Anti-inflammatory Effects of Empagliflozin and Gemigliptin on LPS-Stimulated Macrophage via the IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 Signalling Pathways

Background Sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors are glucose-lowering drugs whose anti-inflammatory properties have recently become useful in tackling metabolic syndromes in chronic inflammatory diseases, including diabetes and obesity. We investigated whether empagliflozin (SGLT2 inhibitor) and gemigliptin (DPP-4 inhibitor) improve inflammatory responses in macrophages, identified signalling pathways responsible for these effects, and studied whether the effects can be augmented with dual empagliflozin and gemigliptin therapy. Methods RAW 264.7 macrophages were first stimulated with lipopolysaccharide (LPS), then cotreated with empagliflozin, gemigliptin, or empagliflozin plus gemigliptin. We conducted quantitative RT-PCR (qRT-PCR) to determine the most effective anti-inflammatory doses without cytotoxicity. We performed ELISA and qRT-PCR for inflammatory cytokines and chemokines and flow cytometry for CD80, the M1 macrophage surface marker, to evaluate the anti-inflammatory effects of empagliflozin and gemigliptin. NF-κB, MAPK, and JAK2/STAT signalling pathways were examined via Western blotting to elucidate the molecular mechanisms of anti-inflammation. Results LPS-stimulated CD80+ M1 macrophages were suppressed by coincubation with empagliflozin, gemigliptin, and empagliflozin plus gemigliptin, respectively. Empagliflozin and gemigliptin (individually and combined) inhibited prostaglandin E2 (PGE2) release and COX-2, iNOS gene expression in LPS-stimulated RAW 264.7 macrophages. These three treatments also attenuated the secretion and mRNA expression of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, and proinflammatory chemokines, such as CCL3, CCL4, CCL5, and CXCL10. All of them blocked NF-κB, JNK, and STAT1/3 phosphorylation through IKKα/β, MKK4/7, and JAK2 signalling. Conclusions Our study demonstrated the anti-inflammatory effects of empagliflozin and gemigliptin via IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 pathway downregulation in macrophages. In all cases, combined empagliflozin and gemigliptin treatment showed greater anti-inflammatory properties.


Introduction
Certain aspects of chronic low-grade inflammation in obesity and metabolic syndrome-related diseases, such as hypertension, atherosclerosis, and diabetes mellitus, have been studied [1]. Some antidiabetic drugs exert anti-inflammatory properties that can be mediated by directly or indirectly regulating the inflammatory response, and recent studies show that sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors may exert potential antiinflammatory functions [2][3][4].
Sodium-glucose cotransporters are a family of active glucose transporter proteins expressed in bacteria and animals, including 12 human genes [5,6]. SGLT1 is expressed in numerous organs, including the small intestine, kidney, brain, heart, and immune cells [7][8][9][10][11], while SGLT2 is predominantly expressed in the kidney [12]. SGLT2 inhibitors were initially known to improve hyperglycemia through glycosuria by competitively inhibiting both SGLT1 and SGLT2 [13]. These antidiabetic agents have been recently reported to promote M2 macrophage polarization with an anti-inflammatory phenotype and to attenuate the production of proinflammatory cytokines on diabetic cardiomyopathy and nephropathy in mice [14,15]. Two recent studies have shown evidence of SGLT2 protein expression in RAW 264.7 (mouse macrophage cell line) [16] and Kupffer (hepatic stellate macrophage) cells [17].
Combination therapy of SGLT2 and DPP-4 inhibitors has a stronger glucose-lowering effect than either monotherapy in type 2 diabetes patients due to different mechanisms and complementary effects [34]. This treatment strategy is expected to provide additional anti-inflammatory effects to reduce disease progression and risk of complications. However, the molecular mechanisms of SGLT2 and DPP-4 inhibitors for inflammatory disease have not been fully verified. Furthermore, it is rare to investigate the anti-inflammatory effect after direct treatment of macrophages with a combination of these two agents. Therefore, we evaluated the impact of SGLT2 inhibition, DPP-4 inhibition, and simultaneous SGLT2 and DPP-4 inhibition on proinflammatory response in LPS-stimulated macrophages and their potential mechanism.

