In the present network pharmacological analysis, database-based screening of biological targets of 42 volatile active components from DME revealed final set of 117 target genes. Results of the GO enrichment and KEGG analysis of these target genes suggested that they are mainly involved in the regulation of inflammatory response, apoptotic process, regulation of cell proliferation, neutrophil extracellular trap formation, arachidonic acid metabolism, phospholipase D, HIF-1, and VEGF signalling pathways. Multiple signalling pathways like PI3K-Akt, TNF, HIF-1, and VEGF signalling pathways have been found to play a vital role in the therapeutic mechanism of DME against RA. These pathways are triggered by pro-inflammatory cytokines, resulting in immune-mediated inflammation that further leads to an aggravation of RA. In the arachidonic acid metabolism pathway, the PTGS2 gene is overexpressed by the modulation of TNF-α and nuclear factor κ beta (NF-κB) signalling pathways during the inflammatory cascades. Overexpression of the COX-2 enzyme, encoded by PTGS2, in the synovial tissues of RA patients causes the infiltration of inflammatory cells and synovial hyperplasia, resulting in joint degradation [63, 64]. Uncontrolled proliferation of synovial fibroblasts is characteristic of the pathology of RA. RA synovial fibroblasts (RASF) often exhibit hypoxia, and one of the main regulators of this is a hypoxia-inducible factor (HIF)-1 [4, 65, 66]. Along with pro-inflammatory cytokines, synovial cellular infiltration and invasive behaviour are triggered by VEGF, which is a critical regulator of angiogenesis in RASFs under hypoxic conditions [65]. Researchers recently discovered that blocking HIF-1 with RNA interference (RNAi) lowers the expression of inflammatory cytokines like IL-6, IL-1, and TNF in peripheral blood serum and synovial cell culture in a collagen-induced arthritis Wistar rat model [67]. Therefore, inhibiting the expression of PTGS2 and HIF signalling pathways in synovial cells might be therapeutically beneficial in reducing the inflammatory response in RA. Network pharmacology analysis also highlighted major phytoconstituents of DME having therapeutic effects such as 1,3,4,5-Tetrahydroxycyclo-hexanecarboxylic acid, n-Hexadecanoic acid, Octadecanoic acid, and Ergost-5-en-3-ol, (3 beta 24r) which have multiple targets in multiple pathways. This result supports the findings of Wink et al. (2015) which indicated that the activity of a medicinal plant was due to synergistic interactions of several phytoconstituents [68]. Our H-C-T-P network analysis established that DME may intervene in the inflammatory pathways through “multi-component-single target” or “multi-component multi-target” interactions to minimize the release of pro-inflammatory mediators and inflammatory cytokines. This finding further justifies the usage of DME as traditional medicine in the treatment of the chronic inflammatory disease like RA.
PPI network analysis revealed AKT1 as a central target protein in RA. It is well known that RA patients’ synovial tissues have abnormally active PI3K/AKT signaling pathways [69, 70]. Additionally, activation of the PI3K/AKT signaling pathway results in the aberrant production of various downstream effector molecules that have anti-apoptotic and pro-survival effects. AKT also activates the NF-κB pathway, and upon its’ activation, NF-κB additionally activates a vast spectrum of downstream genes and effectors molecules like COX-2 that further contribute to synovial proliferation and inflammation [71]. A previous study showed that curcumin, a plant-derived tetra-terpenoid obtained from Curcuma longa, can inhibit the activation of PI3K/AKT pathways by suppressing the insulin like growth factor or IGF1 (the upstream cytokine of PI3K/AKT pathway) in RA fibroblast-like synoviocyte (RA-FLS) cell line [72]. Inhibition of the PI3K/AKT also leads to decrease in TNF-α, IL-6, MMP-2, and MMP-9 production, which suppresses cell proliferation and invasion, and promote apoptosis in cultured RA-FLS [72]. From our H-C-T-P network and docking results it was found that the compounds like Gamma-Tocopherol, Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, Benzenepropanoic acid, 4-hydroxy-, methyl ester, 1,3,4,5-Tetrahydroxycyclo-hexanecarboxylic acid can interact with the AKT1 and can inhibit it. Several studies also reported the modulatory role of PI3K/AKT pathway and anti-inflammatory role by these compounds [73–75]. Thus, it is plausible to infer from these previous data that the inhibition of AKT1 might significantly attenuate the PI3K/AKT signalling-mediated chronic inflammation progression during RA. Apart from AKT1, the PPI analysis displayed that 10 signalling pathways were directly associated with RA development, suggesting that DME could modulate these pathways against chronic inflammation like RA. The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that promote ligand-dependent transcription of target genes which can regulate inflammation [76]. Most of the anti-inflammatory effects of the PPARs act by inhibiting NF-κB and activator protein (AP)-1, which ultimately repress the expression of downstream genes like PTGS2, inducible nitric oxide synthase (iNOS), IL-6, and IL-12 that are involved in the inflammatory responses [76]. PPARα has also been reported to control the duration and magnitude of the inflammatory response through its ability to induce the expression of genes encoding proteins that are involved in the catabolism of pro-inflammatory lipid mediators [77]. It was reported that compounds like squalene, benzeneacetic acid, and endogenous fatty acid like n-hexadecanoic acid, and octadecanoic acid act as a potential PPARA agonist in both in vivo and in vitro condition [78–80]. Our results indicate that 9,12-Octadecadienoic acid (Z, Z), 2-hydroxy-1-(hydroxymethyl) ethyl ester present in DME possibly interact with PPARA and PPARG in a significant way to control inflammation.
