The dichlorofluorescin (DCF) assay is a cell-based method used to investigate the scavenging of radicals in addition to cell-free assays. Within this assay, a fluorescent dye binds to free radicals inside cells if respective radicals are not detoxified beforehand by antioxidative substances or proteins. To determine scavenging of radicals, DCQAs as single substance were applied to different cell types of both, human and rodent origin together with a radical forming oxidant. These studies revealed PoDs in the range of 1–3 µM for 1,3-DCQA (Pérez-García et al., 2000; Pagano et al., 2016; Tong et al., 2017). The range of PoDs for 3,4- and 3,5-DCQA were quite large, spanning 0.01–200 µM (Kang et al., 2011; Kim et al., 2012; Liang and Kitts, 2018), and only one study was available that applied 4,5-DCQA with a PoD of 200 µM (Liang and Kitts, 2018). Due to the wide range of PoD values, a comparison across DCQA isoforms cannot be made (Figure 4B). It is noteworthy that the PoD of 200 µM detected by Liang and Kitts (2018) represents the lowest applied concentration. Therefore, it is possible that lower concentrations also induce an antioxidant effect detected by the DCF assay.

The inhibition of lipid peroxidation by oxidants determined by measurement of malondialdehyde (MDA) is an adverse process leading to cellular damage with subsequent pathologies. The detection of MDA was used to investigate the antioxidative potency of DCQAs mainly after applying a noxious substance such as tert-butylhydroperoxide (TBHP) or lipopolysaccharide (LPS). The inhibition of TBHP- or LPS- induced lipid peroxidation by DCQAs as single substance has been investigated (Figure 4B) in human hepatocytes and mitochondria as well as microsomes from rat liver homogenate (Gebhardt and Fausel, 1997; Psotová et al., 2003; Zha et al., 2007). Gebhardt and Fausel (1997) applied TBHP to primary hepatocytes to induce oxidative stress. Subsequently, selected compounds, including 1,3-DCQA, were applied to the cell culture to investigate the antioxidant activity of the test items. The study found that 1,3-DCQA had an EC₅₀ value of 23.6 µM, which was the lowest compared to caffeic acid, chlorogenic acid, and cynaroside with EC₅₀ values of 45, 35.3, and 62.4 µM, respectively (Gebhardt and Fausel, 1997). Psotová et al. (2003) assessed the antioxidant activity by investigating TBHP-induced lipid peroxidation in the mitochondrial fraction of rat liver homogenate dependent on the co-incubation with phenolic substances. The results indicated that 1,3-DCQA exhibited an IC₅₀ value of 1.96 mM, whereas protocatechuic, caffeic, rosmarinic, ferulic and chlorogenic acid revealed IC₅₀ values of > 2, 0.59, 0.09, > 2, and 2.37 mM, respectively (Psotová et al., 2003). Lipid peroxidation in microsomes derived from rat liver homogenate following ascorbate-Fe²⁺-induced oxidative stress was investigated for its prevention by 3,5-DCQA. Results indicated a significant and dose-dependent decrease in MDA levels already at 5 µM 3,5-DCQA (Zha et al., 2007). Regarding this endpoint, 3,5-DCQA seemed to be more active than 1,3-DCQA. Besides in vitro, Wang and Xiao (2019) induced acute lung injury to mice using LPS and treated this model with 5, 10, and 20 mg of 3,5-DCQA via intraperitoneal injection. Within this study, MDA level in lung tissue of the acute lung injury model strongly increased and dose-dependently decreased after application of 3,5-DCQA. The decrease was significant after applying 5 mg and returned to basal MDA levels at 20 mg 3,5-DCQA (Wang and Xiao, 2019). In addition to testing single substances, DCQAs containing extracts were tested in vivo for their effect on lipid peroxidation by determining MDA concentration (Yin et al., 2018; Li et al., 2019). This strengthened the observation, that DCQA-rich extracts induce the antioxidant activity in vivo.

