Anti-Inflammatory Potential of Arglabin in LPS-Activated RAW 264.7 Macrophages

To defend the body from harmful stimuli like the infectious invasion of viruses and poisons, the human immune system has the ability to initiate an inflammatory response.¹ An array of defense systems known as the inflammatory response work to remove dangers, encourage tissue healing and reestablish physiological balance.² The five primary indicators of inflammation, which serve as the body’s “natural defense system” against illness and damage, are temperature, redness, malfunction, edema and soreness. While some degree of inflammation is necessary for good health, too much inflammation can harm host cells and result in illnesses.³ However, research has demonstrated a broad spectrum of diseases, including autoimmune disorders, cardiovascular disorders, chronic respiratory disorders, neurological disorders and cancers, are linked to the shift of inflammation into a chronic or dysregulated state.² Particularly, the development of numerous metabolic illnesses, such as obesity and insulin resistance, is directly linked to persistent inflammation. Macrophages, a major immune system subgroup, play a vital part in the body’s reaction to infection and injury.⁴

Macrophages have a major impact on the development of inflammatory diseases and are essential in regulating immunological responses. Through Toll-Like Receptors (TLRs), macrophages are able to detect stimuli and initiate signaling pathways, including NF-κB and MAPK.² Metamorphism is the first stage of every inflammation, followed by exudation defense and proliferative healing. The primary cause of chronic inflammation is hyperplasia, which typically involves lymphocytes and plasma. The primary pathogenic symptom is cell infiltration.⁴ Activated macrophages secrete a significant amount of pro-inflammatory mediators (NO), cytokines (TNF-α and IL-6) and chemokines (IL-8) during chronic inflammation.¹ TNF-α is an essential cytokine that controls the immune system’s inflammation. It is capable of producing Interleukin-6 (IL-6) and Interleukin-1 (IL-1). Monocyte Chemoattractant Protein-1 (MCP-1) and Cyclooxygenase-2 (COX-2) create Prostaglandin-Endoperoxide synthase 2 (PGE2), which is a common pro-inflammatory cytokine that can control immunological responses.2

Remarkably, in chronic inflammation, activated macrophages start the inflammatory response by producing more NO, ROS and inflammatory cytokines like IL-6 and TNF-α.⁵

The development of an inflammatory model is important practically for screening inflammatory medications and treating inflammatory disorders because the features of these conditions are complex and challenging to address.⁶ Around the world, established cell lines are frequently employed for both in vitro and in vivo investigations. This is because there is an infinite supply of cells through successive passages that have the same genotype and phenotype. Authors now use greater caution when interpreting data from studies that are limited to using established cell lines, nevertheless. The most interesting topic is undoubtedly about the properties of the cells and how stable and comparable they are across different labs and even different stages. Macrophages are a fascinating class of cells. Their ability to polarize, differentiate and become osteoclasts, Kuppfer cells, or dendritic cells has made them renowned for their flexibility. As a result, macrophages are a widely varied kind of cell. Although they are present in every organ and tissue in the body, their phenotype varies greatly based on the physiological condition. Macrophages can swiftly adapt to novel stimuli and are highly sensitive to environmental changes.⁵ RAW 264.7 cells are generated from BALB/c mouse-derived Abelson leukemia virus-transformed cell line and they resemble monocytes and macrophages. It is said that these cells are a suitable model for macrophages. They are able to carry out both phagocytosis and pinocytosis. RAW 264.7 cells produce more NO and improve phagocytosis in response to LPS stimulation. Furthermore, through antibody-dependent cytotoxicity, these cells can destroy target cells.⁷

Receptors located on the membranes of B cells, dendritic cells and macrophages detect and combine LPS to promote local inflammation, trigger the production of inflammatory factors by immune cells and stimulate innate immunity.⁶ Gram-negative bacteria have a large distribution of Lipopolysaccharide (LPS) in their outer cell walls. As a result, LPS triggers a host inflammatory response, which causes the production of more pro-inflammatory mediators, cytokines and chemokines by the immune system. LPS exposure causes macrophages infected with microorganisms to release cytokines and chemokines, which trigger the immune system and cause inflammation. Therefore, one of the main goals of therapeutic approaches for the treatment of inflammatory illnesses is to prevent LPS-induced macrophage activation.¹

