IL-17a (RMSD < 2 Å). RMSD is the average movement of atoms from one point to another at specific time intervals. In other words, this value indicates the average distance between ligands and macromolecules (Fatriansyah et al., 2022). However, even though the value of the binding affinity energy for molecular quercitrin binding (−7.3 kcal/mol) is better than that of quercetin (−6.4 kcal/mol) when confirmed using dynamic simulations; it appears that quercetin is a more stable binding to the IL-17a ligand, whereas 800ps ligand quercitrin still has fluctuations.

Figure 6. Visualization of binding and attachment sites of compounds with TNF-α macromolecules. (A) Quercitrin. (B) Hiravanone. (C) Ononine. (D) Quercetin. [Click here to view]

Mice weight monitoring chart

In the first and second weeks, all groups of mice showed normal weight gain. Entering the third week, the group of mice treated with cigarette smoke showed smaller weight gain than those not exposed to cigarette smoke. However, this is not due to COPD but because the nicotine in cigarettes can reduce feed intake in mice. Nicotine can affect the hypothalamic melanocortin system, which plays a role in synaptic mechanisms affecting test animals’ appetites (Upadhya et al., 2020).

Figure 7. RMSD IL-17a with quercitrin. [Click here to view]

Figure 8. RMSD IL-17a with quercetin. [Click here to view]

Figure 9. Mice weight monitoring chart. [Click here to view]

The weight chart in Figure 9 shows that the negative group lost weight until the 14th week due to COPD. A Japan cohort study showed that a low body mass index or is associated with an exacerbation process that causes a decrease in FEV1 and the mortality rate of COPD patients. Factors that cause low body mass index include oxidative stress resulting in systemic inflammation, shortness of breath (disruption of the supply of oxygen to cells) and shortness of breath thereby disrupting metabolic rates and reduction of skeletal muscle mass due to an imbalance of oxidants-anti-oxidants which increases muscle proteolysis (Sun et al., 2019).

Exposure to 6–10 cigarettes for 35 days, accompanied by LPS induction, causes weight loss, pulmonary function due to fibrosis, and a low survival rate in mice (Gotts et al., 2017).

Evaluation of COHb levels

Table 6 shows that in carboxyhemoglobin levels in the eighth week, cigarette smoke induction can increase the concentration of COHb in the blood. COHb is a complex form between hemoglobin and carbon monoxide (CO) from exogenous and endogenous sources. The bond between hemoglobin and CO is more stable than that between oxygen and hemoglobin. The presence of CO can come from endogenous and exogenous. Endogenous CO is produced from the enzyme heme oxygenase 1 (HO-1) due to the response of hypoxia, inflammation, and oxidative stress. COHb concentrations can also increase due to the production of proinflammatory cytokines and nitric oxide in the respiratory tract due to viral infections and ROS originating from neutrophils in bacterial infections. This event was also found in patients with asthma and pneumonia, so it can be said that increased COHb is associated with inflammation of the airways and lung parenchyma (Yasuda et al., 2005). Exogenous sources of CO come from imperfect oxidation processes of carbon-containing materials, which have a toxic effect because they can act as molecules that can signal apoptosis and oxidative endothelial damage.

Table 6. COHb concentration after treatment. [Click here to view]

Table 7. IL-17a concentration after treatment. [Click here to view]

The difference in COHb reduction in the extract-treated mice is because 50% Kersen extract acts as an antioxidant (Pertiwi et al., 2020), suppressing the oxidation process but only affecting endogenous CO. In this study, exposure to cigarette smoke was still given to all treatment groups so that exogenous CO sources remained. This hypothesis is strengthened by a cohort study proving that baseline COHb levels of smokers were two times higher than nonsmokers, and COHb concentrations increased after smoking (Schimmel et al., 2018).

HISTOLOGY OF LUNGS

Hematoxylin and eosin stains are the most commonly used staining units. Hematoxylin is not directly used as a dye but requires metal assistance (metal cations such as aluminum) to connect with tissues. The ionic bonds between the tissue and the dye are very important. The hematoxylin will later be positively charged so that it can bind to nucleic acids (negatively charged) to form a blue color. Meanwhile, eosin is an anion that is acidic and negatively charged so that it can bind to positively charged components, such as amino acids, in the cytoplasm to produce a pink color (Wick, 2019).

HE staining measures the degree of inflammation, such as inflammatory cell infiltration, alveolus dissemination, and bronchial wall thickening. The infiltrated inflammatory cells originate from the immune system activation due to cigarette exposure and the induction of LPS, which produces free radicals. Stimulated agents such as macrophages, dendritic cells, and neutrophils stimulate the production of various cytokines, chemokines, protease enzymes, myeloperoxidase, and elastase (Wick, 2019).

