Inflammation studies are currently one of the principal hubs of scientific research worldwide. Looking for novel substances for alleviating inflammatory ailments is still a subject of intense interest. Considering the associated adverse effects with the presently used anti-inflammatory medications, improvement of drugs from natural compounds is the product strategy for the treatment of inflammatory response (Cordaro et al. 2020, Zhang et al. 2020).
For a preliminary screening and understanding of the anti-oedematous and anti-inflammatory effects of TQ, the model of xylene-induced ear edema and inflammation was utilized. In agreement with previous reports (Akhtar &Shabbir 2019, Cui et al. 2020, Liu et al. 2020), the application of xylene on the ear edema in our study resulted in excess release of inflammatory mediators, inflammatory cell infiltration, local vasodilatation, capillary permeability, and edema. In contrast, pre-treated mice with TQ displayed a significant decrease in ear edema that indicates the suppressive effect of TQ on capillary permeability, edema development, and inflammation in mice ears. Demirel et al. (Demirel et al. 2018) reported that topically applied TQ resulted in a marked decrease in tissue edema in acute otitis externa in rats.
Pain is an unpleasant sensation that is associated with inflammation and generated by PGE2, histamine, serotonin, bradykinin, and inflammatory cytokines like TNF-a, IL-1b, IL-6, and IL-8 in the peritoneal fluid (Liu et al. 2020). For assessment of the peripheral antinociceptive effect of TQ, acetic acid-triggered abdominal writhing was established. Injection of acetic acid-induced tissue damage and release of inflammatory mediators such as histamine, bradykinin, and PG that in turn arouse nociceptors (Karim et al. 2019, Paradee et al. 2021). This process involves the activation of COX that catalyzes the conversion of arachidonic acid to PGE2 at the peritoneal receptors (Moharram et al. 2021). However, TQ antagonized acetic acid-induced-shriveling in the abdominal muscles that advocates its peripheral anti-nociceptive effect via the inhibition of inflammatory mediators and the PG pathway. It was formerly stated that administration of TQ relieved notably the formalin-induced pain in rats and the antinociception was mediated through the NO/cGMP/KATP channel pathway (Parvardeh et al. 2018). Also, N. sativa fixed oil, including TQ possesses appreciable anti-inflammatory and analgesic effects (Mahboubi et al. 2018). Amin et al. (Amin et al. 2014) found that TQ alleviated markedly the neuropathic pain resulting from chronic constriction of the sciatic nerve in rats.
Carrageenan mediated-paw edema is a well-known experimental model for the acute inflammatory response in addition to screening the anti-inflammatory potential of natural compounds and synthetic chemicals (Liu et al. 2020). In this model, carrageenan injection in the mice's rear paws resulted in intense inflammatory events with a biphasic phenomenon (Mehrzadi et al. 2021). The early phase (0 to 1 h post-injection) is characterized by over secretion of serotonin, bradykinin, and histamine. These mediators initiate tissue edema through the encouragement of the local blood flow and an increase in the capillary permeability (Zahra et al. 2020). The delayed phase (after 1 h and peaked after 8 h) is associated with leukocytic migration and prostaglandin (Zhang et al. 2020). As previously reported (Mehrzadi et al. 2021, Zahra et al. 2020), our study revealed a visible increase in the paw volume after intraplantar injection of carrageenan as compared to that of the control untreated paws. However, pre-treatment of mice with TQ in doses of 50 and 100 mg/kg decreased significantly the edema volume of the paws injected with carrageenan. Likewise, indomethacin exhibited a significant anti-edema effect and reduced paw volume of the induced paws. These results imply the potent anti-inflammatory effect of TQ that is probably because of the inhibition of mediators of inflammation.
In accordance with former studies, our results revealed notable increases in WBCs count and platelet levels in carrageenan-injected mice in comparison with the control (Akhtar &Shabbir 2019, Shabbir et al. 2018). These results indicated the activation of the immune system against tissue injury caused by foreign agents (Akhtar &Shabbir 2019). Treatment with TQ normalized the levels of WBCs and platelets, which reflects its potential immunomodulatory effect. Our results are in harmony with earlier reports (Gülmez et al. 2017a, Nemmar et al. 2011), which revealed the normalization of disordered hematological markers by TQ in experimental models of otitis media and arthritis in rats.
