Empagliflozin Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Rats by Modulating Sesn2/AMPK/Nrf2 Signaling and Targeting Ferroptosis and Autophagy

Pulmonary fibrosis (PF) is a life-threatening disorder that severely disrupts normal lung architecture and function, resulting in severe respiratory failure and death. It has no definite treatment. Empagliflozin (EMPA), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, has protective potential in PF. However, the mechanisms underlying these effects require further elucidation. Therefore, this study aimed to evaluate the ameliorative effect of EMPA against bleomycin (BLM)-induced PF and the potential mechanisms. Twenty-four male Wister rats were randomly divided into four groups: control, BLM treated, EMPA treated, and EMPA+BLM treated. EMPA significantly improved the histopathological injuries illustrated by both hematoxylin and eosin and Masson’s trichrome-stained lung tissue sections, as confirmed by electron microscopic examination. It significantly reduced the lung index, hydroxyproline content, and transforming growth factor β1 levels in the BLM rat model. It had an anti-inflammatory effect, as evidenced by a decrease in the inflammatory cytokines’ tumor necrosis factor alpha and high mobility group box 1, inflammatory cell infiltration into the bronchoalveolar lavage fluid, and the CD68 immunoreaction. Furthermore, EMPA mitigated oxidative stress, DNA fragmentation, ferroptosis, and endoplasmic reticulum stress, as evidenced by the up-regulation of nuclear factor erythroid 2-related factor expression, heme oxygenase-1 activity, glutathione peroxidase 4 levels, and a decrease in C/EBP homologous protein levels. This protective potential could be explained on the basis of autophagy induction via up-regulating lung sestrin2 expression and the LC3 II immunoreaction observed in this study. Our findings indicated that EMPA protected against BLM-induced PF-associated cellular stress by enhancing autophagy and modulating sestrin2/adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor 2/heme oxygenase 1 signaling.


Introduction
Pulmonary fibrosis (PF) is a chronic, progressive, age-related interstitial lung disease with a high morbidity and mortality rate. Its pathological features include excessive collagen deposition, enlarged interstitial spaces between alveoli, thickened alveolar walls, and inflammatory cell infiltration, resulting in widespread scarring, decreased lung compliance, and even lung failure [1]. There is currently no treatment, and animal models of PF are still crucial tools for investigating its etiopathogenesis. Although the etiology of PF is complex and variable, the accepted hypothesis is that it is caused by abnormal wound healing of alveolar epithelial cells in response to repeated injury stimulation [2]. Bleomycin (BLM)-induced PF is the most widely accepted model for developing new anti-fibrosis strategies and evaluating drug efficacy due to its low cost, ease of induction, and good reproducibility [3].
Ferroptosis is a recently described nonapoptotic form of programmed cell death that differs mechanistically and phenotypically from other well-known types of regulated cell death [4]. Biochemically, it is characterized by excessive reactive oxygen species (ROS) as a result of increased iron metabolism and lipid peroxidation, with no involvement of caspase or Bax activation. Morphologically, ferroptotic cells have smaller mitochondria, condensed mitochondrial membrane densities, vanishing mitochondrial cristae, and a ruptured outer mitochondrial membrane [5]. Glutathione peroxidase 4 (GPX4) is a key regulator of ferroptosis. It reduces lipid hydroperoxide production during lipid peroxidation, which is involved in oxidative damage to cellular membrane structure [6]. Recent studies have documented the role of ferroptosis in the pathophysiology of several conditions, including neurodegeneration, ischemic organ injury, atherosclerosis, and cancer [7]. Notably, recent studies have demonstrated that inhibiting ferroptosis can alleviate experimentally induced lung injuries such as lipopolysaccharide-induced acute lung injury [8] and radiationinduced lung fibrosis [9]. However, the role of ferroptosis in BLM-induced PF remains unknown. Therefore, we hypothesize that ferroptosis is a possible underlying mechanism.
Nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important bidirectional role in cancer prevention and progression. As a well-known cytoprotective factor, it plays an important role in protecting cells from oxidative damage caused by increased reactive oxygen species (ROS) levels. When activated by ROS, Nrf2 induces the expression of antioxidant enzymes by binding to the promoters of target genes containing antioxidant response elements, thereby contributing to the synthesis and renewal of glutathione (GSH), a major GPX4 cofactor, and detoxifying molecules [10,11]. Therefore, Nrf2 activation plays a protective role under physiological conditions. However, Nrf2 activation in cancer cells promotes cancer progression and metastasis, as well as chemoresistance by inactivating drug-mediated oxidative stress and protecting cancer cells from drug-induced cell death. Selective Nrf2 modulators can be used as adjuvant therapy after conventional chemotherapy, targeted therapy, and immunotherapy [11]. Targeting the Nrf2 pathway in cancer cells could pave the way for the development of more effective anticancer drugs or improve the efficacy of existing drugs by reducing drug resistance [12]. Furthermore, Nrf2 expression can be reactivated by natural or synthetic compounds via downregulating histone deacetylases and DNA methyltransferases, inhibiting Nrf2 promoter methylation and protecting normal cells from ROS damage and tumorigenesis [13]. Growing evidence suggests a link between Nrf2 and ferroptosis. In addition, many genes that inhibit iron overload and lipid peroxidation have been identified as Nrf2 target genes [14,15]. Adenosine monophosphate-activated protein kinase (AMPK) is a key bioenergetic sensor and metabolic regulator. Recent studies indicated that AMPK activation could enhance Nrf2 nuclear translocation [16].
Sestrin2 (Sesn2) is a stress-inducible protein that is strongly up-regulated by various stressors. It has protective effects on various physiological and pathological states, mainly by regulating oxidative stress, endoplasmic reticulum (ER) stress, autophagy, and inflammation [17]. It has recently been shown to efficiently affect the pathological process of ferroptosis [18]. In addition, emerging evidence suggests that Sesn2 plays a pivotal role in fibrosis progression in many organs, including the liver and heart [19,20]. Furthermore, Sesn2 induction has antioxidant and anti-inflammatory effects in alleviating cigarette-smoke-induced pulmonary injury [21].
Empagliflozin (EMPA) is a sodium-glucose cotransporter 2 (SGLT2) inhibitor that was developed as a glucose-lowering agent for type 2 diabetes mellitus. It controls glucose levels by lowering glucose uptake by renal proximal tubular cells without causing hypoglycemia, thereby improving insulin resistance [22]. Beyond its antidiabetic effect, recent research has shown that EMPA has beneficial effects on the cardiovascular, renal, hepatic, and pulmonary systems, which may be due to its ability to reduce inflammation, oxidative stress, fibrosis, apoptosis, and mitochondrial dysfunction [23][24][25][26]. Although EMPA has recently been shown to ameliorate BLM-induced PF by activating Nrf2 [27], more research is required to elucidate the mechanisms underlying EMPA's lung protective effects against BLM. Therefore, the current study aimed to investigate EMPA's regulatory effect on the Sesn2/AMPK/Nrf2/heme oxygenase 1 (HO-1) signaling pathway as well as its modulatory role in ferroptosis, inflammation, ER stress, and autophagy in the context of BLM-induced PF.

