Natural medicine has been used for the treatment of various diseases since ancient times. Natural compounds contributed tremendously to immunomodulatory effects by influencing immune cells and their secretions (Ortuño-Sahagún et al., 2017). The immune system is greatly challenged by various life-threatening disorders such as cancer and metabolic disorders. Previous studies have reported that phytochemicals like propyl gallate can show immunomodulatory properties via the NFκB pathway and affect the migration of malignant glioma (T. Yang et al., 2017). The relevance of finding new immunomodulatory molecules with minimal side effects might help in better treatment or complement the existing immunomodulatory therapies thereby finding strategies to prevent/treat such immune-related disorders. Inflammation is a key pathology linking multiple disorders. Inflammatory cascade once activated produces inflammatory cytokines which when overproduced need to be suppressed by immunomodulatory molecules. Lipopolysaccharide (LPS) induced activation of macrophages is considered a widely accepted inflammatory model to study immunomodulatory effects of various molecules (S. Han et al., 2017). Herein, methyl 6,12-dimethyltridecanoate isolated from T. ornata was checked for immunomodulatory and anti-inflammatory potential via in silico and in vitro experimental analysis.
In silico molecular docking studies provide initial information about a drug-receptor interaction by providing binding conformation and affinity. Herein, methyl 6,12-dimethyltridecanoate isolated from T. ornata was docked with inflammatory proteins TNFα, NFκB, and COX-2. ET showed binding energy of -2.63, -15.2, and − 2.02 kcal/mol with TNFα, NFκB, and COX-2 proteins respectively. Standard anti-inflammatory drug dexamethasone showed comparable binding energy of -3.74, -20.05, and − 4.1 kcal/mol against TNFα, NFκB, and COX-2. TNFα is naturally produced by activated macrophages and can be activated by NFκB. COX-2 is another protein involved in immune cell activation and inflammation. Studies have revealed that activation of TNFα dependent signaling pathway plays a key role in the control and activation of COX-2 (Yu et al., 2016). Previous reports suggest that compounds from Ficus religiosa like Z-3-hexanol and z-3-hexenyl acetate showed a potent binding score against COX-2 indicating anti-inflammatory properties (Utami et al., 2020). This study corroborates with the current study suggesting the anti-inflammatory/immunomodulatory activity of ET from T. ornata.
Oxidative imbalance is a major outcome in inflammation-related disorders. Oxidative stress-induced inflammation may lead to chronic inflammatory scenarios via differential expression of inflammatory genes. Various proteins, lipid, DNA/RNA have been identified as the targets of oxidative stress and modification in these targets may lead to mutagenesis (. El-Kenawi, Ruffell, 2017). Excessive generation of reactive oxygen species (ROS) for a prolonged time may even alter the function of DNA/some proteins with irreversible damage leading to cell death (Hussain et al., 2016). Therefore, oxidative stress markers such as LPO, SOD, Catalase, and GSH were analyzed in the supernatant of LPS induced/uninduced RAW 264.7 cells. LPO (malondialdehyde), a highly reactive enol is the end product of lipid peroxidation. Herein, LPO levels were increased around 1.7-fold upon LPS induction, in comparison to the normal control (NC). LPO levels were diminished to around 1.3 and 1.5-fold respectively upon pre-treatment with MD and HD of ET. Both LD and HD of ET exhibited results comparable to dexamethasone, the steroidal drug. The current study substantiates the finding of a previous study wherein, Tilapia viscera hydrolysate suppresses malonaldehyde levels (Riyadi et al., 2020). Super oxides are the product of oxidative damage and if not regulated may cause cellular damage. Superoxide dismutase is an enzyme that catalyzes the conversion of superoxides to molecular oxygen and water. Lower SOD levels due to oxidative stress might be alarming and may lead to cellular and tissue damage. Moreover, SOD is also reported to attenuate LPS induced inflammation indicating the importance of oxidative markers during inflammatory pathways (Bowler et al., 2004). Catalase and GSH are other enzymes that protect against oxidative damage (Rezayian et al., 2019). Herein, SOD, catalase, and GSH levels were reduced by 1.6, 1.7, and 1.6-fold respectively upon LPS induction compared to the NC. SOD levels were increased around 1.4-fold in the ET HD pretreatment group with results comparable to dexamethasone, a steroidal anti-inflammatory agent. Our findings correlate to the previous literature suggesting an increase in SOD levels in LPS induced inflammatory model upon treatment with a phenolic and flavonoid-rich fraction of Salvia officinalis (Kolac et al., 2017). Moreover, catalase levels were also increased by 1.7 and 2-fold in ET MD and HD pretreatment groups compared to the LPS control. GSH levels were increased around 1.3-fold in ET MD and HD pretreatment groups compared to the LPS control. HD of ET showed results similar to the dexamethasone suggesting protection against oxidative stress induced damage. Our study is in line with a previous report which showed protective effects of paeoniflorin in LPS induced reaction by detoxification of catalase and GSH (Kim et al., 2010). Our study reports similar findings confirming protection against the LPS induced oxidative damage and indicates anti-inflammatory potential.
Various plant and marine algal isolates such as polyphenols, curcumin, resveratrol, alkenes, and fatty acid esters have been studied against inflammatory disorders (Recio et al., 2012; Pramitha et al., 216). Linoleic acid ester and 13-hydroxy linoleic acid ester from plant sources have been reported as biologically active anti-inflammatory compounds that suppress the secretion of cytokines in the LPS stimulated model (Kolar et al., 2019).
In the present study,
mRNA levels of pro-inflammatory (IL6) and anti-inflammatory (IL10) mediators were checked to study the anti-inflammatory potential of ET. Herein, IL6 levels were increased around 1.3-fold upon LPS induction compared to the NC group, which were reversed to near normal upon pre-treatment with LD, MD, and HD of ET. Conversely, 1L10 expressions were reduced by 1.5-fold on LPS induction, which were reversed to normalcy upon pretreatment with HD of ET. ET showed comparable results as that of dexamethasone, a proven glucocorticoid medication suggesting anti-inflammatory and immunomodulatory potential.
LPS induced ROS can alter the production of iNOS and NFκB proteins, which are major regulators of the inflammatory pathway. Activated NFκB regulates various events, resulting in autoregulation of inflammatory cascade. NFκB regulates the transcription of TNFα and IL6 which are involved in the pathogenesis of various inflammatory disorders (Fiordelisi et al., 2019). Previous studies suggest upregulation of iNOS and NFκB expression upon LPS induction (Lee et al.,2002). Similarly, in this study, iNOS and NFκB expressions were upregulated by 1.2 and 1.3-fold in the LPS induced group compared to the NC. Conversely, iNOS expression levels were downregulated around 1.2-fold in LD and HD groups upon pretreatment with ET. Similarly, NFκB expression levels were downregulated by1.2, 1.6, and 2.6-fold upon pretreatment with LD, MD, and HD of ET in comparison to the LPS control group confirming the immunomodulatory activity. Our study is in line with a previous study wherein, Orixa japonica extract inhibited LPS induced iNOS and NFκB in RAW 264.7 macrophages (Kang et al., 2011). The present study hereby establishes that methyl 6,12-dimethyltridecanoate (ET) isolated from Turbinaria ornata inhibits LPS induced inflammation via iNOS and NFκB pathway (Fig. 6).