CRP, a sensitive acute-phase protein, is a key inflammatory mediator that is secreted by hepatic tissue after being stimulated with cytokines such as IL-1, IL-6, and TNF-α (Hamsa &Kuttan 2011). It indicates the extent of tissue inflammation and its increase is a predictive risk factor for the occurrence of organ damage (Jisha et al. 2019). In this study, carrageenan injection increased the CRP level in paw tissue that indicating a strong inflammatory response of the injured tissue and this agrees with former findings (Jisha et al. 2019, Zhang et al. 2020). On the contrary, the level of CRP displayed a notable decline in TQ-treated groups which explains its powerful anti-inflammatory property. Treatment of arthritic rats with TQ decreased significantly the levels of CRP in an experimental model of rheumatoid arthritis (Arjumand et al. 2019).
In inflammatory conditions, excess ROS is the culprit of cellular and organ damage (El-Shitany &Eid 2019a, Mehrzadi et al. 2021). In agreement with former authors (Alblihed 2020, Mehrzadi et al. 2021), marked increments were noticed in the levels of MDA accompanied by notable decreases in GSH levels and SOD, CAT, GR, and GPx activities in the paw tissues of carrageenan-injected mice. In addition, carrageenan injection resulted in notable increases in the levels of Nrf2 in the cytoplasm with decreases in the nucleus in the paw tissue of the model group. RT-PCR analysis that was performed by El-shitany and Eid revealed that carrageenan injection significantly decreased the Nrf2 expression of mRNA (El-Shitany &Eid 2019b). Remarkably, the treatment of mice with both TQ and indomethacin before carrageenan counteracted ROS overproduction and lessened the oxidative deleterious effects of carrageenan in mice paw tissue. These results are in accordance with former reports (Al Aboud et al. 2021, Dera et al. 2020, Dur et al. 2016, Kundu et al. 2013). Because of its potent radical scavenging ability, TQ alleviates the cellular oxidative stress and lessens the consequent lipid peroxidation (Al Aboud et al. 2021). Supporting former studies (Dera et al. 2020), the antioxidant efficacy of TQ may refer to the elevation of Nrf2 levels and its nuclear translocation along with blockage of Keap1 and induction of HO-1 expression. Our finding revealed that TQ at both tested doses decreased the cytoplasmic Nrf2 and increased its nuclear levels in the paw tissue. Normally, Nrf2 is attached to Keap1 in the cytosol, but under stress conditions of excess ROS production, it moves to the nucleus and binds to the antioxidant response element (ARE), and initiates the activation of cytoprotective enzymes (Albarakati et al. 2020, El-Khadragy et al. 2021, Yuan et al. 2020a). It was reported that TQ treatment abrogated the lung inflammation in a rat model of lung fibrosis through enhancement of the Nrf2/HO-1 signaling pathway (Ahmad et al. 2020). Modulation of Nrf2 signalling by TQ administration was also reported previously in different animal models (Amin et al. 2021, Hamdan et al. 2019, Sabir et al. 2022).
Furthermore, excessive ROS generation is strongly related to the progression of inflammation following carrageenan injection by activating immune cells and secretion of pro-inflammatory mediators (Alblihed 2020). In our study, carrageenan injected mice displayed noteworthy elevations in levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in paw tissue in comparison with control mice and this agrees with former studies (Alblihed 2020, Cui et al. 2020, Mehrzadi et al. 2021). These inflammatory cytokines are produced by macrophages and monocytes, fibroblasts, and endothelial cells in situations of inflammatory reactions and cellular stress (Zhang et al. 2020). TNF-α, a pro-inflammatory cytokine, is involved in the regulation of the secretion of other inflammatory mediators (Liu et al. 2020, Mehrzadi et al. 2021). Also, IL-1β is a pro-inflammatory cytokine, that enhances both local and systemic immune reactions (Mehrzadi et al. 2021). Furthermore, IL-1β and TNF-α trigger the expression of various enzymes, such as COX-2 with subsequent production of prostaglandins as PGE-2 (Alblihed 2020, Zhang et al. 2020). COX-2/PGE-2 signaling pathway plays an important role in various inflammatory diseases (Alblihed 2020). In the present study, intraplantar injection of carrageenan-induced the production of inflammatory mediators such as COX-2 and PGE-2, as reported in former studies (El-Shitany &Eid 2019a, Mitrea et al. 2020, Zhang et al. 2020). In contrast, TQ treatment attenuated notably all these inflammatory mediators in paw tissue, which indicates its potent anti-inflammatory effect and this is in harmony with previous authors (Kundu et al. 2013, Su et al. 2016, Wang et al. 2015). TQ was reported to decrease the protein and gene expression of COX-2 in mice skin exposed to phorbol ester (Kundu et al. 2013). The reduction of these pro-inflammatory cytokines represents a key target to control and regulate the development of acute and chronic inflammation (Alblihed 2020).