A lot of work has gone into finding compounds or herbs that can cure a number of ailments and natural goods are crucial in the identification of innovative newly created chemical entities and lead compounds.⁶ Natural compounds generated from plants have been utilized in various healthcare systems and have been scientifically assessed for a range of bioactivities. Natural medications are recommended since they have less side effects than synthetic drugs because of their associated negative effects. One such molecule is arglabin, a sesquiterpene γ-lactone belonging to the guaianolides class that was isolated from the Artemisia glabella plant species, which is native to Central Kazakhstan.⁸ Early in the 1980s, arglabin was separated from the plant’s aerial portion.⁹ Arglabin has a wide range of pharmacological applications. Numerous applications exist for this chemical, such as antibacterial, neuroprotective and anticancer properties.^8,9

Sesquiterpene γ-lactones are an interesting class of natural compounds that have attracted a lot of attention because of their wide range of biological activities, which include antibacterial, fungicidal, antifeedant, growth-regulating, anthelmintic, immunomodulatory and antitumor properties.¹⁰ Arglabin has a wide range of pharmacological applications. Numerous applications exist for this chemical, such as antibacterial, neuroprotective and anticancer properties.⁸ Arglabin is a white crystalline substance with a chemical structure based on the bicyclo decane skeleton (molecular formula: C₁₅H₁₈O₃).¹¹ It has been observed that arglabin and its derivatives are cytotoxic to a number of cancer cell lines.¹²

The aim of this research was to evaluate the anti-inflammatory properties of AGN in LPS-stimulated RAW 264.7 macrophage cells. To date, the anti-inflammatory action and ROS modulation of AGN have not been extensively studied. In LPS-induced RAW 264.7 cells, we examined the impact of AGN on TNF-α, IL-6, NO and ROS production, cytotoxicity and PGE2 expression.

Prior to the start of the study, all chemicals, reagents, kits and equipment were purchased commercially and made available.

The authorized center provided the RAW 264.7 murine macrophage cell line. The growth medium used for the cells was DMEM enhanced with 1% penicillin/streptomycin and 10% FBS. Every two days, cells were sub-cultured and kept in an incubator with 5% CO₂ and 37°C temperature. The RAW 264.7 cell line was used in all tests and it was in passages 9-12.² Different concentrations of AGN dissolved in DMSO were used to treat the cells, whereas vehicle alone (<0.2% DMSO) was used as the control.⁸

Arglabin’s effects on Raw 264.7 macrophage viability Following treatment with LPS and different doses of AGN, the 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl Tetrazolium bromide (MTT) assay was employed to assess the viability of AGN on the RAW 264.7 cells. In a 48-well plate, macrophages (3×10⁴ cells/well) were planted. Following a 24 hr period, the cells were subjected to a 30 min pretreatment with 0, 2.5, 5, 10, 15 and 20 μM AGN, followed a 24 hr incubation time in 5% CO₂ condition at 37°C. 1 mg/mL of MTT solution was used to treat the cells for 2 hr after which the culture media was removed. Following the removal of the MTT solution, to dissolve the formazan crystals, 200 μL of DMSO was applied. A microplate reader was used to determine absorbance at 570 nm.³

Using the Griess test, the amounts of NO in the cell culture supernatants were determined.³ In a 96-well plate, 100 μL of RAW 264.7 cells were seeded at a density of 2 × 10⁵ cells/mL. After that, the cells were cultured in an incubator for 24 hr at 37°C and 5% CO₂. Cells were pretreated with AGN and Positive control Dexamethasone (Dex) for 1 hour and then cultured for an extra 24 hr before being stimulated with LPS (1 μg/mL). After incubation, 100 μL of the supernatant and 100 μL of Griess reagent were combined in a 96-well plate. A spectrophotometer was used to detect the absorbance at 540 nm wavelength following a 15 min incubation period.²

The ROS-sensitive fluorescent indicator DCFH-DA was utilized to quantify the levels of intracellular ROS.¹ A 24-well plate containing Raw 264.7 macrophages (6×10⁴ cells/well) was planted and cultured for 24 hr. Subsequently, the cells underwent a 30 min pretreatment with AGN (10, 15 μM and Dex), followed by an LPS treatment (0.1 μg/mL) and a 24 hr incubation period at 37°C in an incubator with 5% CO₂. After 30 min of DCF-DA treatment at 37°C, the cells were given three HBSS washes. Using a fluorescent microscope, ROS levels were found and FACS analysis was performed.³

