Reduction of cardiomyocyte hypertrophy and inflammationDAPA reduces ISO-induced cardiomyocyte hypertrophy by decreasing ROS in co-treatment and post-stimulation conditions

Cardiomyocyte hypertrophy worsens cardiac functions and increases cardiovascular risks in patients with cardiovascular diseases. DAPA shows promise in reducing cardiovascular events in T2DM patients with pre-existing cardiovascular conditions [24,25,26,27].

To investigate the effect of DAPA on ISO-induced cardiomyocyte hypertrophy, we treated cardiomyocytes with both ISO and DAPA for 24 and 48 h, followed by an MTT assay, the colorimetric change was quantified using spectrophotometry and correlated with cell number. The ISO was administered at concentrations ranging from 2.5 to 20 µM, with the 20 µM concentration showing the most significant effect; thus, it was chosen to induce cardiomyocyte hypertrophy (Fig. 1A). Similarly, we conducted MTT assays for DAPA at 5, 10, and 20 µM concentrations. Since no significant differences were observed among these concentrations, we used 10 and 20 µM concentrations to evaluate their effects on cardiomyocytes (Fig. 1B). Treatment with ISO at both 10 µM (p < 0.0001) and 20 µM (p < 0.0001) for 24 and 48 h resulted in significant hypertrophy compared to the control group (Fig. 1C and D). Moreover, co-treatment of cardiomyocytes with ISO and DAPA for 24 h significantly reduced cell hypertrophy (Fig. 1E and F). Notable differences in cell structure were observed between control and ISO-treated cells (p < 0.0001), as well as between ISO-treated cells and cells treated with 10 µM (p < 0.0001) and 20 µM (p < 0.0001) of DAPA. Furthermore, when cardiomyocytes were first stimulated with ISO for 24 h to induce hypertrophy and then treated with DAPA (Fig. 1G and H), we observed a highly significant (p < 0.0001) reduction in hypertrophy in the pre-stimulated cardiomyocytes. Significant differences were noted between the control and ISO groups (p < 0.0001), as well as between the ISO group and those treated with 10 µM (p < 0.0001) and 20 µM (p < 0.0001) of DAPA. These findings indicate that DAPA protects against cardiomyocyte hypertrophy, whether administered simultaneously with ISO or after hypertrophic stimulation. In addition, we performed gene and protein expression study for the hypertrophy markers, atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP). We observed that ISO stimulation significantly increased the expression of ANP (gene expression: p = 0.0079; protein expression: p < 0.0001, Fig. 1I and N) and BNP (gene expression: p = 0.0012; protein expression: p = 0.0032,), compared to the control. However, co-treatment with DAPA and ISO mitigated these increases, as shown by the reduced levels of ANP (gene expression: p = 0.0095; protein expression: p < 0.0001) and BNP (gene expression: p = 0.0042; protein expression: p = 0.0133, Fig. 1J and N). This highlights DAPA’s role in preventing hypertrophy in ISO-induced cardiomyocytes [28, 29]. To our knowledge, various SGLT2 inhibitors have been investigated for cardiomyocyte-induced hypertrophy in humans and animal models [9, 28, 29]; however, no direct in-vitro studies have been conducted on cardiomyocytes stimulated by ISO.

Hypertrophic stimuli induce cardiomyocyte hypertrophy, often accompanied by increased ROS (Reactive oxygen species) and inflammation regulated by the AKT pathway. Activating the AKT pathway controls ROS levels and influences hypertrophy, fibrosis, and inflammation; thereby attenuating cardiac inflammation [30,31,32,33,34,35,36]. We examined ROS production in cardiomyocytes exposed to ISO stimulation, with or without prior treatment with DAPA. We found significant differences in ROS levels between groups treated with ISO alone and those receiving ISO + DAPA, highlighting the potential of DAPA in modulating oxidative stress and inflammation in cardiomyocytes. We investigated ROS production in cardiomyocytes exposed to ISO stimulation for 24 h, with and without prior treatment with DAPA for the same duration. ROS levels were measured at 2- and 4-hours post-stimulation. We found significant differences in ROS production between groups treated with ISO alone and those receiving ISO + DAPA at both 2 h (10 µM: p = 0.0016, 20 µM: p = 0.0006) and 4 h (10 µM: p < 0.0001, 20 µM: p < 0.0001) (Fig. 1O). These findings suggest that DAPA protects against cardiomyocyte dysfunction by reducing ROS levels [37]. Additionally, we explored the potential protective effect of DAPA by administering it to cardiomyocytes for 24 h before ISO stimulation. No significant differences were observed at 2 h, but a notable increase in ROS production was seen at 4 h in the DAPA pre-treated group (20 µM: p < 0.0001) compared to the control group. There was also a further significant increase in ROS production in the DAPA + ISO group (10 µM: p < 0.0001, 20 µM: p = 0.0009) (Fig. 1P). These findings indicate complex interactions between DAPA and ISO signaling pathways, with the timing of DAPA administration influencing ROS levels. Variations in signaling pathways and experimental conditions may also play a role. Further studies, with longer-duration DAPA treatment, may be required.