Materials and Methods
2.1. Cell Culture and Reagents. RAW 264.7 murine macrophage cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The macrophages were cultured in high-glucose Dulbecco's Modified Eagle's medium (DMEM; Welgene Inc., Daegu, South Korea) supplemented with 10% (v/v) fetal bovine serum (FBS; Grand Island, NY, USA) and antibiotics (10 μg/mL streptomycin, and 100 IU/mL penicillin) at 37°C and 5% CO 2 in a humidified atmosphere of 95% air. These cells were cultured to ≥85% confluence before treatment with empagliflozin (AdooQ Bioscience, Irvine, CA), gemigliptin (LG Life Sciences Ltd., Seoul, South Korea), lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma-Aldrich, St. Louis, MO, USA), or dexamethasone (Sigma-Aldrich). Empagliflozin and gemigliptin were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and added to the cell culture at the desired concentrations. The final DMSO concentration did not exceed 0.1%, and all samples were incubated with the same amounts of DMSO. LPS and dexamethasone were dissolved in phosphate-buffered saline (PBS; Welgene Inc.), and all additives were used as cotreatments following a 2-h period of cell starvation.
2.2. Cell Viability Assay. A Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan) was used to measure the cytotoxicity of the regents on RAW 264.7 macrophages. After incubation at 37°C for 24 h in 96-well plates at a density of 5 × 10 4 cells/well, the cells were treated with reagents at 37°C for 48 h. Then, the cells were washed, and 10 μL CCK-8 solution was added to each well, followed by incubation at 37°C for 2 h. The absorbance at 450 nm was determined by a microplate absorbance reader (Bio-Rad, Hercules, CA, USA). Treated cell viability was assessed as a percentage of the absorbance values compared to the control (untreated) cells.

Flow Cytometry
Analysis. RAW 264.7 macrophages were collected after treatment for 48 h, and the cells were washed in PBS/2% FCS (flow cytometry staining buffer). The Fc region was blocked by incubating with anti-FcRII/III monoclonal antibodies (mAbs, clone 2.4G2; Invitrogen, Carlsbad, CA) and 10% normal mouse serum for 15 min on ice. Then, the cells were stained for 45 min on ice with FITC anti-CD80 (B7-1; Invitrogen) and fixed in PBS/1% paraformaldehyde. Twenty thousand cells were analysed on a FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA), and data were analysed using FlowJo Vx (TreeStar Inc., Ashland, OR, USA).

RNA Isolation and Quantitative Real-Time Reverse
Transcriptase-Polymerase Chain Reaction (qRT-PCR) Analysis. The total RNAs were extracted using the RNAiso Plus reagent (Takara Bio, Otsu, Japan) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from RNA using avian myeloblastosis virus (AMV) reverse transcriptase (BEAMS Biotechnology, Seongnam, South Korea), and random 9-mer primers, then amplified qPCR using primer sets (Takara Bio Inc., Shiga, Japan) specific for mouse COX- . Quantitative real-time PCR was performed using SYBR Green Master Mix (Takara Bio Inc., Tokyo, Japan) on a Takara TP-815 instrument. Relative expression levels were determined in comparison with the control, using GAPDH as an internal control.

Statistical Analysis.
All experiments were conducted in triplicates, and the data were presented as the means ± standard deviation. The results were analysed using Student's t-test, and results with p values of ≤0.05 were considered statistically significant.