A 60 mm cell culture was seeded with 4 mL per well and 2×10⁵ cells/mL. The cells were grown in an incubator for 24 hr at 37°C and 5% CO₂. Supernatants were obtained after these cells were cultured for a further 20 hr with LPS (1 μg/mL) stimulation after being pretreated with AGN (10, 15 μM and Dex) for one hour. As directed by the manufacturer, ELISA kits were utilized to quantify the PGE2 content in the supernatants.²

Using an ELISA assay kit, the release of cytokines that promote inflammation, namely IL-6, IL-1β and TNF-α was evaluated. A 24-well plate was seeded with cells (1.5×10⁵ cells/well) and left to incubate for 24 hr. The cells underwent a 1 hr pretreatment with AGN at different doses (10, 15 μM and Dex) followed by a 24 hr treatment with LPS (1 μg/mL). The supernatants were collected in accordance with the guidelines provided by the manufacturer and ELISA equipment was used to measure the levels of IL-6, IL-1β and TNF-α.^12,13

The X-ray crystallographic structure of EGFR signalling pathway are identified as critical targets for AGN anti-inflammatory anti-cancer action. The complex of EGFR (PDB code: 4URO) with novobiocin (resolution: 2.59 A; R-value free: 0.246; was discovered.^16–18 Target protein was retrieved from the PDB database. The criteria for choosing PDBs were (a) minimum resolution and (b) the conformation of the docked ligand being the same as in the crystallized structure after the redocking procedure.¹⁹ The PDB file chosen for the molecular docking-based virtual screening study were processed by removing water molecules, adding hydrogen atoms and finally being prepared by Biovia Discovery Studio.

The compound AGN and its target protein, the EGFR (4URO) gene, were uploaded into the PyRx AutoDock VINA.²⁰ The target protein was converted into macromolecules, which changed the atomic coordinates into pdbqt format. To perform molecular docking, the grid box was centered on the crystal structures and all other parameters were left as defaults. The docking results were screened for binding affinity and then all possible docked conformations were generated for the compound. Following analysis with Discovery Studio and PyMOL, only those conformations that specifically interact with the active-site residues of EGFR targeted protein were chosen. Biovia Discovery Studio was employed to explore detailed interactions and their types, including hydrogen bonds, halogens, alkyl and the van der Waals interactions formed between Arglabin and the target EGFR gene in the treatment of human anti-cancer.

Molecular descriptors are the deciding factor for the PK properties and toxicity of a compound. In silico ADMET properties predict the likelihood of compounds being used as human therapeutic agents.²² The admetSAR online server was utilised for calculating the ADMET properties of the compound arglabin. The compound and the target were tabulated in Table 4. It is important for compound to have a promising ADMET profile. The BBB, HIA, aqueous solubility, Caco-2 cell permeability, CYP450 inhibition and Ames’s toxicity was also calculated.^23–28

Table 3:
Bioactivity prediction of the selected inhibitors by Molinspiration.

Table 4:
Pharmacodynamics profile of the selected inhibitors admetSAR.

The information is based on three distinct experiments, was presented as mean±SD. GraphPad Prism was used to statistically analyze the results. A noticeable difference between the groups was indicated by p<0.05. Software called SPSS was used to perform statistical analysis. Tukey’s test is used after a one-way Analysis of Variance (ANOVA) to determine any significant differences among the groups.

A common innate immune response that shields the body from outside stressors is an inflammatory reaction.² Numerous medical disorders, such as atopy, multiple sclerosis, metabolic syndrome, headache, multiple sclerosis, Alzheimer’s disease and Parkinson’s disease are primarily caused by the advancement of inflammation.¹ Numerous illnesses, including arthritis, arthrophlogosis and asthma, can cause inflammation.²⁹

Figure 3:
Measurement of Reactive Oxygen Species production (ROS). The values are presented as mean± SD of three independent experiments. * p<0.05, compared to the control group. # p<0.05 compared to the LPS-treated group.