Effects of EMPA on BLM-Induced Histopathological Alterations
Histopathological examination of hematoxylin and eosin (H and E)-stained sections from both the control and EMPA-treated groups revealed normal lung tissues with alveolar sacs and alveoli separated by thin interalveolar septa. They exhibited type I pneumocytes with flat nuclei, and a small number of type II pneumocytes with centrally rounded vesicular nuclei lined the alveoli ( Figure 1A,C). However, lung tissue sections from the BLMtreated group revealed disrupted lung architecture with marked thickening of interalveolar septa and significant mononuclear cellular infiltrations. Many of the alveolar epithelial lining cells showed vacuolar cytoplasmic necrosis and pyknotic nuclei. Some collapsed alveoli and severe interstitial hemorrhage could be seen ( Figure 1B). However, the normal lung tissue, alveolar sacs, and alveoli were all preserved in the EMPA+BLM-treated group, except for mild thickening of the alveolar septa and mild interstitial hemorrhage ( Figure 1D). In addition, the histopathological score significantly increased in the BLM-treated group as compared to the control groups. In contrast, EMPA improved the histopathological score more effectively in group IV ( Figure 1E). The scoring of lung fibrosis in the BLM-treated group revealed significantly more fibrotic changes in the interalveolar septa, blood vessel walls, and bronchioles when compared to the control group. Furthermore, the EMPA+BLMtreated group showed a significant reduction in lung tissue fibrosis score when compared to the BLM-treated group ( Figure 1F).
Furthermore, histopathological examination of Masson's trichrome-stained lung tissue sections from the control and EMPA-treated groups revealed negligible collagen fibers in the alveolar walls (Figure 2A,C). However, the BLM-treated group revealed an increased amount of collagen fibers ( Figure 2B). Notably, EMPA administration to BLM-treated rats significantly reduced the amount of collagen fibers ( Figure 2D). The morphometric measurements of Masson trichrome-stained sections revealed that the BLM-treated group had a significant increase in the mean area percentage of Masson trichrome when compared to the control and EMPA-treated groups, while the EMPA+BLM-treated group had a considerable decrease when compared to the BLM-treated group ( Figure 2E). The control and (C) empagliflozin (EMPA)-treated groups show thin-walled alveoli (red arrows) and alveolar sacs (s). The thin-walled alveoli are made up of flat type I pneumocytes (black arrows) with densely pigmented nuclei and type II pneumocytes (dashed arrows) with big, rounded nuclei. (B) The bleomycin (BLM)-treated group reveals a disrupted architectural arrangement of the lung with markedly thickened alveolar septa (thick arrows) and extensive mononuclear cellular infiltration (curved arrows). Some alveoli appear collapsed (stars). Many of the alveolar epithelial lining cells exhibit vacuolar cytoplasmic necrosis with pyknotic nuclei (thin arrows). Severe interstitial hemorrhage (Hg) can be observed. (D) The EMPA+BLM-treated group shows many thin-walled alveoli (arrows) and alveolar sacs (S) with normal histology. However, there are still a few collapsed alveoli, minor alveolar wall thickening (thick arrows), and mild interstitial hemorrhage (Hg). Magnification is ×400; scale bar = 50 μm. (E) The histopathological and (F) fibrosis scoring among various studied groups (n = 6 rats in each group). * indicates a p < 0.05, ** indicates a p < 0.01, and *** indicates a p < 0.001. Furthermore, histopathological examination of Masson's trichrome-stained lung tissue sections from the control and EMPA-treated groups revealed negligible collagen fibers in the alveolar walls (Figure 2A,C). However, the BLM-treated group revealed an increased amount of collagen fibers ( Figure 2B). Notably, EMPA administration to BLMtreated rats significantly reduced the amount of collagen fibers ( Figure 2D). The morphometric measurements of Masson trichrome-stained sections revealed that the BLM-treated The EMPA+BLM-treated group shows many thin-walled alveoli (arrows) and alveolar sacs (S) with normal histology. However, there are still a few collapsed alveoli, minor alveolar wall thickening (thick arrows), and mild interstitial hemorrhage (Hg). Magnification is ×400; scale bar = 50 µm. (E) The histopathological and (F) fibrosis scoring among various studied groups (n = 6 rats in each group). * indicates a p < 0.05, ** indicates a p < 0.01, and *** indicates a p < 0.001. group had a significant increase in the mean area percentage of Masson trichrome when compared to the control and EMPA-treated groups, while the EMPA+BLM-treated group had a considerable decrease when compared to the BLM-treated group ( Figure 2E).