Empagliflozin and Gemigliptin Inhibited PGE 2
Production and Attenuated COX-2 and iNOS mRNA Expression in LPS-Activated RAW 264.7 Macrophages. LPS induces the macrophage activation and the release of proinflammatory mediators, such as PGE 2 and nitric oxide (NO), by upregulation of COX-2 and iNOS mRNA expression [37][38][39]. We confirmed that PGE 2 production increased significantly in RAW 264.7 macrophages stimulated with LPS compared to cells without LPS induction. In contrast, empagliflozin and gemigliptin (individually and combined) inhibited PGE 2 production in LPS-activated RAW 264.7 macrophages (Figure 2(a)). COX-2 mRNA and protein expression also increased markedly after LPS stimulation. Consistent with the decrease in PGE 2 protein, the LPSinduced upregulation of COX-2 mRNA expression was attenuated after treatment with empagliflozin or gemigliptin ( Figure 2(b)). mRNA expression of another important inflammatory enzyme, iNOS, was also downregulated by empagliflozin or gemigliptin (Figure 2(c)). Consequently, COX-2 and iNOS proteins were reduced in LPS-stimulated RAW 264.7 macrophages treated with empagliflozin or gemigliptin, indicating that the reduction of PGE 2 protein was due to the suppression of the gene responsible for expression (Figures 2(d) and 2(e)).

Empagliflozin and Gemigliptin Reduced LPS-Induced
Proinflammatory Cytokines and Chemokines in RAW 264.7 Macrophages. We confirmed increased production of inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, in the supernatant of LPS-treated RAW 264.7 macrophages [40]. These cytokines were reduced when the macrophages were treated with empagliflozin or gemigliptin, and an additional reduction was observed in empagliflozin plus gemigliptin-treated macrophages (Figure 3(a)). LPSstimulated RAW 264.7 macrophages naturally upregulated the mRNA expression of proinflammatory cytokines (TNF-3 Journal of Immunology Research α, IL-1β, and IL-6) and chemokines (CCL3, CCL4, CCL5, and CXCL10). Treatment with empagliflozin or gemigliptin reversed the mRNA overexpression of these cytokines and chemokines in LPS-induced RAW 264.7 macrophages (Figures 3(b) and 3(c)). The effect of the empagliflozin plus gemigliptin combination was significantly greater than that of empagliflozin or gemigliptin alone.
3.4. Impact of Empagliflozin and Gemigliptin on NF-κB, MAPK, and STAT Signalling Pathways in RAW 264.7 Macrophages. LPS activated various transcription factors associated with the inflammatory response in RAW 264.7 macrophages. Therefore, we conducted Western blot analysis of NF-κB, MAPKs, and STAT signalling pathways to clarify the potential mechanisms of action of SGLT2 and DPP-4 inhibitors in the LPS-induced release of proinflammatory mediators, such as cytokines and chemokines [41][42][43]. We also used dexamethasone, a well-known anti-inflammatory reagent, as a positive control to compare the efficacy of empagliflozin and gemigliptin [44,45].
NF-κB is a crucial transcription factor for proinflammatory mediators in macrophages [46]. NF-κB phosphorylation was suppressed in RAW 264.7 macrophages treated with empagliflozin or gemigliptin. We also confirmed that empagliflozin and gemigliptin (individually and combined) inhibited IKK phosphorylation, which is a central regulator of NF-κB activation, in LPS-activated RAW 264.7 macrophages (Figure 4(a)).
MAPKs mediate biological processes and cellular responses to external stimuli, such as LPS [42,47]. The JNK, p38, and ERK phosphorylation significantly increased in RAW 264.7 macrophages after LPS induction. Neither empagliflozin nor gemigliptin affected p38 and ERK phosphorylation; meanwhile, empagliflozin and gemigliptin (individually and combined) markedly attenuated JNK phosphorylation in RAW 264.7 macrophages activated by LPS. This effect of JNK phosphorylation was more pronounced in empagliflozin treatment (Figure 4(c)). To further determine the MAPK kinases involved in the JNK pathway inhibition, we examined MKK4 and MKK7, which cooperate in JNK activation. Empagliflozin significantly suppressed both MKK4 and MKK7 phosphorylation in RAW 264.7 macrophages stimulated by LPS, while gemigliptin mainly inhibited MKK7 activation (Figure 4(d)).
During inflammation, a host defense mechanism against various harmful stimuli, activated macrophages play an important role by producing several proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, and inflammatory mediators, including NO and PGE 2 [48]. However, chronic low-grade inflammation may result in metabolic syndrome, diabetes, cardiovascular disease, and cancer [49][50][51][52]. Currently, antidiabetic drugs are in the spotlight for their antiinflammatory properties [53]. There is a meta-analysis to show C-reactive protein and proinflammatory cytokine IL-6 reduction in people treated with at least one SGLT2 or DPP-4 inhibitor among patients confirmed with coronavirus infection [54].
This divergence of empagliflozin according to the cell lines could be explained by the different distributions of SGLT1 and SGLT2, and the different concentrations used 1 μM in HUVECs vs. 80 μM in RAW 264.7 macrophages. Xu et al. [17] recently confirmed SGLT2 protein expression in RAW 264.7 macrophages, and in another study, the selectivity for SGLT2 of empagliflozin (half maximal inhibitory concentration, IC 50 3.1 nM) was similar to that of canagliflozin (IC 50 2.7 nM), but the selectivity for SGLT1 (IC 50 8,300 nM) was much weaker than that of canagliflozin (IC 50 710 nM) [56].
We also confirmed that empagliflozin inhibited NF-κB phosphorylation like other SGLT2 classes. Additionally, we investigated another inflammatory signalling STATs pathway to elucidate an anti-inflammatory mechanism. Empagliflozin inhibited STAT1 and STAT3 phosphorylation and also suppressed its upstream JAK2 activation. Subsequently, we verified that empagliflozin blocked IKK, which is upstream kinase of NF-κB. However, it only suppressed JNK signalling without affecting p38 and ERK and naturally inhibited MKK4 and MKK7, which are upstream molecules of JNK. Empagliflozin inhibited the MKK4/JNK pathway more significantly than gemigliptin.