Figure 4:
Enzyme-linked immunosorbent assay for PGE2 using Elisa Kit. The values are presented as mean± SD of three independent experiments. * p<0.05, compared to the control group. # p<0.05 compared to the LPS-treated group.

Figure 5:
Inhibition of LPS-induced pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) production by AGN in LPS stimulated RAW 264.7 cells. The values are presented as mean±SD of three independent experiments. * p<0.05, compared to the control group. # p<0.05 compared to the LPS-treated group

Anti-inflammatory medication development and research have made considerable use of inflammation models created by exposing macrophages to Lipopolysaccharide (LPS) exposure.¹³

LPS in Gram-negative bacteria’s outer membrane stimulates macrophages, which in turn causes them to generate large amounts of inflammatory mediators and pro-inflammatory cytokines when Gram-negative bacteria infect cells. Thus, it is common practice to test anti-inflammatory drugs using the inflammatory effects of Lipopolysaccharide (LPS) on macrophages.¹ An immunological response is primarily brought on by the activation of macrophages and Lipopolysaccharides (LPS) are a common agent that triggers a macrophage-mediated inflammatory response.² Therefore, RAW 264.7 macrophage cells were used for the examination in the current study. Numerous studies have demonstrated that when LPS is added to RAW 264.7 cells, which are macrophages generated from rodents, the levels of NO, PGE2 and other components related to inflammation rise. Arachidonic acid is converted by LPS-producing macrophages into PGE2, an inflammatory mediator.²

It may be possible to shield host tissues from the damaging impact of chronic or extreme inflammation by employing strategies for inflammation mitigation. The negative effects of anti-inflammatory medications that are currently on the market vary according on the patient, the length of treatment and the dosage.³ Plants have long been the source of a vast array of medicinally active ingredients that prolong human life. Many of these components belong to a unique class of natural chemicals known as Sesquiterpene Lactones (SLs) and have an α-methylene-γ-lactone structural motif. The Asteraceae family has a large number of these chemicals, which are recognized to have potential biological and medicinal properties. These properties include contraceptive, antihelminthic, antishistosomal and anticancer properties. Plant extracts high in SLs have drawn a lot of interest in the treatment of human illnesses. Due to their unique features, SLs in cancer clinical trials are able to selectively target cancer and tumor stem cells while avoiding normal ones. Arglabin from Artemisia glabella, artemisinin from Artemisia annua L and dimethyl amino-arglabin are used to treat lung, liver and ovarian malignancies in phase I and II clinical trials.

Figure 6:
The docking pose for the AGN enzymes complex was selected based on the binding affinity.

These are the SL medicines, or their derivatives, that are now undergoing clinical testing.³⁰

The active ingredient of the novel anticancer medication created in Kazakhstan is arglabin, a sesquiterpene lactone of the guaian type. According to toxicological research, the medication in lyophilized dosage form does not alter the morphological makeup of bone marrow or peripheral blood at maximally tolerated levels. Neither does it impact the functional state of the kidneys, liver, heart, or lungs. Subcutaneous, intramuscular, intravenous and abdominal injections of the medication do not cause local irritation. There were no discernible pyrogenic or allergic properties. Furthermore, arglabin is neither teratogenic, mutagenic, or embryotoxic. The medication remains in the body for about 22 hr, which is a rather long retention time. Arglabin enters the peripheral tissues quickly after exiting the central compartment (blood). The lungs and spleen contain the highest quantities of the medication within the first hour, while the liver and skeletal muscles contain the largest concentrations within 3 hr. The medication is more heavily concentrated and held in the liver for a longer duration of time compared to the other tissues. The medication can break through Blood Brain Barrier (BBB).³¹ The low water solubility of arglabin has a detrimental effect on the compound’s bioavailability and, in turn, its pharmacological efficacy. Therefore, enhancing the compound’s water solubility through structural change makes sense as a way to increase its bioavailability.¹¹

LPS-induced macrophage activation increases excessive NO output by breaking down L-arginine.¹ Short-lived free radical NO is an endogenous messenger that plays a number of roles in host defense, neurotransmission and vascular homeostasis. Nitric Oxide Synthase (NOS) converts L-arginine into NO.⁷ iNOS in macrophage cells controls the production of NO in response to immune system stimuli that cause inflammation in mammals.¹ Thus, in RAW 264.7 cells, the current study showed that arglabin significantly downregulated NO, which was elevated as a result of LPS. NO, which is generated by the catalysis of L-arginine by inducible Nitric Oxide Synthase (iNOS), is a crucial signal molecule in the body. Overabundance of NO can cause inflammatory disorders to develop. Therefore, one of the key strategies to manage the inflammatory response is to effectively restrict the excessive release of NO.⁶ First, we found that arglabin inhibited NO production.

