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Introduction

Lung ischemia-reperfusion (I/R) injury is a common complication that occurs following lung transplantation, cardiopulmonary bypass, pulmonary embolism thrombectomy, and other surgical procedures (1,2). Despite improvements in medical procedures, the incidence of lung I/R injury remains high as has the clinical mortality rate following, especially in lung transplantation (3).

During the lung I/R process, excessive levels of inflammatory mediators and reactive oxygen species (ROS) are released into the circulation, with a crucial role in the sequence of events leading to lung failure (4). Indeed, both inflammatory response and oxidative stress have been found to contribute to the pathogenesis of multi-lung injuries and the inhibition of inflammation and oxidative stress was shown to ameliorate lung injuries (5-7).

Sulforaphane (SFN) is an isothiocyanate derived by the hydrolysis of glucosinolates via the enzyme myrosinase and is enriched in cruciferous vegetables, particularly in broccoli sprouts (8). SFN plays a vital role in redox homeostasis, exerting a cytoprotective action against oxidative stress via activation of Nrf2-related pathways (9-11). Recent studies have shown that the beneficial effects of SFN in I/R-related diseases are due to its antioxidant and anti-inflammatory properties (12). For example, SFN could protect against I/R injury in the liver by activating the Nrf2/ARE pathway (13). Furthermore, the cardioprotective effects of SFN against oxidative stress in cardiomyocytes undergoing I/R were mediated by activation of the Nrf2/HO-1 pathway (14).

Oxidative stress and inflammatory responses underlie several pathological conditions, including I/R injury, and metabolic and age-related diseases. Natural compounds such as SFN may trigger a cellular self-defense mechanism that can effectively mitigate oxidative stress commonly associated with several diseases (15). During the lung I/R process, excessive levels of inflammatory mediators and ROS are released into the circulation, ultimately leading to lung failure; however, the molecular mechanism of SFN on lung I/R injury remains unclear. Therefore, here, the hypothesis that SFN may protect the lung against I/R-induced oxidative stress and inflammation via regulation of the Nrf2-related antioxidant pathway was assessed, based on the following evidence: i) Lung I/R injury is closely associated with oxidative stress and inflammation (6,16); ii) oxidative stress and subsequent apoptosis are involved in lung function disorder (17); iii) SFN was shown to exert a protective effect against oxidative stress via the Nrf2-related antioxidant pathway (18).

The present study aimed to explore the effects of SFN against lung I/R-induced inflammation and oxidative stress and the potential mechanisms involved.

Materials and methods

Animal management and ethical statement

Male Wistar rats (200-250 g) aged 8-9 weeks were obtained from the animal research center at Lvye Pharmaceutical Co., Ltd. in Shandong, China (certificate number SYXK 2018-0028). Rats were housed in a standard environment with a regular light/dark cycle and free access to water and standard chow. The project was approved by the Ethics Committee of Yantai Mountain Hospital, Yantai, China (approval no. 2021-12.). The rats received humane care and all efforts were made to alleviate suffering.

Establishment of the lung I/R model

The rat model of lung I/R injury was induced as described previously (19). Pentobarbital sodium (50 mg/kg, i.p.) was used to fully anesthetize rats, which were then placed on a homeothermic table to maintain the core body temperature at 37˚C. The left pulmonary hilum was clamped with a noninvasive arterial clip, resulting in complete ischemia and hypoxia of the left lung for 1 h. Next, the vascular clamp was released for 2 h to restore ventilation and perfusion to the left lung. The rats were anesthetized with ether and sacrificed by exsanguination at the end of the experiment. Death was confirmed by a lack of autonomous respiration, no reflexive responses, and a lack of a heartbeat.

SFN was obtained from MilliporeSigma (cat. no. S4441), and a stock solution of 5 mM was prepared in DMSO (cat. no. #D2650, MilliporeSigma; final concentration <0.1%). Stock solutions were stored at -25˚C. The stock solution was diluted using 0.9% NaCl solution into 30 mg/kg as previously reported (20). The rats were divided into three equal and random groups (n=10): Sham group, rats subjected to the same thoracotomy procedure but without a hilar block; SFN group, rats subjected to lung I/R injury given SFN (30 mg/kg/day) by intraperitoneal injection for 7 consecutive days before the I/R model was established; I/R group, rats subjected to lung I/R injury were given the same volume of 0.9% NaCl solution. Fig. 1B shows a schematic diagram of the grouping and interventions.