Glutathione (GSH) is a peptide with well-established antioxidant properties in cells. It is present in two forms: the reduced form, GSH, and the oxidized form, glutathione disulfide (GSSG). The ratio of GSH to GSSG is a crucial parameter in assessing cellular oxidative stress levels (Franco and Cidlowski, 2012). The impact of DCQAs as single substance on the cellular GSH level has been assessed in various studies (Figure 4B) in vitro (Kim et al., 2005; Kang et al., 2011; Liang and Kitts, 2018). Within these studies, the cellular GSH content of cells derived from liver (HepG2), colon (Caco-2), and bone tissue (SH-SY5Y) was determined after an oxidative stimulus and subsequent DCQA incubation. The PoDs obtained were determined to be 5 µM (Kang et al., 2011), 25 µM (Kim et al., 2005), and 200 µM (Liang and Kitts, 2018) for 3,5-DCQA as well as 200 µM for 3,4- and 4,5-DCQA (Liang and Kitts, 2018). It must be noted, however, that the PoD of 200 µM across these studies is due to the circumstance of 200 µM being the lowest applied concentration in the respective study by Liang and Kitts (2018). Hence, the range of PoDs from 5–200 µM for 3,5-DCQA can be explained by the choice of concentrations in one specific study. Due to the wide range of applied concentrations and limited data on 1,3-, 3,4-, and 4,5-DCQA no comparison on the activity of DCQA isoforms can be made. In addition to in vitro experiments, 4,5-DCQA was administered orally at doses of 5, 10, and 20 mg/kg bw to mice of a NASH model over the period of 4 weeks. The GSH level in the liver decreased significantly in the NASH model compared to a normal mice group, whereas the additional treatment with 4,5-DCQA resulted in a dose-dependent increase of liver GSH starting at 5 mg/kg bw. At 20 mg/kg bw the basal GSH level was nearly reconstituted (Liu et al., 2019b). Even though this model does not represent a lung-specific disease model, the authors suggest a similar effect during inflammation of the respiratory tract after systemic application of DCQAs. Furthermore, the effect of DCQA-containing extract on cellular GSH levels was determined in oxidative-stimulated keratinocytes (HaCaT) and hepatocytes derived from both human and rats origins (Gebhardt and Fausel, 1997; Yin et al., 2018; Li et al., 2019; Gao et al., 2021; Zheleva-Dimitrova et al., 2023). Overall, the in vitro and in vivo data indicates that DCQAs reconstitutes the cellular GSH level after applying an oxidative trigger.

Another mechanism of antioxidative defense is the expression of antioxidative proteins such as superperoxide dismutase (SOD), catalase (CAT), or heme oxygenase (HO-1). The protein levels of different antioxidant acting proteins have been evaluated in vitro and in vivo applying single substances as well as DCQA-containing extracts. Cao and colleagues (2017) applied 100 µM 3,5-DCQA to kidney epithelial cells (Vero cells) after infecting the cell culture with EV71 virus. Viral inoculation resulted in a decreased expression of GSH metabolic enzymes, i.e., glutathione reductase (GR), glutathione peroxidase (GPX), and glucose-6-phosphate dehydrogenase (G6PD) (Rahman et al., 1999). Treating the cells with 100 µM 3,5-DCQA returned basal protein levels of GSH homeostasis-related proteins (Cao et al., 2017). In addition, the impact of 3,5-DCQA on the expression of HO-1 on 3T3-L1 cells after treatment with a hormonal cocktail (MDI) was investigated. Expression of HO-1 on a protein level was elevated after incubation with 10 µM 3,5-DCQA compared to a MDI-treated control (Raineri et al., 2021). Regarding in vivo studies, within a mouse model of acute lung injury induced by LPS applying 3,5-DCQA by intraperitoneal injection at doses of 5, 10 or 20 mg SOD levels in the lung were clearly decreased within the acute lung injury model and was elevated statistically significant after treatment of 10 and 20 mg 3,5-DCQA (Wang and Xiao, 2019). The effect on antioxidative proteins (SOD, GPX, CAT) was also investigated using DCQA-containing extracts in vivo (Kwon et al., 2019; Li et al., 2019). Taken together, these data suggest that DCQAs induce antioxidant activity not only through their ability to scavenge free radicals, but also by inducing the expression of proteins critical for cellular antioxidant defense mechanisms. As summarized by Zuo and Wijegunawardana, ROS play an important role in the pathogenesis of respiratory diseases such as COPD, asthma, and inflammation-associated disease as their further enhance ROS production (Zuo and Wijegunawardana, 2021). Therefore, not only scavenging of ROS by DCQA but also the activation antioxidative stress response can have a beneficial impact in the treatment of respiratory diseases.