Focusing on proteins linked to extracellular redox as a cancer treatment strategy has attracted increased and persistent interest in recent years. In fact, crucial cellular processes, interactions between cells in the microenvironment and the operation of ion channels and cell surface receptors are all regulated by the extracellular as well as intracellular redox state. Crucially, thiol-based antioxidants were found to decrease arglabin’s biological activity. Remarkably, ROS can also cause reversible alteration of redox-sensitive cysteines in physiological settings. Nevertheless, arglabin causes a delay in the generation of ROS in cancer cells, coinciding with the damage caused in lysosome and mitochondria.¹² One small but extremely reactive molecule is ROS. ROS buildup can trigger a variety of defense mechanisms in intracellular activities. Moreover, ROS are byproducts of regular metabolism in cells. Overabundance of ROS can upset the equilibrium between the pro- and anti-oxidative systems.⁶ They are organic byproducts of oxygen metabolism and are crucial for maintaining homeostasis and cell signaling. However, in some pathological and infectious conditions, excessive ROS formation can lead to oxidation of nucleic acid and protein and can have detrimental impacts on the structure of cells. New studies have revealed that ROS can cause inflammation by inducing the secretion of several cytokines through the activation of the MAPK signaling pathway.¹ Inflammation is one of the many additional clinical disorders that is closely linked to increased ROS generation. Intracellular ROS generation is accelerated by the entrance of macrophages, which is facilitated by inflammatory mediators and cytokines. Additionally, ROS take part in the inflammatory signaling cascade and function as secondary messengers. Hence, arglabin’s capacity to scavenge free radicals may be the reason behind its potent suppression of ROS in RAW 264.7 macrophages induced with LPS. The substantial anti-inflammatory effects of arglabin may be explained by the inhibition of pro-inflammatory mediators and cytokines that arglabin mediated ROS production inhibition may possibly potentially block. Mononuclear macrophages secrete TNF-α, which triggers the cytokine pathway in inflammatory reactions, which induces macrophages to produce IL-6.⁶ One of the most significant pro-inflammatory cytokines, TNF-α, has a wide range of cellular actions and is essential for the initiation and maintenance of inflammation. IL-6 is another cytokine that stimulates inflammation. These cytokines are implicated in tissue damage and multiple organ failure and are in charge of the acute phase response.²⁹ Arglabin has a strong immunomodulatory potential and restores the manufacture process of cytokines that inhibit inflammation, such as TNF-α, IL-4 and IL-1β. It also supports research findings on the system of interferon and cytokines in breast cancer patients.³¹ Immune regulation of inflammation can be achieved by inhibiting this, according to numerous prior research.² Our study findings showed that arglabin reduced TNF-α and IL-6 levels.

Arglabin effectively prevents the production of prostaglandin PGE2, which is primarily generated by the enzyme Cyclooxygenase-2 (COX-2). PGE2 is involved in several physiological and pathological processes and acts as an inflammatory mediator and immunological regulator.⁶

In the previous investigation, it was demonstrated that suppression of growth and reduced viability by arglabin in different cancer cells in human, includes cells from advanced prostate cancer that were originating from several metastatic locations and had variable tolerance to androgens. Notably, this compound successfully suppressed both anchorage-dependent and anchorage-independent 2D and 3D prostate cancer cell formation and proliferation after long-term treatment.³¹ In our present work, arglabin found to have significant effect on cell viability dose dependently.