Effects of EMPA on BLM-Induced Inflammatory Status
The examination of CD68 immunohistochemical (IHC)-stained sections revealed a few CD68-positive brownish macrophages that were distributed in alveolar walls in both the control and EMPA-treated groups ( Figure 3A,C). Nevertheless, the BLM-treated group revealed numerous cells with a very strong CD68 immunoreaction ( Figure 3B). On the other hand, the EMPA+BLM-treated group showed numerous CD68-positive macrophages with a strong positive immunoreaction ( Figure 3D). Morphometric analysis of the

Effects of EMPA on BLM-Induced Inflammatory Status
The examination of CD68 immunohistochemical (IHC)-stained sections revealed a few CD68-positive brownish macrophages that were distributed in alveolar walls in both the control and EMPA-treated groups ( Figure 3A,C). Nevertheless, the BLM-treated group revealed numerous cells with a very strong CD68 immunoreaction ( Figure 3B). On the other hand, the EMPA + BLM-treated group showed numerous CD68-positive macrophages with a strong positive immunoreaction ( Figure 3D). Morphometric analysis of the mean percentage of positive CD68 immunoreactions revealed a highly significant increase in the BLM-treated group when compared to the control group. However, the EMPA+BLMtreated group revealed a significant decrease when compared to the BLM-treated group ( Figure 3E). mean percentage of positive CD68 immunoreactions revealed a highly significant increase in the BLM-treated group when compared to the control group. However, the EMPA+BLM-treated group revealed a significant decrease when compared to the BLMtreated group ( Figure 3E).  Likewise, the total leucocytic count (TLC) ( Figure 3F), as well as the percentages of neutrophils and lymphocytes in the bronchoalveolar lavage fluid (BALF) ( Figure 3G,H), were significantly higher in the BLM-treated group than in the control and EMPA-treated groups, while the macrophage percentage was significantly lower ( Figure 3I). Meanwhile, concomitant EMPA administration to BLM-treated rats significantly restored the leucocytic parameters to near-control values ( Figure 3). In the BLM-treated group, there were significant increases in both BALF tumor necrosis factor-alpha (TNF-α) and high mobility group box 1 (HMBG1) levels when compared to the control and EMPA-treated groups. EMPA, on the other hand, attenuated inflammatory responses, as evidenced by significant reductions in TNF-α and HMBG1 levels in BALF ( Figure 3J,K). These findings suggested that EMPA could attenuate the BLM-induced inflammatory status.

Effects of EMPA Treatment on BLM-Induced Lung Fibrosis
When compared to the control and EMPA-treated groups, BLM treatment resulted in a significant increase in the lung index in the BLM-treated group. However, this increase was ameliorated by concomitant EMPA administration to the BLM-treated rats ( Figure 4A). In the BLM-treated group, there were significant increases in the fibrotic markers transforming growth factor beta 1 (TGF-β1) and hydroxyproline levels in lung tissues when compared to the control and EMPA-treated groups. EMPA, on the other hand, attenuated fibrotic responses, as evidenced by significant reductions in TGF-β1 and hydroxyproline levels in lung tissues ( Figure 4B,C). These findings suggested that EMPA could attenuate BLMinduced lung fibrosis.
that are close to the control (×400, scale bar = 50 μm). (E) A morphometric analysis of the mean area percentage of CD68 immune reactivity; (F) the total leucocytic count; (G) neutrophil percentages; (H) lymphocyte percentages; (I) macrophage percentages; (J) bronchoalveolar lavage fluid (BALF) tumor necrosis factor-alpha (TNF-α); and (K) BALF high mobility group box 1 (HMBG1) among various studied groups (n = 6 rats in each group). Data are presented as mean and standard deviation (error bars) and analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for multiple comparisons. ** indicates a p < 0.01 and *** indicates a p < 0.001. Likewise, the total leucocytic count (TLC) ( Figure 3F), as well as the percentages of neutrophils and lymphocytes in the bronchoalveolar lavage fluid (BALF) ( Figure 3G,H), were significantly higher in the BLM-treated group than in the control and EMPA-treated groups, while the macrophage percentage was significantly lower ( Figure 3I). Meanwhile, concomitant EMPA administration to BLM-treated rats significantly restored the leucocytic parameters to near-control values ( Figure 3). In the BLM-treated group, there were significant increases in both BALF tumor necrosis factor-alpha (TNF-α) and high mobility group box 1 (HMBG1) levels when compared to the control and EMPA-treated groups. EMPA, on the other hand, attenuated inflammatory responses, as evidenced by significant reductions in TNF-α and HMBG1 levels in BALF ( Figure 3J,K). These findings suggested that EMPA could attenuate the BLM-induced inflammatory status.