DAPA reduces ISO-induced cardiomyocytes by activating the AKT pathway, enhancing GLUT1 expression, and downregulating pro-fibrotic markers

Studies have demonstrated the reduction of hypertrophy, fibrosis, and inflammation, along with enhanced antioxidant and mitochondrial function, through PI3K/AKT signaling activation in cardiac cells with various hypertrophy induction methods, although not with ISO specifically [10, 15]. In the current study, we assessed the gene expression directly and indirectly linked to the AKT pathway. Cardiomyocytes were treated with ISO for 6 h, followed by 24 h of DAPA treatment, and gene expression analysis was conducted. The results indicate that PI3K expression significantly increased in both the ISO + DAPA (gene expression: p = 0.0104; protein expression p = < 0.0001) and DAPA (gene expression p = 0.0096) groups compared to the ISO-alone group, suggesting that DAPA enhances AKT signaling (Fig. 2A). However, no differences were observed in AKT gene expression (Fig. 2B). In contrast, our protein expression study showed a significant decrease in the pAKT/AKT ratio in the ISO-treated group (p = 0.0011) compared to control, while the addition of DAPA to ISO-treated cells significantly increased pAKT/AKT expression (p = 0.0001) (Fig. 2K). The increase in pAKT/AKT expression when DAPA was added to ISO-treated cells suggests that DAPA enhances the phosphorylation of AKT, rather than altering its total expression. This implies that DAPA may facilitate the activation of AKT through upstream mechanisms, such as promoting PI3K activity, leading to increased phosphorylation of AKT. AKT activation is regulated through phosphorylation, therefore, DAPA’s effect on increasing pAKT/AKT indicates its role in modulating this signaling pathway, potentially by enhancing the phosphorylation state of AKT, rather than through changes in total AKT expression [38]. This increase in PI3K/AKT expression is associated with reductions in fibrosis markers, such as pSMAD. The expression of pSMAD was notably elevated when treated with ISO alone compared to the control (gene expression: p = 0.0075; protein expression: p = 0.0059) (Fig. 2C, L and S). However, this elevated pSMAD expression was significantly reduced after adding DAPA to the ISO treatment (gene expression: p = 0.0342; protein expression: p = 0.0433). These findings suggest that DAPA not only enhances PI3K/AKT signaling but also mitigates the pro-fibrotic effects induced by ISO. No significant differences were observed in αSMA gene expression in the ISO + DAPA-treated group. Interestingly, there was a significant reduction at the protein level (Fig. 2D, M, and S). The discrepancy between the unchanged gene expression and the significant decrease in αSMA protein expression (p = 0.0198) could be attributed to post-transcriptional or post-translational modifications. DAPA could exert effects that stabilize or degrade the αSMA protein independently of gene transcription, which is not uncommon in signaling pathways where protein regulation can occur at multiple levels. Furthermore, we screened for the apoptotic marker p38, and no significant differences were observed between the studied groups in both gene and protein expression analyses (Fig. 2E, N, and S), suggesting that the observed effects of DAPA are not attributable to changes in apoptosis. This further supports the notion that DAPA’s action is more focused on modulating fibrosis and hypertrophy rather than apoptosis.