Pro-inflammatory mediators
Only a few in vivo study reports on the antiinflammatory effects of the combination of SGLT2 and DPP-4 inhibitors. Dual therapy, empagliflozin and linagliptin, synergistically ameliorated hepatic fibrosis and inflammation by reducing mRNA expression of proinflammatory cytokines in mouse liver [59]. Another combination therapy with dapagliflozin and saxagliptin ameliorated diabetic cardiomyopathy and diabetic nephropathy in mice by inhibiting proinflammatory cytokines and NLRP3/ASC inflammasome production [15,64]. However, no studies have investigated whether the combination of SGLT2 and DPP-4 inhibitors directly affects the inflammatory response and its signalling pathways involved in macrophages. We clarified for the first time that the dual therapy, empagliflozin and gemigliptin, has stronger anti-inflammatory effects in RAW 264.7 macrophages.

Conclusions
In summary, we verified that empagliflozin and gemigliptin individually possess anti-inflammatory activity by reducing PGE 2 and proinflammatory cytokine production, resulting from the inhibitory effects of COX-2, iNOS, cytokine, and chemokine mRNA expression in RAW 264.7 macrophages. These results were mediated by blocking the IKK and NF-κB, MKK4/7 and JNK, and JAK2 and STAT1/3 signalling pathways. MKK4/JNK phosphorylation was prominently inhibited by empagliflozin, while JAK2-STAT1/3 activation was primarily suppressed by gemigliptin. All these antiinflammatory effects were enhanced when empagliflozin was combined with gemigliptin compared to empagliflozin or gemigliptin treatment alone. This study is the first to show the direct effect of the anti-inflammatory mechanisms of empagliflozin and gemigliptin on macrophages and presents the stronger anti-inflammatory properties of combination therapy.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon reasonable request.