Effects of EMPA Treatment on BLM-Induced Lung Fibrosis
When compared to the control and EMPA-treated groups, BLM treatment resulted in a significant increase in the lung index in the BLM-treated group. However, this increase was ameliorated by concomitant EMPA administration to the BLM-treated rats ( Figure 4A). In the BLM-treated group, there were significant increases in the fibrotic markers transforming growth factor beta 1 (TGF-β1) and hydroxyproline levels in lung tissues when compared to the control and EMPA-treated groups. EMPA, on the other hand, attenuated fibrotic responses, as evidenced by significant reductions in TGF-β1 and hydroxyproline levels in lung tissues ( Figure 4B,C). These findings suggested that EMPA could attenuate BLM-induced lung fibrosis.

Effects of EMPA on Redox Status, Ferroptosis, and ER Stress Status
BLM treatment significantly increased malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels while reducing GSH and GPX4 levels and HO-1 activity in the BLM-treated group compared to the control and EMPA-treated groups. However, these changes were reversed by EMPA treatment (Figure 5A-E). Similarly, Nrf2 relative expression level was found to be significantly down-regulated in the BLM-treated group compared to the control and EMPA-treated groups. However, concomitant EMPA administration to BLM-treated rats significantly upregulated Nrf2 relative expression levels compared to those treated only with BLM ( Figure 5F). Furthermore, the levels of C/EBP homologous protein (CHOP), a mediator of ER stress-induced apoptosis, were significantly increased after BLM treatment compared to the control and EMPA-treated groups. However, the coadministration of EMPA attenuated the BLM effect on CHOP levels ( Figure 5G). These findings suggested that EMPA could attenuate the BLM-induced oxidative and ER stress and ferroptosis in the BLM rat model. pared to the control and EMPA-treated groups. However, concomitant EMPA administration to BLM-treated rats significantly upregulated Nrf2 relative expression levels compared to those treated only with BLM ( Figure 5F). Furthermore, the levels of C/EBP homologous protein (CHOP), a mediator of ER stress-induced apoptosis, were significantly increased after BLM treatment compared to the control and EMPA-treated groups. However, the coadministration of EMPA attenuated the BLM effect on CHOP levels ( Figure  5G). These findings suggested that EMPA could attenuate the BLM-induced oxidative and ER stress and ferroptosis in the BLM rat model.

Effects of EMPA on Autophagy Regulation
The examination of LC3 II IHC-stained sections revealed many cells with strong positive LC3 II immunoreactions in their cytoplasm in both the control ( Figure 6A) and EMPA-treated groups ( Figure 6C). However, the BLM-treated group revealed a few cells with a very weak LC3 II immunoreaction ( Figure 6B). Nonetheless, sections from rats cotreated with EMPA showed numerous LC3 II-positive cells with a strong positive immunoreaction ( Figure 6D). Furthermore, morphometric analysis of the mean percentage of positive LC3 II immunoreaction revealed a highly significant decrease in the BLM-treated group when compared to the control group, whereas concomitant EMPA administration to BLM-treated rats revealed a significant increase when compared to the BLM-treated rats ( Figure 6E). Likewise, Sens2 relative expression levels were found to be significantly downregulated in the BLM-treated group compared to the control and EMPA-treated

Effects of EMPA on Autophagy Regulation
The examination of LC3 II IHC-stained sections revealed many cells with strong positive LC3 II immunoreactions in their cytoplasm in both the control ( Figure 6A) and EMPA-treated groups ( Figure 6C). However, the BLM-treated group revealed a few cells with a very weak LC3 II immunoreaction ( Figure 6B). Nonetheless, sections from rats cotreated with EMPA showed numerous LC3 II-positive cells with a strong positive immunoreaction ( Figure 6D). Furthermore, morphometric analysis of the mean percentage of positive LC3 II immunoreaction revealed a highly significant decrease in the BLM-treated group when compared to the control group, whereas concomitant EMPA administration to BLM-treated rats revealed a significant increase when compared to the BLM-treated rats ( Figure 6E). Likewise, Sens2 relative expression levels were found to be significantly downregulated in the BLM-treated group compared to the control and EMPA-treated groups. However, concomitant EMPA administration to BLM-treated rats significantly up-regulated Sens2 relative expression levels compared to those treated only with BLM ( Figure 6F). Indeed, the p-AMPK levels in lung tissues were significantly lower in the BLMtreated group than in the control group. Meanwhile, concomitant EMPA administration to BLM-treated rats significantly increased lung p-AMPK levels compared to those treated only with BLM ( Figure 6G). These findings suggested that EMPA could promote autophagy in BLM rat model. groups. However, concomitant EMPA administration to BLM-treated rats significantly up-regulated Sens2 relative expression levels compared to those treated only with BLM ( Figure 6F). Indeed, the p-AMPK levels in lung tissues were significantly lower in the BLM-treated group than in the control group. Meanwhile, concomitant EMPA administration to BLM-treated rats significantly increased lung p-AMPK levels compared to those treated only with BLM ( Figure 6G). These findings suggested that EMPA could promote autophagy in BLM rat model.

Effects of EMPA on DNA Damage
DNA samples from both the control and EMPA-treated groups showed normal, distinct bands (3162 and 3061 bp, respectively) in the DNA ladder assay. The BLM-treated group, in contrast, displayed an apoptotic fragmented DNA pattern. When EMPA was administered concurrently with BLM, DNA fragmentation was reduced, protecting against BLM's adverse effects (Figure 7). group, in contrast, displayed an apoptotic fragmented DNA pattern administered concurrently with BLM, DNA fragmentation was r against BLM's adverse effects (Figure 7).