SGLT2 inhibitors, like DAPA, influence cellular ion balance by reducing sodium reabsorption in the kidneys, which can impact NHE1 activity. This interaction is crucial for their cardioprotective effects, including improvements in glucose metabolism, reduced inflammation, and reduced oxidative stress within cardiovascular cells [39,40,41]. To delve deeper, we further assessed the gene expression of SGLT2, NHE1, and GLUT1 in ISO-induced cardiomyocytes. We observed an increase in SGLT2 expression following ISO stimulation (Fig. 2G, P, and S) compared to the control (gene expression: p = 0.0095; protein expression p = 0.0256). However, the addition of DAPA to the ISO-treated cardiomyocyte significantly reduced SGLT2 expression in the ISO + DAPA group (gene expression: p = 0.0237; protein expression p = 0.0.347). This reduction could be due to DAPA’s ability to restore inflamed cardiac cells, influencing the pathway’s responsiveness to inhibitors, particularly the NHE1 pathway. NHE1 regulates intracellular pH and cell volume by exchanging intracellular hydrogen ions for extracellular sodium ions. This pathway is crucial in ROS production and inflammatory responses within cardiovascular cells. By modulating NHE1 activity, DAPA helps reduce oxidative stress and inflammation, providing long-term cardioprotective effects [42,43,44]. Our results indicated that DAPA exerts its effects by influencing the activity of key signaling molecules involved in oxidative stress and inflammation. The significant induction of NHE1 expression (Fig. 2H, Q, and S) upon ISO stimulation (gene expression: p = 0.0031; protein expression: p = 0.0012) was attenuated with the addition of DAPA (gene expression: p = 0.0035; protein expression: p = 0.0.0027), suggesting that DAPA helps to stabilize NHE1 activity, thereby reducing ROS production and inflammation over time. Further, the activity of NHE1 can be influenced by changes in cellular sodium levels resulting from SGLT2 inhibition, which may impact intracellular pH and calcium regulation. We observed a significant increase in GLUT1 expression in the ISO + DAPA (p = 0.0118) compared to the ISO-alone group. Additionally, there was an increase in GLUT1 expression in the ISO-treated group (p = 0.0002) compared to the control group (Fig. 2I, R and S). The increase in GLUT1 expression with ISO + DAPA treatment suggests a cellular adaptation to maintain glucose homeostasis despite the reduced glucose reabsorption due to SGLT2 inhibition. This enhanced glucose uptake could potentially modulate cellular metabolism and contribute to the observed DAPA’s effects on NHE1 activity. The interplay between these transporters and metabolic pathways highlights the complex mechanisms by which DAPA exerts its effects, potentially offering new insights into its therapeutic benefits and the regulation of intracellular pH and calcium levels [45]. Furthermore, we analyzed the expression of SGLT1 but did not find any significant differences between the treated groups (Fig. 2F, O, and S). One possible reason for this could be that the effects of DAPA, a selective SGLT2 inhibitor, are primarily exerted through SGLT2 rather than SGLT1. While SGLT1 is also involved in glucose transport, its expression and role may not be as strongly influenced by DAPA in this context. Additionally, the expression of SGLT1 might be regulated by different mechanisms that are not directly affected by the PI3K/AKT or hypertrophy-related signaling pathways. Therefore, the lack of significant changes in SGLT1 expression suggests that DAPA’s cardioprotective effects might be more related to its impact on SGLT2 or other signaling cascades, rather than on SGLT1 specifically [46].

DAPA inhibits ISO-induced cardiomyocyte inflammation and modulates antioxidant responses

Inflammation and oxidative stress play important roles in cardiomyocyte dysfunction. We investigated the effects of DAPA on inflammation and antioxidant expression. Our results showed that NLRP3 expression increased following ISO induction but was significantly reduced after DAPA treatment (gene expression: p = 0.0032; protein expression: p = 0.007) (Fig. 3A, F, and K), indicating that DAPA exerts a potent anti-inflammatory effect. Moreover, the marked upregulation of NLRP3 in the ISO-alone group was accompanied by an increase in NRF2 (Nuclear factor erythroid 2-related factor 2) expression in the DAPA-treated group (Fig. 3B). Given that the AKT/PI3K pathway is crucial for NRF2 activation [45], our results demonstrate that NRF2 expression significantly increased following DAPA stimulation (Fig. 3B, G, and K). However, this increase was mitigated when ISO was introduced (gene expression: p = 0.025; protein expression: p < 0.0001). Importantly, the reduction in NRF2 expression caused by ISO was effectively restored with the addition of DAPA to the ISO-treated group (gene expression: p < 0.0001; protein expression: p < 0.0001). These findings suggest that DAPA has anti-inflammatory properties and enhances the antioxidant response by promoting NRF2 activation, thereby countering the detrimental effects of ISO-induced stress. We further measured the expression of antioxidant and cytoprotective genes such as NQO1 (NAD(P)H Quinone Dehydrogenase 1) and HO-1 (Heme Oxygenase 1). NQO1 gene expression was reduced in the ISO-treated group (p = 0.0116), but no significant differences were observed in the protein expression study (Fig. 3C, H, and K). This discrepancy may be due to the post-transcriptional regulation of NQO1, where factors such as mRNA stability or translational efficiency could influence protein levels without affecting mRNA expression directly. In contrast, HO-1 expression was significantly elevated in the DAPA + ISO treated group (gene expression: p = 0.0425; protein expression: p = 0.0032) (Fig. 3D, I, and K). The impaired expression of these antioxidant and cytoprotective genes in the ISO-treated group suggests that ISO induces oxidative stress and disrupts cellular defense mechanisms. DAPA appears to counteract these effects by enhancing NRF2 activation, which in turn upregulates the expression of NQO1 and HO-1. This restoration of gene expression indicates that DAPA not only mitigates the oxidative damage caused by ISO but also supports cellular resilience by promoting the synthesis of key antioxidant and cytoprotective genes.