The widening of the air cavity due to alveolar damage in COPD conditions is caused by the process of apoptosis in alveolar septal capillary cells, which are very susceptible (Wang et al., 2020). Administration of antioxidants can increase α-1antitrypsin, a serine (serine protease inhibitor) that protects the lungs by inhibiting protease-producing neutrophils, thus preventing apoptosis and emphysema (Rangasamy et al., 2004). As previously explained, COPD is a manifestation of a protease-anti-protease imbalance, where an excess protease will destroy the alveolar septum, thereby widening the air cavities and reducing the portion of air exchange in the alveoli.

The HE staining on Figure 10 and Table 8 showed that inflammation was significantly different in the Sham group, with the greatest degree in the CS + LPS group (raw data is in the appendix) followed by the CS + LPS + extract Muntingia calabura (EMC) 14 mg/30 gBW group (****p < 0.0001; *p < 0.05). Whereas in the CS + LPS + Budesonide group and CS + LPS + EMC 3.5mg/30gBW, there was a decrease in the degree of inflammation, the cavities between the alveoli were more compact, and the thickness of the bronchial walls decreased and did not differ significantly from the Sham group. That is, administering 50% ethanol extract of Kersen leaves at a dose of 3.5 mg/30 g BW can affect the progression of inflammation in lung tissue. However, in this study, there was no dose-dependent relationship. The higher dose does not show any significant improvement.

PAS staining consists of periodic acid reacted with Schiff’s reagent (fuchsin in acid). Periodic acid will oxidize components that have free hydroxyl groups or alkyl amine groups to become dialdehydes. The aldehyde reacts with Schiff’s reagent to form an insoluble magenta complex. This magenta complex colors components containing carbohydrates such as glycogen, glycoproteins, and proteoglycans. PAS detects goblet cells and mucin (glycoprotein) in lung tissue in COPD (Hauber and Zabel, 2009).

Figure 10. Hematoxylin and eosin staining. Score the degree of inflammation from H&E staining. (A) Sham. (B) CS + LPS. (C) CS + LPS + Budesonide 1 mg/KgBW. (D) CS + LPS + EMC 3.5 mg/30 g BW. (E) CS + LPS + EMC 7 mg/30 g BW. (F) CS + LPS + EMC 14 mg/30 g BW. Scale = 50 μm. [Click here to view]

Figure 11. PAS staining. PAS staining to evaluate the presence of mucin-glycoprotein, which is marked with a magenta spot (white arrow). (A) Sham. (B) CS + LPS. (C) CS + LPS+ Budesonide 1 mg/KgBW. (D) CS + LPS + EMC 3.5 mg/30g BW. (E) CS + LPS + EMC 7 mg/30g BW. (F) CS + LPS + EMC 14 mg/30 g BW. Scale = 50 μm. [Click here to view]

Table 8. Degree of inflammation hematoxylin and eosin stain. [Click here to view]

Mucus is produced by secretory cells, in this case, the goblet cells in the airways function as traps for particles and micro-organisms (these goblet cells are a form of differentiation from epithelial cells). After the mucus task is completed, the trapped mucus and particles will be removed by ciliated cells, commonly called mucous ciliary clearance. However, both of these functions are disrupted in COPD conditions. Overproduction of mucus causes resistance to ciliary movement. Wu et al. research proved that cigarette exposure causes epithelial cells to secrete IL-17a, directly stimulating MUC5B expression through the NFκB and Act-1 signaling pathways (Wang et al., 2020).

Figure 11 and Table 9 showed components containing glycoprotein were seen in magenta in the CS + LPS group (*p < 0.05 for the Sham group) and the CS + LPS group (MCE 7 mg and 14 mg/30 g). Meanwhile, no glycoprotein component was found for the Sham and CS + LPS MCE 3.5 mg/30 g group (#p < 0.05 for the CS + LPS group). So it can be said that administering 50% ethanol extract of Kersen leaves at a dose of 3.5 mg/30 g BW can reduce mucus production in COPD conditions. However, in this study, there was no dose-dependent relationship. The higher dose does not show any significant improvement.

Masson’s trichrome staining to evaluate the presence of blue (wavy) collagen is shown in Figure 12 and Table 10. The three principles of staining in this staining can stain smooth muscle, collagen fibers, and erythrocytes. In principle, the dye first reacts with acidic components, then when phosphoric acid reacts, components with low permeability/less porous will turn red, and at the same time, aniline blue enters the collagen component (Bordoloi et al., 2020).

Table 9. Mean positive area values of mucin-glycoprotein detection periodic acid Schiff stain. [Click here to view]

Figure 12. Masson’s trichrome staining of cigarette smoke and LPS-induced mice and the treatment. The extent of collagen disposition is indicated by the presence of blue areas in Masson’s Trichrome staining. (A) Sham. (B) CS + LPS. (C) CS + LPS + Budesonide 1 mg/KgBW. (D) CS + LPS + EMC 3.5 mg/30 g BW. (E) CS + LPS + EMC 7 mg/30 g BW. (F) CS + LPS + EMC 14 mg/30 g BW. Scale = 50 μm. [Click here to view]

The collagen disposition in COPD reflects changes in lung function due to the development of emphysema and mechanical stress around the bronchi. Increased collagen around the alveoli is also caused by damage to parenchymal fibers due to changes in the ratio of collagen type I to collagen type III. The presence of protease activity (MMP-9, MMP13) causes collagen and elastin fibers to become embedded in a shape (Xing et al., 2021). Other studies state that in the extracellular matrix, type I and several types IV collagen fragments are degraded by metalloproteinases (MMP) and are associated with the severity of COPD (Calvi et al., 2012).