Even before the first clinical signs and symptoms appear in RA, activation of the innate system leads to an increase of effector molecules involved in RA-FLS aggression. Specific TLR2, TLR3, and TLR4 are highly expressed in the synovial fluid of RA patients, and their stimulation results in the activation of NF-κB pathway that leads to an increase in the production and upregulation of pro-inflammatory cytokines (such as TNF-α, IL-1, and IL-12), Type I interferon (Type I IFN), and several MMPs within the disease joint [63, 64]. Our differential expression analyses revealed that TLR4, PTGS2, VEGFA, MMP-9, and ESR1 were notably upregulated in the synovial samples of RA patients. Activated NF-κB regulates genes that contribute to inflammation, including TNF-α, IL-6, IL-8, iNOS, and PTGS2 (also known as COX-2) [81]. In the inflamed synovium of RA, unregulated inflammatory condition promotes synovial hyperplasia and angiogenesis [4]. ACE (Angiotensin-converting enzyme) is increased in RA-FLS that catalyses the formation of angiotensin II from its inactive precursor, angiotensin I, and then raises the angiotensin II concentration in RA patients that leads to synovial hypoxia by hypoxia-inducible factor (HIF-1α) [82]. Studies reported that induction of HIF-1α with TLR signalling also enhances the production of inflammatory cytokines (IL-6, IL-8, TNF-α), MMPs (MMP- 1, MMP-3, MMP-9), and VEGF, thereby escalating the inflammatory state within the RA synovium [65]. A positive-feedback regulation between HIF-1α and VEGF triggers angiogenesis in hypoxic conditions [65]. Several reports have demonstrated that the synergistic effects of plant extracts can modulate and downregulate inflammatory cytokines like iNOS, TNF-α, IL-6, and even COX-2 in animal models [17, 18, 83]. The apoptotic proteins that trigger the aberrant proliferation of RA synovial fibroblasts (RASFs) can be inhibited by RASFs [4, 84]. The apoptosis mechanism in osteoclasts of RA joint is regulated by the Bcl-2 protein family [85]. They directly contribute to the release of cytochrome c from the mitochondria, which triggers the activation of caspase (CASP)-3 and caspase-9 [86]. NF-κB, however, controls the overexpression of Bcl proteins in RA and this inhibition could potentially prevent RASFs and macrophages for undergoing apoptosis [87]. These changes in RA synovium have been linked to altered apoptotic response of synovial and inflammatory cells [4]. Our differential expression data also shows the down regulation of CASP-3 in the synovial samples of RA patients, confirming further the accuracy of the key target proteins identified using PPI network analysis. In summary, by interacting with these targets, active ingredients in DME may contribute to the treatment of RA.