It has been reported that both IL-1β and TNF-α trigger the production of inducible nitric oxide synthase (iNOS), resulting in the development of inflammatory responses via the production of NO (Alblihed 2020, Mehrzadi et al. 2021). Our results revealed high NO levels in paw tissue injected with carrageenan which is similar to other reports (El-Shitany &Eid 2019a, Zhang et al. 2020). NO is an intercellular molecule that maintains the tissue homeostasis of numerous physiological functions (Alblihed 2020). However, high NO levels under pathological circumstances liberate peroxynitrite radicals that result in severe cellular damage such as oxidation of DNA and peroxidation of lipid molecules with subsequent tissue oxidative injury and inflammatory events (Zhang et al. 2020). Besides, earlier reports stated that high NO enhances the liberation of arachidonic acid from the cellular membrane which accordingly activates COX-2 and PGE-2 formation (Alblihed 2020, Zhang et al. 2020). Further, elevated NO levels increase the COX-2 half-life via the production of free radicals and inhibition of COX-2 auto inactivation (Alblihed 2020). Similar to previous reports (Saghir et al. 2019, Wang et al. 2015), TQ pre-treatment decreased the NO levels in inflamed tissue which is assumed by the regulation of inflammatory cytokines and the iNOS signalling pathway.
Regulation of inflammatory reaction is a complex process related to several pathways such as the NF-κB signalling pathway that controls the expression of various pro-inflammatory mediators (El-Shitany &Eid 2019a). In accordance with former studies (El-Shitany &Eid 2019a, Liu et al. 2020, Mitrea et al. 2020), this study found a significant increment in paw NF-κB levels in the carrageenan group compared to the control. During pathological conditions, NF-κB translocates into the nucleus to control the release of different inflammatory mediators (Cui et al. 2020). Accordingly, the elevations in levels of inflammatory cytokines such as TNF-α, IL-1β, IL-6, NO, COX-2, and PGE2 are attributed to the activated NF-κB in the inflamed paw tissue following carrageenan injection (Alblihed 2020, Cui et al. 2020, Zhang et al. 2020). This in turn explains the anti-inflammatory ability of TQ as evidenced by low levels of NF-κB in TQ-treated mice. Former authors stated the ability of TQ to subside tissue inflammatory response by deactivation of NF-κB and MAPKs signaling (Arjumand et al. 2019, Dera et al. 2020, Wang et al. 2015). Also, TQ was reported to lessen the nuclear translocation and the DNA binding of NF-kB by hindering the phosphorylation and consequent degradation of IkB-α in mice skin exposed to phorbol ester (Kundu et al. 2013).
Acute inflammation is characterized by adhesion and infiltration of white blood cells, particularly neutrophils. MPO is a protein secreted from neutrophils and is a reliable index to assess the degree of neutrophil infiltration (Abdel-Lateff et al. 2020). Its reaction with H2O2 and halides results in hypochlorous acid that possesses a strong oxidant with microbicidal activities (Alblihed 2020). However, a high level of MPO is indicative of tissue injury in acute inflammatory response (Cui et al. 2020). In harmony with former reports (Abdel-Lateff et al. 2020, Cui et al. 2020, Zhang et al. 2020), this study revealed marked increases in MPO activity and MCP-1 levels in paw edema tissues. This suggests that carrageenan resulted in infiltration of neutrophils in inflammatory sites as confirmed by histological alterations. MCP-1 is a vital chemokine that controls the immigration and infiltration of monocytes/macrophages at the inflamed foci (Zhang et al. 2020). Elevated MCP-1 levels were recorded following carrageenan injection in a model of acute pleurisy; demonstrating the infiltration of leukocytes in the inflamed tissues (Lansley et al. 2017). In this study, mice that received TQ displayed low levels of MPO and MCP-1 in inflamed paw tissues and this is similar to former reports (Al Aboud et al. 2021, Karaca et al. 2017). TQ administration decreased the levels of hepatic MCP-1 in sepsis injury in BALB/c mice and MCP-1 levels of murine microglia cells treated with lipopolysaccharide (Taka et al. 2015, Wang et al. 2019). These findings further demonstrate the TQ protection against inflammation through blocking the migration and infiltration of immune cells in inflamed tissue as validated by the inhibition of MPO and downregulation of proinflammatory cytokines including MCP-1.