Table 10. Masson’s trichrome stain collagen area value. [Click here to view]

From the above results, it can be seen that at a high EMC dose (14 mg/30 gBW), it gives the smallest collagen area value, followed by a dose of 3.5 mg and then a dose of 7 mg/30 gBW. However, there was no significant difference compared to the CS + LPS group.

The stimulation due to CS + LPS results in acute exacerbations of COPD. In line with research by Yang et al. (2021) it was shown that exposure to large areas of collagen in the second and third months increased,.

Evaluation of TNF-α expression by western blotting

TNF-α is known to suppress tropoelastin in lung fibroblasts and regulate MMPs such as MMP-2, MMP-9 which have elastolytic activity, thus preventing MMP overproduction if not inhibited, will cause emphysema characteristics in the lungs (Mukhopadhyay et al., 2006).

Previous studies have proven that Kersen fruit in vitro using LPS induction can reduce the Expression of TNF-α, IL-1ß, and IL-6 in macrophages through inactivation of the NF-κB pathway, MAPKs p38 and c-Jun NH2-terminal kinase 1/2 (JNK1/2), as well as Janus kinase 2/signal transducers and activators of transcription 1/3, and activation of nuclear factor erythroid-2-related factor 2 (Nrf2) followed by a decrease in the induction of HO-1 production (Lin et al., 2017).

TNF-α is a cytokine that is also produced by macrophages. In the IL-17 signaling pathway, IL-17a plays a role in the de novo transcription of cytokines, one of which is TNF-α. Figures 13 and 14 showed by western blot analysis, it can be said that the administration of 50% ethanol extract of Kersen leaves can improve COPD characteristics, including parameters of body weight, lung histopathology, and expression of IL-17a and TNF-α in the IL-17 signaling pathway through a model COPD animals, at an optimal dose of 3.5 mg/30 gBW.

The ideal therapy strongly affects the target pathway with minimal effect on other pathways to reduce side effects. Drugs will have pharmacological effects by binding to specific receptors, and the magnitude of the biological response depends on the concentration of the drug-target complex formed (Singh et al., 2017). In this study, the concentration at a low dose, namely 3.5 mg/30 gBW, had formed a complex between the receptor and the ligand component of the extract, which, if the concentration were increased, there would be saturation of the receptor so that it would not provide improvement in COPD conditions.

Figure 13. Detection of TNF-α protein level expression from mice lung homogenates. [Click here to view]

Figure 14. Quantitative TNF-α expression relative to β-actin by ImageJ. [Click here to view]

There are other possibilities because Kersen extract has cytotoxic activity (Chen et al., 2005), so the dose of the extract taken from natural materials with cytotoxic potential can be reduced to get benefits from the extract components. The cytotoxic potential indicates that the components in the extract can be developed into antimicrobial or anticancer agents.

CONCLUSION

Sixteen target genes can be intervened by compounds contained in 50% ethanol extract of Kersen leaves, namely TNF-α, IL6, IL1B, RELA, CXCL8, CCL2, IL10, NFKBIA, IL4, CXCL1, CSF3, IL17A, TNFRSF1A, IKBKB, RELB, and SIRT6.

The content in the 50% ethanol extract of Kersen leaves can intervene in the pathogenesis of COPD in the IL-17 signaling pathway and stably bind to IL-17a for 10ns. So IL-17a (upstream gene) and TNF-α (downstream gene) were selected for in vivo confirmation.

Administration of ethanol extract of Kersen leaves can improve COPD characteristics, including weight parameters, lung histopathology, and expression of IL-17a and TNF-α in the IL-17 signaling pathway through COPD animal models at optimal doses of 3.5 mg/30 gBW.

ACKNOWLEDGEMENTS

This research is funded by the Directorate of Research and Development, Universitas Indonesia, under Hibah PUTI 2022 (Grant No. NKB-544/UN2.RST/HKP.05.00/2022).

AUTHORS’ CONTRIBUTIONS

AB devised the project and the main conceptual ideas and designed the experiment. FF designed the experiment. NN worked out almost all of the technical details and performed the experiments. NN proposed the experiment in discussions with FF and AB NN, and AB wrote the manuscript.

CONFLICTS OF INTEREST

We assure you that there are no conflicts of interest related to this study.

ETHICAL APPROVALS

Ethics Committee has approved all protocols, Faculty of Medicine, University of Indonesia with No. KET-583/UN2. F1/ETIK/PPM.00.02/2022

DATA AVAILABILITY

All data generated and analyzed are included in this research article.

PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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