Figure 1 SFN attenuates I/R-induced lung lesions in rats. (A) Schematic representation of the chemical structure of the isothiocyanate sulforaphane. (B) Schematic of experimental design. (C) Representative images of H&E stained lung sections. Scaler bar, 100 µm. H&E staining showed that the infiltration of inflammatory cells was reduced and the alveolar wall thinned gradually in the lungs of the SFN group compared with those of the I/R group. (D) Quantification of lung injury scores. (E) The W/D ratio, (F) PPI, and (G) MPO values of the lungs. Data are presented as the mean ± SD. **P<0.01, Sham vs. I/R group; #P<0.05, ##P<0.01, I/R vs. SFN group. SFN, sulforaphane; I/R, ischemia/reperfusion; H&E, hematoxylin and eosin; i.p. intraperitoneal; W/D, wet/dry; PPI, pulmonary permeability index; MPO, myeloperoxidase.

Blood samples were taken from the femoral artery after reperfusion. Subsequently, the animals were sacrificed with an intravenous overdose of pentobarbital sodium (100 mg/kg). The left lungs were removed for further examination.

Hematoxylin-eosin staining

The middle of the left lung was immediately fixed in 10% formalin and maintained at 4˚C for 24 h. Tissues were dehydrated after 24 h, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin for 30 min at room temperature. All images were taken using a Nikon Eclipse 80i microscope (magnification, x400; Nikon Corporation). The extent of lung damage was evaluated blindly using a histological scoring system as described previously (21).

Blood gas analysis

Arterial blood samples were obtained for blood gas analysis. A blood gas analyzer was used to record pH, the partial pressure of oxygen (PaO2), and the partial pressure of carbon dioxide (PaCO2) in a 0.5 ml sample of arterial blood drawn from the abdominal aorta (RapidPoint 500, Siemens AG).

Detection of lung tissue wet-to-dry (W/D) weight ratio

Wet weight was determined by immediately weighing freshly harvested left upper lung lobe samples. The lung tissue sample was then dried until its weight remained constant. Finally, the dry weight of the lung tissue sample was determined. The lung tissue's W/D weight ratio was calculated by dividing the wet weight by the dry weight.

Detection of pulmonary permeability index

The pulmonary permeability index (PPI) was measured as previously reported (22). To determine total plasma protein, plasma supernatants were obtained and stored at -70˚C. Bronchoalveolar lavage of the lung was performed with 1 ml normal saline. The bronchoalveolar lavage fluid (BALF) was centrifuged at 3,000 x g at 4˚C for 15 min. The BALF supernatant was then stored at -70˚C for the Bradford assay to detect total BALF protein concentration. PPI was calculated by dividing the protein concentration in the BALF by the protein concentration in the plasma.

Detection of myeloperoxidase activity

A total of 100 mg lung tissue was mixed with 1 ml RIPA lysate, homogenized with a glass homogenizer, placed on ice for 30 min to fully lyse the cells, and then centrifuged at 13,000 x g at 4˚C for 15 mi to collect supernatants. The lung tissue myeloperoxidase (MPO) activity was measured using a colorimetry assay kit (cat. no. #A044-1-1, Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

Detection of antioxidant capacity

The lung tissues were homogenized and centrifuged to obtain the supernatant as above for the following experimental detections. The activities of glutathione peroxidase (GSH-Px; cat. no. #A005-1-2), superoxide dismutase (SOD; cat. no. A001-3-2), and catalase (CAT; cat. no. #A007-1-1) in lung tissues were assessed by colorimetry using commercial kits (all from Nanjing Jiancheng Bioengineering Institute) as described previously (23).