Effects of EMPA Treatment on the Electron Microscopic Alterations in
Electron microscopic examination of the ultrathin sections of the vealed normal ultrastructure features of the lung. Type I pneumocytes in shape, with clearly demonstrated flat nuclei filling a large part of th II pneumocytes were cuboidal in shape with euchromatic, rounded n microvilli. The most distinguishing feature of these cells was the pre cytoplasmic lamellar bodies. Alveolar macrophages were detected w nuclei and prominent nucleoli, a large number of lysosomes in their cy tiple pseudopodia on their surface in both the control and EMPA-trea 8A,D). However, ultrathin sections from the BLM-treated group revea bations of type II pneumocytes, with irregularly shrunken nuclei, emp

Effects of EMPA Treatment on the Electron Microscopic Alterations in the Lung Specimens
Electron microscopic examination of the ultrathin sections of the lung specimens revealed normal ultrastructure features of the lung. Type I pneumocytes appeared flattened in shape, with clearly demonstrated flat nuclei filling a large part of the cytoplasm. Type II pneumocytes were cuboidal in shape with euchromatic, rounded nuclei and scattered microvilli. The most distinguishing feature of these cells was the presence of numerous cytoplasmic lamellar bodies. Alveolar macrophages were detected with large, indented nuclei and prominent nucleoli, a large number of lysosomes in their cytoplasm, and multiple pseudopodia on their surface in both the control and EMPA-treated groups ( Figure 8A,D). However, ultrathin sections from the BLM-treated group revealed marked perturbations of type II pneumocytes, with irregularly shrunken nuclei, empty lamellar bodies, disturbed mitochondria, and detached microvilli in the alveolar space, as well as thick collagen fibrils and edematous fluid in the interstitial tissue ( Figure 8B,C). Concomitant EMPA and BLM treatment improved lung ultrastructure, resulting in type II pneumocytes with large euchromatic nuclei. Few mitochondria remained disrupted, and the microvilli were mostly intact. Some lamellar bodies were partially empty, while others had lung surfactant strands ( Figure 8E).

Discussion
As novel evidence, this study clearly demonstrates the protective role of EMPA in BLM-induced PF in experimental animals by ameliorating oxidative stress, ferroptosis, inflammation, ER stress, and fibrosis, as well as improving histopathological changes and autophagic activity in lung tissues. This protective potential may be related to the activation of the Sesn2/AMPK/Nrf2/HO-1 signaling pathway.
The BLM model is one of several PF animal models that is widely used due to its superior characteristics. According to the current study, BLM caused pulmonary inflammation and fibrosis, as evidenced by lung histopathological findings, significant elevations in various inflammatory mediators such as TNF-α, HMG1, and BALF TLCs, and a very strong CD68 immunoreaction revealed by immunostaining, as well as the lung index, TGF-β1 levels, and the primary building block for collagen content, hydroxyproline, which is consistent with previous findings [28]. These pathological alterations were markedly reversed by EMPA, consistent with Kabel et al. [27], and supported by EMPA's antiinflammatory and anti-fibrotic potential demonstrated in different models of organ fibrosis in the absence of diabetes [29,30].
A growing body of evidence suggests that ROS plays an important role in the pathogenesis of PF. High levels of ROS promote TGF-β1-induced fibrosis and the fibroblastto-myofibroblast transition, as well as inflammatory cytokine secretion [28]. Furthermore,