We also examined the expression of endothelial nitric oxide synthase (eNOS) in cells treated with ISO and ISO + DAPA. As shown in our gene and protein expression analyses (Fig. 3E, J, and K), eNOS expression significantly decreased following ISO treatment (gene expression: p = 0.0020; protein expression: p = 0.0055) and reversed when DAPA was added to the ISO treatment (gene expression: p = 0.0001; protein expression: p = 0.0032). The decrease in eNOS expression due to ISO may stem from the oxidative stress and inflammatory response ISO induces, negatively affecting eNOS expression and function. ISO is known to cause endothelial dysfunction by reducing nitric oxide availability, essential for vascular health and function. The restoration of eNOS expression with DAPA suggests that DAPA may activate signaling pathways that enhance eNOS expression, possibly via the PI3K/AKT pathway, which is known to positively regulate eNOS [47, 48]. Therefore, DAPA appears to counteract ISO’s negative effects and supports endothelial function by maintaining eNOS expression and activity.

Additionally, we assessed the secretion levels of key pro-inflammatory markers, analyzing the expression of inflammatory cytokines IL-1β (Interleukin 1 beta), IL-6 (Interleukin), and TNFα, which are crucial in both physiological and pathological processes [49]. For IL-1β, there were significant effects from ISO and DAPA treatment, with a significant interaction between the two (p = < 0.05), suggesting that DAPA’s effect on IL-1β expression depends on the presence of ISO (Fig. 3L). Similarly, IL-6 secretion level was significantly affected by ISO and DAPA, with a significant interaction (p = < 0.05), indicating that DAPA’s impact on IL-6 expression is also condition-dependent (Fig. 3M). For TNFα, we observed significant effects from both ISO and DAPA with a notable interaction between the two (p = < 0.05), suggesting that DAPA’s influence on TNFα expression varies based on ISO treatment (Fig. 3N). These results highlight the important roles of both ISO and DAPA in modulating IL-1β, IL-6, and TNFα levels, with significant interactions warrant further investigation. Understanding the regulation of these inflammatory cytokines is essential for exploring their role in disease progression and treatment responses, particularly regarding DAPA effects on inflammation and metabolic health [50].

DAPA effects on human aortic endothelial cellsDAPA reduces TNFα-induced inflammation in AECs via modulating the AKT pathway

Given the limited data and the lack of molecular studies, we investigated the role of DAPA on AECs, particularly to identify any similarities in pathways affected by DAPA in both AECs and ISO-stimulated cardiomyocytes. To determine the optimal concentration of DAPA for treating AECs we conducted an MTT assay with various treatments: TNFα alone (Fig. 4A), DAPA alone (Fig. 4B), pre-treatment with TNFα for 24 h followed by DAPA (Fig. 4C), and co-treatment with TNFα + DAPA for 24 h (Fig. 4D). The optimal concentrations were identified as 100 ng/ml for TNFα (determined by EC50) and 1 µM for DAPA, which were used in all subsequent experiments.