IL-1β represents an IL-1 type cytokine and therefore a major mediator of innate immune reactions. Indicated as pro-inflammatory cytokine, its activation results in cellular cascades via NF-κB and MAPK pathways and subsequent expression of various target genes, e.g., IL-6, COX-2 or IL-1b (Weber et al., 2010). The alleviation of an IL-1β-mediated inflammatory response by DCQAs has been evaluated applying 1,3-, 3,5-, and 4,5- DCQA to several rodent- (RAW264.7, BMDMs, J774A.1, BV2) and human-based (EA.hy926, MH7A) cells on a transcriptional and protein level after incubation with stimuli, i.e., LPS, IFN-γ, TNFα, or ox-LDL. mRNA expression of IL-1β after stimuli was decreased by all applied DCQAs with PoDs ranging from 1–290 µM for 1,3-DCQA, of 10–200 µM for 3,5-DCQA, and of 10 µM for 4,5-DCQA (Kim D. B. et al., 2022; Park et al., 2022; Wu et al., 2022; Zheng et al., 2022; Tang et al., 2023; Yang et al., 2023). Comparing this data, the lowest observed PoD is similar for all DCQAs, however a relatively wide range for 1,3- and 3,5-DCQA is apparent. Expression of IL-1b after induced inflammation was verified in vitro on a protein level (Figure 6A) in MH7A cells and bone marrow-derived macrophages (BMDMs) for 3,5-DCQA (PoDs: 10–20 µM) and 4,5-DCQA (PoD: 10 µM) after incubation with LPS or ox-LDL (Noh et al., 2022; Zheng et al., 2022; Yang et al., 2023). Here, no relevant difference in PoDs between DCQAs and thus no difference in biological activity, was observed. A decrease of IL-1β after a pro-inflammatory stimuli was also confirmed in vivo for 3,5-DCQA. In a lung specific model, a decrease in IL-1β secretion after intraperitoneal injection of 5 mg to an acute lung injury model in mice was observed (Wang and Xiao, 2019; Tang et al., 2023). This further indicates the anti-inflammatory properties of DCQAs in vitro and in vivo.

IL-17A is involved in various pathological outcomes, including acute and chronic respiratory diseases (Gurczynski and Moore, 2018). Yang et al. (2023) observed a decrease of the IL-17A protein level in MH7A and RAW264.7 cells after a pro-inflammatory trigger with TNFα or LPS, respectively, and subsequent 3,5-DCQA treatment with 20 µM and higher (Yang et al., 2023). Furthermore, IL-17 is able to induce TNFα in epithelial, endothelial, and fibroblastic cells. The lung is one of many organs which is affected by TNFα within inflammatory diseases, e.g., chronic bronchitis, COPD, and asthma. TNFα-dependent inflammation is supposed to be based on deregulation of leukocytes and lymphocytes recruitment as well as depletion of the cellular antioxidant GSH and hence the induction of oxidative stress (Mukhopadhyay et al., 2006). Therefore, TNFα secretion is one parameter to evaluate the anti-inflammatory potential of DCQAs. The impact of DCQAs on TNFα within stimulated cell systems has been investigated in various studies (Figure 6A). 3,4- and 4,5-DCQA were applied to stimulated RAW264.7 resulting in a decrease of TNFα secretion (Kim et al., 2020; Jang et al., 2021). Furthermore, primary rat chondrocytes were treated with 4,5-DCQA, respectively, resulting in a similar observation of decreased TNFα protein level (Jang et al., 2022; Park et al., 2022). Taken together, PoDs of 50 μM and 4–10 µM were determined for 3,4- and 4,5-DCQA, respectively. This indicates, that 4,5-DCQA seems to obtain a higher biological activity regarding inhibition of TNFα secretion compared to 3,4- and 3,5-DCQA. An impact of 3,5- and 4,5-DCQA on the TNFα expression was also observed within in vivo studies. In more detail, TNFα protein expression was markedly decreased in lung tissue as well as in bronchoalveolar lavage fluid (BALF) of an acute lung injury mice model after intraperitoneal injection of 5 mg and 25 mg/kg bw 3,5-DCQA, respectively (Chen et al., 2016; Wang and Xiao, 2019). Thus, in vivo data confirms the potential of DCQAs to inhibit TNFα, and therefore an important pro-inflammatory mediator.