Discussion
As novel evidence, this study clearly demonstrates the protective role of EMPA in BLM-induced PF in experimental animals by ameliorating oxidative stress, ferroptosis, inflammation, ER stress, and fibrosis, as well as improving histopathological changes and autophagic activity in lung tissues. This protective potential may be related to the activation of the Sesn2/AMPK/Nrf2/HO-1 signaling pathway.
The BLM model is one of several PF animal models that is widely used due to its superior characteristics. According to the current study, BLM caused pulmonary inflammation and fibrosis, as evidenced by lung histopathological findings, significant elevations in various inflammatory mediators such as TNF-α, HMG1, and BALF TLCs, and a very strong CD68 immunoreaction revealed by immunostaining, as well as the lung index, TGF-β1 levels, and the primary building block for collagen content, hydroxyproline, which is consistent with previous findings [28]. These pathological alterations were markedly reversed by EMPA, consistent with Kabel et al. [27], and supported by EMPA's anti-inflammatory and anti-fibrotic potential demonstrated in different models of organ fibrosis in the absence of diabetes [29,30].
A growing body of evidence suggests that ROS plays an important role in the pathogenesis of PF. High levels of ROS promote TGF-β1-induced fibrosis and the fibroblast-tomyofibroblast transition, as well as inflammatory cytokine secretion [28]. Furthermore, high ROS levels induce lipid peroxidation, which triggers ferroptosis and subsequent fibrosis-related diseases such as PF [9,31,32]. Li et al. demonstrated the effectiveness of ferroptosis inhibition on radiation-induced lung fibrosis by downregulating proinflammatory cytokines mediated by Nrf2 pathway activation [9]. Therefore, we hypothesized that inhibiting ferroptosis could be an effective strategy for ameliorating BLM-induced PF.
GPX4 is a key regulator of ferroptosis. It has been documented that GPX4 overexpression inhibits ferroptosis, whereas GPX4 deletion results in severe pathological phenotypes related to ferroptosis [33]. The current findings demonstrated the main characteristics of ferroptosis, including MDA burden, 4-HNE accumulation, reduced GPX4 levels, and GSH depletion. The decreased GPX4 levels could be explained by GSH depletion, ROS accumulation, and lipid peroxidation, exceeding GPX4 clearance ability [34].
Intriguingly, there was a significant decrease in MDA and 4-HNE levels in the EMPA + BLM-treated group, indicating alleviated oxidative stress and lipid peroxidation, accompanied by significantly increased GSH and GPX4 levels. These findings could imply that EMPA exhibited remarkable free radical scavenging activities, reversed the reduction in antioxidant enzyme activity, and regulated ferroptosis. In line with this, in vivo and in vitro studies demonstrated that EMPA could reduce ROS generation in mitochondria and cytoplasm [35,36]. According to previous studies, EMPA may have the ability to suppress ferroptosis in different animal models [37,38]. To the best of our knowledge, this represents the first study that demonstrated the beneficial effects of EMPA treatment on the regulation of ferroptosis in PF, and we inferred that the reduction in GSH content and inhibition of system Xc-might be the targets of EMPA to act upon, as previously reported [39].
Nrf2 primarily regulates ferroptosis by directly affecting the expression and function of GPX4. In addition to regulating intracellular iron concentration via the Nrf2/HO-1 axis, the Nrf2 signaling pathway promotes cystine/glutamate reverse transport system Xcand GPX4 expression [14]. In the meantime, knocking down Nrf2 and Nrf2-target genes enhanced ferroptosis-mediated cell death [39]. Previous research has shown that upregulating Nrf2 expression and its dependent antioxidant factors, including HO-1, can mitigate oxidative stress-induced pulmonary inflammation and fibrosis in the BLM-induced PF model [40]. In the same context, the present findings showed that BLM reduced Nrf2 expression levels and HO-1 activity, which were significantly associated with GPX4 and GSH levels, supporting the novel role of Nrf2 in PF pathogenesis by regulating ferroptosis [40]. Furthermore, these effects were reversed by EMPA treatment, indicating that EMPA's antioxidant effects are mainly through activating Nrf2 signaling, as evidenced in different models and tissues [25,41,42]. Therefore, we hypothesized that ferroptosis may be one of the pathogenetic mechanisms in BLM-induced PF, and EMPA treatment may regulate ferroptosis to ameliorate PF partly by reducing lipid peroxidation, suppressing oxidative stress, enhancing GSH production, and increasing GPX4 levels via the Nrf2/HO-1 signaling pathway. However, the mechanism by which EMPA activates Nrf2 expression requires further investigations to uncover the upstream regulatory factors.
Autophagy is a fundamental cytoprotective mechanism that maintains cellular homeostasis. It degrades and recycles damaged cytoplasmic constituents in response to cellular stress [43]. There is mounting evidence supporting the pathogenic role of impaired autophagy in PF, as well as the ameliorative effects of autophagy induction on disease progression [44,45]. Sesn2, an important sestrin member, is involved in the positive regulation of autophagy [46]. Evidence suggests that Sesn2 is involved in oxidative-stress-related respiratory diseases [47]. It has also been shown to inhibit fibrogenic and inflammatory cytokine production [48]. However, its regulatory role in autophagy in PF remains unknown.
The current findings revealed that BLM significantly reduced Sesn2 expression as well as p-AMPK levels, which is a downstream target modulated by Sesn2 and one of the classic core molecular regulators of autophagy. These findings were associated with a significant decrease in LC3-II levels revealed by immunostaining, indicating a blockade of autophagic flux, which is consistent with Sosulski et al. [44]. In addition, Yang et al. found that Sesn2 was downregulated in TGF-β-treated cells and BLM-treated mice, whereas its up-regulated expression induced autophagy and alleviated PF [49]. Furthermore, Shi et al. reported that activating AMPK could ameliorate BLM-induced lung fibrosis [50]. In this study, reduced Sesn2 expression could be attributed to reduced Nrf2 expression, which is an essential transcription factor for Sesn2 expression in response to stress [17]. In contrast, Zehender et al. revealed that autophagy activation was required for fibroblast activation in dermal and pulmonary fibrosis [51]. Sesn2 was also found to increase dramatically in LPS-induced acute lung injury [52]. These contradictory results regarding the paradoxical status of Sesn2 expression may be related to the different periods of lung injury, time window, oxidant stress level, and cell phenotype.
Several studies have reported the role of EMPA in inducing autophagy, which is consistent with our findings [53,54]. However, the impact of EMPA on Sesn2 expression has yet to be investigated. In this study, we found that EMPA significantly increased Sesn2 expression and AMPK phosphorylation, as well as increased LC3-II immunostaining, indicating increased autophagy activity. In the same vein, Sun et al. reported that EMPA improved obesity-related cardiac dysfunction via promoting Sesn2-mediated AMPK-mTOR signaling and Sesn2-induced Nrf2 activation by enhancing the autophagic degradation of Keap1 [55].
Furthermore, given the role of AMPK in Nrf2 phosphorylation and nuclear accumulation [56], we could speculate that EMPA-induced Nrf2 expression and ferroptosis regulation depend on enhancing the Sesn2/AMPK signaling pathway. This finding was consistent with previous findings by Lu et al. [37]. Furthermore, EMPA improved autophagy and up-regulated Sesn2 expression by enhancing the SIRT1 signaling pathway [57,58].
Given the close relationship between ER stress and autophagy, as well as the role of ER in the development of lung fibrosis [59], we investigated whether EMPA could protect the lungs via an ER-stress-associated mechanism. CHOP is a transcription factor that is used as a marker for ER stress and cell survival [60]. Our findings revealed that BLM significantly increased CHOP levels in lung tissues, which were significantly ameliorated by EMPA treatment, as previously reported [45,61]. Hence, we could postulate that the potential effect of EMPA on mitigating ferroptosis, inflammation, and ER stress in our model was due to its positive impact on Sesn2 expression, as previously reported [18].
Notably, BLM causes single-and double-stranded DNA breaks, which are linked to accelerated DNA fragmentation [62]. In the present study, DNA fragmentation was assessed via the DNA ladder assay method. BLM treatment caused significant DNA band fragmentation. This finding was consistent with previous findings by Hu et al. [63]. Meanwhile, EMPA coadministration reduced DNA fragmentation. Similarly, Min et al. and Ekici et al. showed that EMPA protected against DNA damage in other animal models [64,65].
There are some limitations to this study. First, the precise molecular mechanism underlying EMPA-mediated regulation of autophagy and ferroptosis has yet to be evaluated. Second, the mechanism of action of EMPA needs further investigation by knocking down or overexpressing the related proteins. Third, whether co-treatment with EMPA affects the tumor-killing potency of BLM, as well as whether EMPA is a safe drug for patients receiving BLM, remains to be determined.