Gene expression analysis demonstrated that DAPA treatment significantly upregulated AKT expression (p = 0.043 vs. control, Fig. 4E). However, AKT expression was significantly reduced following TNFα induction (gene expression: p = 0.0008; protein expression: p < 0.0001) (Fig. 4E, V, and Z). Notably, when DAPA was introduced to TNFα-stimulated cells, AKT and pAKT expression increased significantly (gene expression: p = 0.0034; protein expression: p < 0.0001 for TNFα alone vs. TNFα + DAPA). The AKT pathway significant role in cell survival and metabolism suggests that DAPA helps counteract TNFα-induced stress by promoting AKT activity. The ability of DAPA to restore AKT expression in the presence of TNFα indicates its potential protective effect against inflammation-induced cellular stress, highlighting its therapeutic potential in conditions characterized by inflammatory stress and impaired AKT signaling [51, 52]. Similarly, PI3K expression was increased in the DAPA-treated group compared to controls (p = 0.002), but this increase was significantly reversed after treatment with TNFα (p < 0.0001) (Fig. 4F). However, when cells were co-treated with DAPA + TNFα, the inhibition of PI3K was reversed, with PI3K expression significantly increasing again (p < 0.001 for TNFα-alone vs. DAPA + TNFα groups). The PI3K/AKT pathway is essential for promoting cell growth and survival, and the activation of PI3K leads to the production of PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5) P3), which recruits and activates AKT, thereby promoting survival and growth signals. The ability of DAPA to upregulate PI3K expression even in the presence of TNFα suggests that DAPA enhances the cell’s capacity to resist inflammatory damage and promotes repair mechanisms [53, 54]. Furthermore, we study how TNFα affects GRP78 (Glucose-regulated protein). GRP78 is an endoplasmic reticulum (ER) stress marker, and its expression is linked to the protective effects observed in the PI3K/AKT signaling pathways. We observed an increase in GRP78 expression in the TNFα-alone group compared to the DAPA group (p = 0.0003) (Fig. 4G). Additionally, there was a reduction in GRP78 expression in the co-treated TNFα + DAPA group versus the TNFα-alone group; and despite being not statistically significant (p > 0.05), a net reduction was noted. The reduction in GRP78 with DAPA treatment suggests that DAPA might alleviate the ER stress induced by TNFα, further supporting its protective effect on AECs. This reduction in ER stress could also contribute to the overall cellular resilience against inflammatory damage, enhancing cell survival and function by upregulating critical signaling pathways such as AKT and PI3K.The MAPK pathway is involved in cellular responses to a variety of stress signals, and its activation is associated with inflammation, apoptosis, and cell differentiation [55]. DAPA’s ability to modulate this pathway suggests it might influence AECs’ responses to inflammatory stress by affecting MAPK expression [56]. Our results showed that DAPA treatment increased MAPK expression compared to the control (p = 0.015, Fig. 4H). In contrast, MAPK expression was significantly reduced in the TNFα group (p = 0.0041). Interestingly, when DAPA was added to TNFα-stimulated cells, MAPK expression was elevated, although the increase was not statistically significant in the TNFα + DAPA group. This suggests that DAPA may at least partially restore MAPK levels, potentially influencing inflammatory signaling pathways. These results highlight the complex interplay between DAPA and TNFα and emphasize the need for further studies to elucidate how DAPA modulates MAPK and related inflammatory pathways in AECs. Based on our observations of inflammatory pathway activation and inhibition in AECs, we extended our study to examine a broader set of inflammatory genes involved in regulatory processes. Cells stimulated with TNFα alone showed a significant upregulation of inflammatory genes, including NF-κB-nuclear factor kappa B (p < 0.0001, Fig. 4I), NLRP3-NLR family pyrin domain containing 3 (p = 0.0058, Fig. 4K), IL-1β (p = 0.0091, Fig. 4M), TNFα (gene expression p = 0.0088; protein expression p < 0.0001, Fig. 4M, Y, Z), and IL-6 (p < 0.0001, Fig. 4N). This marked increase underscores the strong pro-inflammatory effects of TNFα. However, treatment with DAPA suppressed this rise in the TNFα + DAPA group, as evidenced by decreased expression of NF-κB (p < 0.0001, Fig. 4I), NLRP3 (p = 0.0020, Fig. 4J), IL-1β (p = 0.0002, Fig. 4M), TNFα (gene expression p = 0.0016; protein expression p < 0.0001, Fig. 4M, Y, Z), and IL-6 (p < 0.0001, Fig. 4N), demonstrating its potent anti-inflammatory effects that counteract the pro-inflammatory actions of TNFα. Additionally, we investigated NRF2, an antioxidant gene, which showed increased expression with DAPA compared to the control group (p = 0.0016) (Fig. 4J). NRF2 expression (p < 0.0001 for TNFα-alone group vs. DAPA group) was reduced in TNFα but this reduction was reversed with the addition of DAPA (p = 0.0002), suggesting the DAPA’s role in mitigating oxidative stress through the NRF2 pathway. NRF2 regulates the expression of antioxidant proteins crucial for protecting against oxidative damage triggered by inflammation and injury. The elevation in NRF2 expression with DAPA suggests that DAPA enhances the cell’s antioxidant defenses, further emphasizing its potential therapeutic benefit in combating inflammation-associated oxidative stress [57, 58].