To further document the potential effect of the active compounds on the key targets, in silico molecular docking analysis followed by MD simulation was performed. Docking results suggest that 1,3,4,5-Tetrahydroxycyclo-hexanecarboxylic acid showed the best ligand efficacy with interactions with TLR4 (-4.556 kcal/mol), AKT1(-7.283 kcal/mol), ACE (-7.451 kcal/mol), IL-6 (-5.091 kcal/mol), and CASP3 (-7.096 kcal/mol) proteins. Previous studies reported that TLR4 activates AKT1, which leads to the activation of NF-κB [88]. NF-κB, a transcription factor that promotes chronic inflammation, is inhibited by derivatives of 1,3,4,5-tetrahy-droxy-cyclo-hexanecarboxylic [89]. So, it is possible that 1,3,4,5-tetrahy-droxycy-clo-hexanecarboxylic acid suppressed the TLR4 mediated AKT1 activation to regulate NF-κB activation. Another phytocompound, 9,12-Octadecadienoic acid (Z, Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester, also showed a strong binding affinity with PPARA (-10.329 kcal/mol) and PPARG (-9.845 kcal/mol. Most of the anti-inflammatory effects of PPARs result from their inhibition of the NF-κB signaling pathway, which also inhibits the production of various genes implicated in the inflammatory response, including iNOS, IL-6, and IL-12 [76]. Additionally, inhibition of NF-κB could simultaneously lead to a decrease in COX-2, TNF-α production, and Caspase (CASP)-3 activation, decreasing the level of pro-inflammatory cytokines and promoting apoptosis in the inflamed synovium [81]. The protein-ligand docked complexes of AKT1, PPARA, and PPARG were subjected to MD simulation to assess the structural alteration of the complexes under dynamic circumstances. From our simulation study, AKT1 formed 13 hydrogen bonds and among those, Arg 86 and Lys 14 were also indicated in our docking studies (Fig. 5B). Similarly, PPARA formed 23 hydrogen bonds during the simulation studies, among them three hydrogen bonds (Tyr 314, His 440, and Tyr 464) were similar to docking studies. PPARG also formed 23 hydrogen bonds in the simulation study, and the hydrogen bonds of PPARG were also in line with docking analysis results. All three protein-ligand complexes developed good stability in the target protein’s active site pocket during the course of the simulation, indicating their modulatory actions. The RMSD values were calculated to analyze the structural alterations of the protein-ligand complexes, and both ligand compounds revealed minimal differences. In addition, all three protein-ligand complexes also had reduced RMSF values, indicating the presence of a secondary structure, such as a helix, which denotes the stability of bound structures.
Our in silico-based approach identified the key targets, key genes, and key pathways that could be modulated by volatile compounds of DME during RA amelioration. The impact of DME on controlling the expression of crucial gene targets, as identified by network-based investigations, has been further confirmed through experimental validation using RT-PCR. Our previous study showed that DME can attenuate inflammatory paw-edema, soft tissue swelling and bone erosion in experimental animal groups via modulating the different hematological, biochemical parameters when orally fed to arthritic rats [29]. Although stimuli like FCA are thought to activate the NF-κB pathway, they also increase the level of reactive oxygen species (ROS) and inflammatory cytokines, which in turn trigger immune cells to release more inflammatory cytokines and enzymes to aggravate arthritis [90]. According to several reports, PI3K/AKT pathway activation upregulates genes that support vascular permeability, thrombogenicity, and inflammatory cascades by activating the NF-κB pathway [72, 91]. Meanwhile, previous studies on adjuvant-induced arthritis model have shown that, activation of PPARG expression is linked with the anti-inflammatory activity by inhibiting Iκβ phosphorylation and thus inhibiting NF-κB nuclear translocation [76, 92]. Our gene expression studies depicted that, in experimental animal models, the active ingredients in DME can inhibit the core target protein AKT and through modulating PPARG, it can ultimately limit the production of Iκβ. The anti-inflammatory characteristics of PPARA and PPARG also result from their ability to prevent NF-κB translocation in the nucleus, which in turn suppresses the expression of downstream inflammatory response-related genes such COX-2, IL-6, and TNF-α inside the RA synovium (Fig. 10). Our current work revealed that pro-inflammatory cytokines (TNF-α and IL-6) and key inflammatory enzymes like COX-2 had their mRNA expression and protein level expression significantly downregulated in arthritic rats after oral DME administration, indicating the plant's immunomodulatory function. Moreover, upon treatment with DME, it significantly restored the oxidative stress parameters. Thus, it is plausible that DME’s reduction of oxidative stress is one of the primary mechanisms for the inhibition of COX-2 and other inflammatory cytokine genes production in RA condition.
This study combined network pharmacology and molecular structure-based approach to elucidate the molecular mechanism of DME and the results suggest that key target proteins related to anti-inflammation can be effectively modulated by multiple active components of DME. One of the probable mechanisms of DME in treatment of RA may be related to the inhibition of AKT1 which may significantly attenuate the PI3K/AKT signalling-mediated chronic inflammation progression during RA (Fig. 10). PPARA and PPARG are the other two significant factors which may be modulated by DME. Additionally, mRNA and protein level analysis from our in vivo study supported the anti-inflammatory anti-rheumatoid properties of DME.