IL-6 represents another pro-inflammatory cytokine with an important role in respiratory diseases associated with inflammation but also in the pathogenesis of asthma and potentially COPD (Rincon and Irvin, 2012). It binds to its receptor IL-6R which subsequently associates with gp130 resulting in an activation of specific JAK tyrosine kinases, following phosphorylation, and activation of transcription factors such as STAT3. Furthermore, IL-6 dependent activation of C/EBPβ via the MAPK pathway is known (Rincon and Irvin, 2012). On a transcriptional level, reduction of LPS-induced IL-6 expression was observed in vitro after treatment with 1,3- and 3,5-DCQA at PoDs of 290 µM and 100 μM, respectively (Park et al., 2022; Wu et al., 2022; Tang et al., 2023). Comparing this data, no relevant difference across cell systems and DCQAs were obvious considering that Wu and colleagues (2022) only used one concentration of 290 µM which explains the higher PoD of 1,3-DCQA. IL-6 expression was further evaluated in vitro on a protein level resulting in a similar observation of decreased IL-6 levels in inflammatory cell models after DCQAs incubation. In more detail, PoDs for reduced IL-6 expression ranging between 25–50 µM for 3,4-DCQA, at 50 µM for 3,5-DCQA, and a range of 2–50 µM for 4,5-DCQA were obtained (Li Y. et al., 2013; Kim et al., 2020; Jang et al., 2021; Park et al., 2022). The comparison of PoDs suggests that protein expression seems to be more sensitive than transcriptional assessment of IL-6 and further that 4,5-DCQA is suggested to be biologically slightly more active than 3,4- and 3,5-DCQA (Figure 6A). IL-6 secretion was also investigated in vivo after treatment of inflammation models with 3,5-DCQA. Most importantly, two studies investigated IL-6 secretion in lung tissue and BALF after intraperitoneal injection of 3,5-DCQA in a LPS-induced acute lung injury model in mice. While the IL-6 protein level in lung tissue was already significantly reduced applying 5 mg 3,5-DCQA (Wang and Xiao, 2019), the IL-6 level in BALF was markedly decreased at a dose of 25 mg/kg bw (Chen et al., 2016). The in vivo data confirms the anti-inflammatory properties in regard of inhibition of IL-6 secretion observed in vitro. The observation of reduced IL-6 in stimulated cell and animal models treated with DCQAs is in line with the observation on the downstream IL-6-dependent activation of STAT3. Treatment with 3,5-DCQA resulted in a decrease in phosphorylated and thus activated STAT3 in stimulated rodent cells at concentrations as low as 100 µM (Park et al., 2022; Tang et al., 2023). Furthermore, 3,5-DCQA at a dose of 30 mg/kg bw reduced the protein level of phosphorylated STAT3 in the spinal cord tissue of a complete Freund’s adjuvant-induced inflammatory model in mice (Park et al., 2022).

Lastly, the protein level of the chemokine CCL2, which is suggested to be involved in regulation of neutrophilic lung inflammation (Mercer et al., 2014), was evaluated in vivo. Wang and Xiao (2019) applied 3,5-DCQA via intraperitoneal injection to an acute lung injury model in mice induced by LPS and observed a decrease of CCL2 secretion after an administration of 5 mg 3,5-DCQA i. p. (Wang and Xiao, 2019).