Our findings demonstrated that arglabin strongly inhibits IL-6, NO, TNF-α, ROS and PGE2, pointing to the potential use of arglabin as an anti-inflammatory drug. Treatment with arglabin is anticipated to have an anti-inflammatory effect on LPS-induced cells, consistent with these results. Therefore, the biological activity of arglabin derivatives will be studied in order to establish the principles of focused search for novel compounds with anticancer, antimicrobial, anti-inflammatory and immunomodulatory characteristics. These principles will then be used to build highly effective medications.³¹

The current study’s objective was to elucidate the compound arglabin’s inhibitory effect on the gene involved in the EGFR signalling pathway. Table 2 lists the physicochemical characteristics of the substances found using SwissADME. According to Lipinski’s Rule of Five, which analyses if a chemical has a specific pharmacological or biological action that would make it an orally active medicine in humans, the compound arglabin demonstrated strong binding affinity as well as drug-like properties. The compounds have molecular weight below 500 Daltons and have less than five hydrogen bond donors and ten hydrogen bond acceptors. The goal of the current in silico investigation was to find effective of anti-cancer compound. Arglabin’s bioactivity was assessed using molinspiration by measuring its activity against a GPCR ligand, a kinase inhibitor, an ion channel modulator, a protease inhibitor, a nuclear receptor ligand and an enzyme inhibitor.

A molecule with a bioactivity score greater than 0.00 is thought to have significant biological activities, while values ranging from -0.10 to 0.00 are thought to be moderately active and values less than -0.10 are thought to be inactive.²⁸ The predicted bioactivity by molinspiration is shown in Table 3. The compound arglabin selected for the present study has an acceptable range of kinase inhibitors, enzyme links, ion channel modulators and protease inhibitors.

The ADMET profile was analysed with the tool admetSAR to determine the pharmacodynamic study of arglabin to understand the drug’s action inside a host’s body. The ADMET study focused on the parameters that can define absorption, distribution, metabolism, excretion and toxicity, Human Intestinal Absorption (HIA), Solubility (LogS), CaCO-2 permeability, P-glycoprotein substrate inhibition, cytochrome substrate/inhibitor, AMES toxicity and acute rat toxicity (LD₅₀).The compound showed optimal solubility with a value of 2.006, which is higher than 4 (>4) and other immensely satisfying results such as being non-toxic and non-carcinogenic, as shown in Table 4. The ability to penetrate the BBB and HIA was also depicted in the admetSAR predictions.³¹ Arglabin is found to be a potentially active compound against the EGFR signalling pathway gene in anti-cancer employing molecular docking. The inhibitory mechanism was looked into and the essential residues in the binding pocket were found. Arglabin has been discovered to have a binding energy of -5.3 kcal/mol, (PDB code: 4URO) (Table 5 and Figure 6). The lower the binding energy, the greater the ligand’s affinity for the receptor. Consequently, the ligand with the greatest affinity might be use as the potential drug for further studies.

Table 5:
Binding interactions of AGN with 4URO.

Following a thorough examination of all docked conformations with arglabin, specific interactions with EGFR (PDB code: 4URO) were discovered, as four hydrogen bonds with ASP 81, SER 128, ASN 54, ARG 144 ARG 200, ARG 84, GLU 58, PRO 87 ALA 98, ILE 102 & ASP 89 of the compounds is present in the active site region of EGFR, as shown in Figure 1. The other interactions, such as Pi-Pi, Pi-Anion, Pi-Alkyl and Van der Waals interactions of the compound arglabin were presented in Figure 1. These interactions help to lock the arglabin inside the substrate-binding pockets and thus effectively inhibit the EGFR signalling pathway gene in anti-cancer activity. After being tested in vitro, the analysis concluded that the arglabin’s binding and therapeutic properties make it a promising candidate for developing a potential anti-cancer inhibitor.

The findings showcase that arglabin exhibited strong anti-inflammatory properties on RAW 264.7 macrophages. By decreasing the relevant expression, arglabin treatment in RAW 264.7 macrophages induced with LPS drastically reduced the production of NO, TNF-α and IL-6. There was a correlation found between arglabin’s capacity to suppress the inflammatory response and a decrease in intracellular ROS generation. Arglabin had a major impact on the viability of cells. The study’s findings are in favor of arglabin as a substitute medication that may be used to treat inflammatory illnesses in a way that is both secure and efficient. To confirm its role as a regulator of macrophage activation and to comprehend the exact molecular processes governing the anti-inflammatory effect using an animal model, more research is required.

The authors declare that there is no conflict of interest.