The adhesion molecules like ICAM-1 (Intercellular Adhesion Molecule 1) and VCAM-1(Vascular cell adhesion molecule 1) are crucial in inflammation and atherosclerosis, owing to their involvement in the process of leukocyte recruitment to the sites of inflammation. Here the expression of ICAM-1 (Fig. 4O) and VCAM-1 (Fig. 4P) was increased in the TNFα-alone group vs. DAPA group (ICAM p = 0.0003, VCAM p = 0.0006) and was reduced in the co-treated TNFα + DAPA group vs. TNFα-alone group (ICAM p = 0.0004, VCAM p = 0.0097). Their reduction with DAPA treatment indicates the DAPA potential to reduce vascular inflammation via its inhibitory effect on endothelial activation and leukocyte adhesion, which are critical steps in the atherosclerotic process [59, 60].

We next investigated the expression of SGLT2, sodium/hydrogen exchanger 1(NHE1), and glucose transporter 1 (GLUT1), given their crucial roles in the relationship between inflammation and oxidative stress, especially in the context of DAPA’s function as an SGLT2 inhibitor. In the TNFα-alone group, there was a significant increase in SGLT2 expression (gene expression: p = 0.0481; protein expression: p = 0.0024 vs. control, Fig. 4R, X, and Z) and its upstream regulator NHE-1 (p = 0.0002 vs. DAPA group, Fig. 4S). Interestingly, while we observed a decrease in protein expression in TNFα + DAPA co-treated group compared to TNFα alone (protein expression: p = 0.0032 vs. TNFα, Fig. 4R, X, and X) but this was not reflected in gene expression levels, suggesting that DAPA may influence post-transcriptional regulatory mechanisms that affect protein stability or translation without significantly altering gene transcription. Further investigation is needed in a greater sample size. The reduction in SGLT2 and NHE-1 expression suggests that DAPA may limit glucose uptake in endothelial cells, potentially lowering glucose-induced oxidative stress [61, 62]. Further, we observed GLUT1 expression (Fig. 4T) significantly increased in both the TNFα-alone group (p = 0.0086 vs. DAPA group) and the TNFα + DAPA co-treatment group (p = 0.0083 vs. DAPA group). GLUT1 is crucial for basal glucose uptake and likely maintains its expression to ensure a steady supply of essential glucose during stress conditions. The lack of GLUT1 expression difference between the DAPA + TNFα co-treatment group compared to the TNFα-alone group implies that while DAPA may impact multiple pathways, it might not influence GLUT1 expression under conditions of TNFα-induced stress nor substantially alter the expression of glucose transporters in the presence of TNFα [63]. Additionally, we also studied the gene expression analysis for SGLT1, and no significant differences were observed between the study groups (Fig. 4Q, W, and Z), aligning with the protein expression results.

Additionally, we study the expression of eNOS (endothelial nitric oxide) which is a pivotal regulator of vascular homeostasis. We found an increase in eNOS expression in the control and DAPA-treated groups (gene expression: p = 0.045) (Fig. 3U). Following TNFα administration, eNOS expression was significantly decreased (p = 0.0001) and then restored when treated with DAPA (p = 0.0086). This suggests that DAPA exerts protective effects by enhancing eNOS expression, thereby facilitating vasodilation and preserving vascular function amid inflammatory conditions. The restoration and elevation of eNOS expression by DAPA underscore its potential to counteract the detrimental effects of inflammation on endothelial function [30, 64]. Overall, DAPA demonstrated a protective role in AECs against TNFα-induced stress by modulating key signaling pathways, including AKT/PI3K, MAPK (Mitogen-activated protein kinases ), and inflammatory pathways. The regulation of adhesion molecules and SGLT2 further supports its potential therapeutic benefits in vascular health.

DAPA effects on extracellular matrix (ECM) remodeling, vascular function, and inflammation in AECs

To validate the observed changes in SGLT2 expression, we conducted immunofluorescence (IF) analysis (Fig.