Besides the inhibition of cytokines, DCQAs impact further inflammatory mediators, such as iNOS expression, NO production, COX-2 expression, and PGE₂ expression. Under physiological conditions the nitric oxide synthase (NOS) produces endogenous NO via a constitutive NOS isoform. During inflammatory processes, however, an inducible NOS (iNOS) isoform is expressed and results in an excess of NO, which can regulate expression of certain cytokines and chemokines (Speyer et al., 2003). An enhanced iNOS expression has been associated with several inflammatory diseases of the lung, e.g., COPD, asthma, and acute respiratory distress syndrome (Huang et al., 2015). The impact of DCQAs on iNOS expression on a transcriptional and protein level within inflammatory triggered cell models has mainly been evaluated applying rodent cell models. mRNA expression was decreased within inflammatory BMDMs, RAW264.7, and J77A.1 cell models after incubation with DCQAs with PoD of 290 µM for 1,3-DCQA (Wu et al., 2022), ranging between 100–200 µM for 3,5-DCQA (Tang et al., 2023), and of 10 µM for 4,5-DCQA (Zheng et al., 2022). On a protein level inflammatory models of RAW264.7 cells, primary rat chondrocytes, and BMDMs were treated with 4,5-DCQA resulting in PoDs of a range between 2–200 μM, respectively (Puangpraphant et al., 2011; Jang et al., 2021; Jang et al., 2022; Park et al., 2022; Zheng et al., 2022). Xia et al. (2014) assessed the impact of 1,3-DCQA treatment in iNOS expression in a cytokine-induced inflammatory model of human coronary artery smooth muscle cells observing a decrease of induced iNOS expression at 10 µM for both, transcriptional and protein expression (Xia et al., 2014). Taken this information together, it is indicated that protein expression is more sensitive than transcription of iNOS without a relevant difference of biological activity between evaluated DCQAs. The expression of iNOS was also evaluated in vivo on a protein level. Here, the protein level in lung was decreased after administration of 5 mg 3,5-DCQA applied via intraperitoneal injection to an acute lung injury model in mice (Wang and Xiao, 2019; Jang et al., 2021). The in vivo data emphasizes potential to inhibit iNOS observed in vitro.

Besides expression of iNOS, the downstream endpoint NO production has been assessed in numerous studies. Excessively produced NO by iNOS is indicated to be involved in constriction, inflammation and remodeling of the lung observed in asthma, hence are associated with adverse effects (Prado et al., 2011). Therefore, reduction of increased NO is suggested to be beneficial for inflammatory respiratory diseases. NO production in vitro was mainly assessed in LPS-stimulated RAW264.7 cells with PoDs ranging from 8–80 µM for 3,4-DCQA, 3–40 µM for 3,5-DCQA, and 4–40 µM for 4,5- DCQA (Chen et al., 2015; Tian D. et al., 2020; Kim et al., 2020; Jang et al., 2021; Li et al., 2022). Within IL-1β-stimulated primary rat chondrocytes a PoD of 10 µM was observed after 4,5-DCQA treatment (Jang et al., 2022). Taken together, no isoform-dependent biological activity was observed for this endpoint.

COX-2 is an inducible isoform of cyclooxygenase which catalyzes the cyclooxygenation of arachidonic acid to prostaglandin G₂ and subsequently H₂. The promotor region of COX-2 contains various transcriptional regulatory elements such as the cAMP response element (CRE) or for NF-κB (Park and Christman, 2006). Hence, an induction or repression of COX-2 can be related to an activation or inhibition of the NF-κB signaling pathway. Protein expression of COX-2 was evaluated in vitro in various studies, mainly applying RAW264.7, BMDMs but also in BV2 cells and primary chondrocytes derived from rats. All cell models were first treated with a pro-inflammatory trigger, i.e., LPS, IL-1β, or ox-LDL and subsequently with 3,5- or 4,5-DCQA as single substance. In all test systems a decrease of COX-2 protein expression compared to the effects of sole inflammation-inducing agent was apparent with PoDs ranging between 2 and 20 µM for 4,5-DCQA (Puangpraphant et al., 2011; Jang et al., 2021; Jang et al., 2022; Park et al., 2022; Zheng et al., 2022). Comparing protein expression PoDs and biological activity across DCQAs, no relevant difference is apparent. Data on COX-2 expression was also available for an in vivo study. Wang and Xiao (2019) observed a reduction of COX-2 protein level in lung tissue after applying 5 mg 3,5-DCQA intraperitoneally to an acute lung injury model in mice. Therefore, the in vitro determined amelioration of COX-2 induction after inflammatory stimuli was confirmed in vivo for the two isoforms tested.

The same pattern as for COX-2 has been observed for a reaction product of COX-2, prostaglandin E₂ (PGE₂). Within LPS- or IL-1β-stimulated rodent cell models 3,4-, 3,5-, and 4,5-DCQA reduced the PGE₂ level with PoDs ranging without differences regarding the isoforms from 2–20 µM (Tian K. et al., 2020; Jang et al., 2021; Jang et al., 2022).