Experimental Design
This experiment was conducted on 24 male Wistar rats obtained from the animal house of Tanta University, Egypt, and weighing 160 to 200 g. Throughout the experiment, rats were kept at 22 ± 2 • C with 12 h dark/light cycles and access to chow and water ad libitum. After a one-week acclimatization period, rats were randomly divided into four experimental groups of six rats each (Figure 9). Group I (the control group) received saline intraperitoneally in the same manner as BLM injections, as well as 1% carboxymethyl cellulose (CMC) orally in the same manner as EMPA. Group II (the BLM-treated group) received BLM (15 mg/kg) intraperitoneally three times per week for four successive weeks in order to induce pulmonary fibrosis [66,67]. Group III (the EMPA-treated group) received EMPA dissolved in 1% CMC orally via oral gavage at a dose of 10 mg/kg/day throughout the experimental period [27,68]. Group IV (the combined EMPA and BLM-treated group) received EMPA (10 mg/kg) orally via oral gavage seven days before BLM administration and continued for four weeks after BLM injection (Figure 9) [67].
ceived EMPA dissolved in 1% CMC orally via oral gavage at a dose of 10 mg/kg/day throughout the experimental period [27,68]. Group IV (the combined EMPA and BLMtreated group) received EMPA (10 mg/kg) orally via oral gavage seven days before BLM administration and continued for four weeks after BLM injection (Figure 9) [67].
At the end of the experiment, the overnight fasted rats were weighed, anesthetized with thiopental sodium (20 mg/kg), and sacrificed. After opening the thoracic cavity, BALF was collected as described below and stored at −80 °C. Lungs were harvested, soaked in ice-cold saline, and weighed, and the lung index was calculated as lung wet weight (mg)/body weight (g). The left lung was then fixed in a 10% formaldehyde solution for histopathological and IHC examination. The right lung was stored at −80 °C until it was used for tissue homogenate preparation, nuclear extraction, microsomal separation, and RNA extraction. Total protein concentrations in lung tissue homogenates, nuclear fractions, and microsomes were determined using Lowry's method and bovine serum albumin as a standard [69].
The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Tanta University, Egypt (approval no. 35760/9/22) and followed the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 85 -23, revised in 1996).

BALF Collection and Measurement of Inflammatory Markers
The thoracic cavity was opened, and the tracheas were exposed and cannulated with a blunt needle attached to a syringe. Bronchoalveolar lavage was performed three times with 0.8 mL of sterile phosphate-buffered saline (PBS). The fluid was then centrifuged at At the end of the experiment, the overnight fasted rats were weighed, anesthetized with thiopental sodium (20 mg/kg), and sacrificed. After opening the thoracic cavity, BALF was collected as described below and stored at −80 • C. Lungs were harvested, soaked in ice-cold saline, and weighed, and the lung index was calculated as lung wet weight (mg)/body weight (g). The left lung was then fixed in a 10% formaldehyde solution for histopathological and IHC examination. The right lung was stored at −80 • C until it was used for tissue homogenate preparation, nuclear extraction, microsomal separation, and RNA extraction. Total protein concentrations in lung tissue homogenates, nuclear fractions, and microsomes were determined using Lowry's method and bovine serum albumin as a standard [69].
The study protocol was approved by the Ethics Committee of the Faculty of Medicine, Tanta University, Egypt (approval no. 35760/9/22) and followed the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 85 -23, revised in 1996).

BALF Collection and Measurement of Inflammatory Markers
The thoracic cavity was opened, and the tracheas were exposed and cannulated with a blunt needle attached to a syringe. Bronchoalveolar lavage was performed three times with 0.8 mL of sterile phosphate-buffered saline (PBS). The fluid was then centrifuged at 2000 rpm for 10 min at 4 • C. Cell pellets were collected and resuspended in 500 µL of sterile PBS for total and differential cell counts [70]. BALF supernatant was collected and used to estimate TNF-α and HMGB 1 levels using enzyme-linked immunosorbent assay (ELISA) kits (RayBiotech Inc., Peachtree Corners, GA, USA, and Sunred biological technology, Shanghai, China, respectively).