The occurrence of inflammation is common in broad span of respiratory diseases, e.g., acute/chronic bronchitis, asthma, or COPD. It was shown that INF-α, INF-γ, TNFα, IL-10 and IL-6 were enhanced in plasma after influenza virus infection (Kaiser et al., 2001). More importantly, IL-6 and TNFα plasma and nasopharyngeal lavage level correlated with symptom scores as well as temperature values (Kaiser et al., 2001). Hence, the reduction of such cytokines by DCQAs can strongly contribute to the reduction of symptoms and play an important role in the treatment of inflammation-related respiratory diseases. Besides, increase of the cytokine IL-17A results in enhanced IL-6 levels, which further increases MUC5AC and MUC5B protein levels (Kim and Criner, 2013). These proteins play a crucial role in the mucus and enhanced expression can be seen as increased mucus production. Hence, decreasing IL-17A as well as IL-6 by DCQAs is a valid treatment option for symptom relief. Furthermore, IL-17 and IL-6 play an important role in Th2-low asthma and therefore a co-treatment with DCQAs might also be beneficial in reducing inflammation during this severe respiratory disease (Ramakrishnan et al., 2019; Habib et al., 2022). As previously stated the induction of iNOS expression correlates with several inflammatory diseases of the lung, e.g., COPD, asthma, and acute respiratory distress syndrome (Huang et al., 2015). Furthermore iNOS-triggered NO inducues mucus secretion in allergic asthma and also results in enhanced ROS levels (Lee et al., 2021). Therefore, reduction of iNOS expression, as observed after treatment with DCQAs, can result in a beneficial effect in treatment of respiratory diseases. Last but not least, COX-2 is expressed in response to various pro-inflammatory cytokines and mediators. Hence, induction of COX-2 is associated with the pathology of inflammation-related respiratory diseases (Rumzhum and Ammit, 2016). Again, the treatment with DCQAs can contribute in a positive way for respective respiratory diseases.

The NF-κB pathway is supposed to play an important part within the pathogenesis of inflammatory diseases. It can be activated by a variety of stimuli such as cytokines, oxidative stress, and UV radiation. It obtains numerous downstream targets, which includes cytokine expression. Evidence on the role of NF-κB signaling pathway in inflammation associated respiratory diseases, e.g., asthma and COPD, is convincing, therefore an impact on this pathway can be a gate opener for the use as therapeutic substance within these diseases (Edwards et al., 2009). As a brief summary, activation of this pathway includes phosphorylation of IκBα by IKKα with subsequently ubiquination of IκBα (Figure 7). This process results in a release of the NF-κB subunits p50/p65 from IκBα followed by their translocation in the nucleus and respective transcriptional activity (Edwards et al., 2009). The impact of DCQAs on the NF-κB pathway subsequent to its activation has been investigated in vitro on several levels (Figure 6B). Tang et al. (2023) evaluated the impact of 3,5-DCQA on the phosphorylation of IκBα in J774A.1 and RAW264.7 cells after stimulation with LPS and IFN-γ. Hereby, a decrease of IκBα phosphorylation after 3,5-DCQA treatment was observed at 100 µM. Furthermore, the same observation was made for 4,5-DCQA after treatment of rodent macrophages with pro-inflammatory triggers at PoDs of 2 and 10 µM (Jang et al., 2021; Zheng et al., 2022). In addition, IL-1β-stimulated and 4,5-DCQ-treated primary rat-derived chondrocytes showed an increase of IκBα with a simultaneous decrease of phosphorylated IκBα in the cytosol at 40 µM 4,5-DCQA (Jang et al., 2022). Furthermore, nucleus translocation of p65 was evaluated in human- and rodent-based cell systems on protein level. Jang et al. (2022) observed simultaneously an increase of cytosolic p65 and decrease of p65 in the nucleus in stimulated RAW264.7 cells and primary rat-derived chondrocytes after treatment with 4,5-DCQA at concentrations of 4 and 20 µM (Jang et al., 2021). A decreased translocation of p65 in the nucleus was also observed within LPS-stimulated EA.hy926 cells after treatment with 5 µM 1,3-DCQA (Kim D. B. et al., 2022). Lastly, a decrease in phosphorylated p65, which is associated with enhanced transcription activity, in stimulated rodent-derived cell models after treatment with 3,5- and 4,5-DCQAs at concentrations of 100 and 10 μM, respectively, was observed (Zheng et al., 2022; Tang et al., 2023). Overall 4,5-DCQA seems to obtain a higher activity in regard of NF-κB deactivation, however data is limited to a few studies applying 3,5-DCQA to investigate this endpoint. Hence, no appropriate comparison regarding this endpoint can be made. Besides in vitro data, on respiratory tract specific study is available evaluating the potential of 3,5-DCQA to inhibit NF-κB activation in vivo. After intraperitoneal injection of 5 mg 3,5-DCQA to an acute lung injury model induced by LPS in mice resulted in a decrease of phosphorylated p65 within lung tissue, suggesting less NF-κB-dependent transcriptional activation (Wang and Xiao, 2019). All in all, these data indicates that DCQAs alleviates the activation of the NF-κB pathway on several levels potentially regardless of the isoform.