Determination of Hydroxyproline Content and TGF-β1 in Lung Tissues
Hydroxyproline was estimated as a biochemical index of fibrosis using a colorimetric assay, as previously described [71]. In brief, samples were hydrolyzed with 6 N HCl for 24 h at 120 • C, then treated with the buffer and chloramine T reagent for 20 min at room temperature before being treated with perchloric acid for 15 min at 60 • C. After cooling, the absorbance of the red complex was measured at 550 nm. TGF-β1 levels were measured in lung tissue homogenates using an ELISA kit (Uscn Life Science Inc., Wuhan, China) according to the manufacturer's protocol.

Assessment of Lung Redox Status and Ferroptosis Markers
The MDA levels in lung tissue homogenates were determined using the thiobarbituric acid method, as previously described by Ohkawa et al. [72]. The 4-HNE levels in lung tissue homogenates were assessed using an ELISA kit (Fine Biotech, Wuhan, China). The GSH levels were measured colorimetrically using a commercial kit supplied by Biodiagnostic, Egypt, according to the manufacturer's instructions. Microsomal separation was performed according to the method of Schenkman et al. [73]. The microsomal HO-1 activity was measured using a modified method of Tenhunen et al. [74], in which the amounts of NADPH consumed were calculated using the molar extinction coefficient (ε = 6.220 M −1 cm −1 ). The GPX4 levels in lung tissue homogenates were assayed using an ELISA kit purchased from MyBioSource (San Diego, CA, USA) according to the manufacturer's protocol.

Determination of Active p-AMPK and CHOP in Lung Tissue Homogenates
The p-AMPK levels in lung tissue homogenates were measured using an ELISA kit purchased from Glory Science Co., Ltd., Del Rio, TX, USA. Nuclear extraction of lung tissues was carried out using a Membrane, Nuclear and Cytoplasmic Protein Extraction Kit (Bio Basic Inc., Markham, ON, Canada), as previously described [75]. The nuclear extracts of lung tissues were then assayed for CHOP levels using an ELISA kit purchased from MyBioSource, San Diego, CA, USA.

DNA Fragmentation by the Ladder Assay
The DNA was extracted and fragmented as described by Verma et al. [70]. The lung tissues' DNA was extracted using the GF-1 Tissue DNA Extraction Kit (Vivantis; Shah Alam, Selangor Darul Ehsan, Malaysia). The high-purity genomic DNA was then eluted in lowsalt or water-based buffers. The DNA purity and concentration were then measured at 260 nm. Following that, a 1.5% agarose gel was loaded with 5 µL of each sample and 1 µL of loading dye (6× DNA Loading Dye, Fermentas International, Burlington, ON, Canada). The voltage was adjusted to 80 volts, and the time was adjusted to 45 min. After the run has been completed, the gel was placed on the gel documentation system (Analytic Jena, Biometra model: Biodoc analyzer) for visualization, imaging, and analysis.

Histopathologic and IHC Assays
Paraffin-embedded blocks were prepared from left lung tissue specimens. For histological examination of lung tissue injury, 5 µm thick sections were stained with H and E and Masson's trichrome stain. For IHC staining, 5 µm thick sections were deparaffinized, rehydrated, and washed in PBS, and endogenous peroxidase activity was inhibited with 3% hydrogen peroxide. The sections were then incubated at 4 • C with the primary antibodies, rabbit monoclonal anti-LC3II (0.5 mg/mL; 1:400; catalog no. ab232940; Abcam, MA, USA), and macrophage CD68 (1 mg/mL; 1:400; Bio-Rad, Alfred Nobel Drive, Hercules, CA, USA). To detect immunoreactivity, the sections were treated with a biotinylated secondary antibody, streptavidin peroxidase conjugate, and diaminobenzidine chromogen. The sections were counterstained with Mayer's hematoxylin and morphometrically analyzed using a Leica Qwin 500 C Image analyzer (Leica Imaging System Ltd., Cambridge, UK), with photomicrographs evaluated. At a magnification of 400×, ten randomly selected nonoverlapping fields from each slide were quantified for the mean alveolar septal thickness in H and E-stained sections, the mean area percentage of collagen fiber content in Masson trichrome-stained sections, and the mean percentage and color intensity of positive IHC reactions for LC3II and CD68 in diaminobenzidine-stained sections.

Electron Microscopy
The lung tissues were fixed in 2.5% glutaraldehyde buffer and then in osmium tetroxide. They were then washed and dehydrated with alcohol. They were inserted in epoxy resin to prepare blocks that were later used for cutting ultra-thin sections, which were stained with uranyl acetate and lead citrate. Photographs were captured by a JEOL 2100 transmission electron microscope (Tokyo, Japan) [78,79].

Statistical Analysis
All data were analyzed using the Statistical Package for the Social Sciences (SPSS) software, Version 20.0. For each group of six rats, the data were expressed as means and standard deviations. The one-way analysis of variance (ANOVA) followed by Tukey's test was used to compare data among groups. A p-value of less than 0.05 was considered statistically significant.

Conclusions
The current study is the first to demonstrate that EMPA has a promising protective effect against BLM-induced PF in rats by enhancing autophagy and mitigating ferroptosis, inflammation, and ER stress via modulating the Sesn2/AMPK/Nrf2/HO-1 signaling pathway. However, further studies are warranted to confirm the protective effect of SGLT2 inhibitor therapy, such as EMPA, against pulmonary complications in patients receiving BLM and to determine the extent to which it can antagonize BLM's therapeutic effect in such patients by promoting chemoresistance.

Data Availability Statement:
The data used in this study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.