The MAPK pathway represents a signaling pathway, which is involved in several cellular processes, including inflammation. The p38 subgroup of mitogen-activated protein kinases (MAPKs), which include also extracellular signal-regulated kinases (ERK) and c-Jun NH₂-terminal kinases (JNK), are a group of MAPKs that are strongly activated by cytokines and chemokines (Figure 7). Further, activation of p38 MAPKs is suggested to be associated with the pathogenesis of COPD (Renda et al., 2008). Several studies investigated the potential of DCQA to alleviate the activation of the MAPK pathway after an induced inflammation by LPS in vitro. The level of phosphorylated p38, and therefore activated p38, was reduced in LPS-triggered RAW264.7 and EA.hy926 cells after treatment with 1,3- and 4,5-DCQA at concentrations of 1 and 4 μM, respectively (Jang et al., 2021; Kim D. B. et al., 2022). Furthermore, the levels of phosphorylated JNK and ERK in LPS-treated RAW264.7 cells were decreased by 3,5- and 4,5-DCQA at 1 and 4 μM, respectively (Jang et al., 2021; Yang et al., 2023). Taken together, these data indicates the potential of DCQAs to alleviate the activation of the MAPK signaling pathway, regardless of the applied isoform.

The NLRP3 (NLR family pyrin domain containing 3) inflammasome is a protein complex that belongs to the group of pattern recognition receptors, which recognize pathogen-associated molecular patterns (PAMPs) as well as damage-associated molecular patterns (DAMPs). These patterns subsequently activate mechanisms to eliminate the respective stimuli, e.g., infections, and/or repair tissue. Activation of the NLRP3 inflammasome occurs as response to various stimuli, e.g., pathogens. Briefly, the activation process itself is a two-step procedure. First, priming occurs via a variety of ligands resulting in an activation of NF-κB with subsequent enhanced NLRP3, pro-IL-1β, and pro-IL18 expression. Secondly, the protein complex of the inflammasome assembles, including the final activation of NLRP3 with subsequent cleavage of pro-IL-1β and pro-IL18 by caspase 1 to the respective active cytokines and their extracellular secretion. During the recent years an association between activation of NLRP3 inflammasome and inflammatory respiratory diseases has been made, including allergic rhinitis, asthma, and COPD (Leszczyńska et al., 2022). The potential of 1,3- and 3,5-DCQA to alleviate the activation of the NLRP3 inflammasome has been evaluated in two in vivo studies. Wang and colleagues (2022) applied 3,5-DCQA via intraperitoneal injection to an acute lung injury model in mice and observed a decrease of NLRP3 protein in lung tissue after injection of 5 mg 3,5-DCQA (Wang and Xiao, 2019). Furthermore, 25 mg/kg bw intraperitoneal injected 1,3-DCQA to an arthritis model in mice induced by monosodium urate resulted in decreased levels of NLRP3 protein in the respective paw tissue (Wu et al., 2022). Taken together, it can be suggested that DCQAs reduce the activation of NLRP3 inflammasome, however data is scarce and further research needs to be done to elucidate the impact of DCQAs on this inflammatory relevant pathway.

Inflammatory-associated pathways such as NLRP3, NF-κB, and MAPK play a crucial role in inflammation-related respiratory diseases such as acute/chronic bronchitis, asthma, or COPD (Mishra et al., 2018; Leszczyńska et al., 2022; Panek et al., 2023). Therefore, a treatment with agents which can decrease the activation of these pathways, such as DCQA, can have a positive impact in the treatment of such respiratory diseases.