Detection of oxidative stress

The lung tissues were homogenized and centrifuged to obtain the supernatant for the following experimental detections. ELISA was used to detect 8-OH-dG (cat. no. ab285254; Abcam Co.US) in lung tissues as described previously (24). Briefly, 40 µl sample dilution buffer was added to 10 µl samples in sample wells. A well was left empty as blank control. 8-OH-dG Biotinylated Detection antibody (50 µl; 1:100) was added to each well and incubated for 45 min at 37˚C. The plate sealer was removed and the wells aspirated and refilled with the wash solution. The washing procedure was repeated three times and the plates dried on absorbent filter papers. SABC working solution (100 µl) was added to each well, the plate covered and incubated at 37˚C for 30 min. Then, the solution was discarded and the plate washed five times with 1X Wash Solution. TMB substrate (90 µl) was added to each well and incubated at 37˚C in dark for 15-30 min. The shades of blue should be seen in the first 3-4 wells by the end of the incubation. Stop solution (50 µl) was added to each well to terminate the reaction. The color in the well changed from blue to yellow. Absorbance at 450 nm was recorded using a VersaMax ELISA Microplate Reader (Molecular Devices, LLC) within 15 min. after adding stop solution. A colorimetric method was used to detect malondialdehyde (MDA; cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute) levels in the lung tissues as previously described (25).

Detection of serum cytokines

Blood samples were collected from the femora artery after reperfusion. The levels of IL-6, IL-1β, and TNF-α in serum were then analyzed using the Pro™ Mouse Cytokine Panel kit (#5827, Bio-Rad Laboratories, USA) according to the manufacturer's instructions.

Western blot analysis

Total protein was extracted from lung tissues. Briefly, 100 mg of lung tissues were homogenized in RIPA buffer for 10 min followed by centrifugation at 13,000 x g for 10 min at 4˚C. The protein concentration was detected using a Bradford Assay kit (cat. no. P0006; Beyotime Institute of Biotechnology). Western blot analyses were performed as described previously (26). Briefly, SDS-PAGE was performed by heating the samples for 8 min at 100˚C and loading 10 µg/lane of the proteins onto a 5-15% linear acrylamide gradient gel. Following transfer to PVDF membranes, the membranes were blocked in 5% BSA (cat. no. SW3015, Beijing Solarbio Science & Technology Co., Ltd.) dissolved in TBST (20%Tween-20) for 2 h at room temperature, and subsequently treated overnight at 4˚C with primary antibodies against the following proteins: Nrf2 (1:3,000; cat. no. #12721), HO-1 (1:3,000; cat. no. #43966), NQO1 (1:3,000; cat. no. #62262), CAT (1:3,000; cat. no. #12980), Histone H3 (1:3,000; cat. no. #4499), Bax (1:3,000; cat. no. #14796), Bcl-2 (1:3,000; cat. no. #498), Cleaved Caspase-3 (1:3,000; cat. no. #9664), and β-actin (1:5,000; cat. no. #4970). All primary antibodies were obtained from Cell Signaling Technology, Inc. The membranes were then incubated with a secondary antibody [1:5,000; anti-rabbit IgG (H+L), cat. no. #14708; Cell Signaling Technology, Inc.) for 2 h at room temperature, followed by TBST washes. Chemiluminescent detection was performed using an ECL kit and a ChemiDoc Touch imaging system (Bio-Rad Laboratories, Inc.). ImageJ (version 1.53; National Institutes of Health was used for densitometry analysis.

TUNEL assay

Apoptosis was determined using a TUNEL assay with a TUNEL test kit (cat. no. T2190, Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. The lung tissues were placed in 10% formalin at room temperature overnight for paraffin embedding and then were sectioned into 5 µm thick slices and processed for TUNEL. Next, tissues were dehydrated, incubated with 0.9% NaCl for 5 min, then rinsed with PBS for 5 min, fixed in 4% paraformaldehyde at room temperature for 15 min, then rinsed twice with PBS (5 min per wash). The sections were mixed with biotinylated nucleotides and terminal deoxynucleotidyl transferase and incubated at 37˚C for 60 min. Following PBS washes, the lung tissues were blocked with 0.3% hydrogen peroxide and incubated with HRP-conjugated streptavidin at room temperature for 30 min, then washed three times with PBS and stained with hematoxylin at room temperature for 3 min. A total of 5 fields of view were automatically selected by Image-Pro Plus version 5.1 (Media Cybernetics, Inc.). The percentage of apoptotic cells was calculated for each field of view. The mean was calculated to obtain the percentage of apoptotic cells and the results are expressed as the apoptotic index (AI), calculated as: AI (%) = (apoptotic nuclei count / total nucleus count) x100%.

Immunohistochemistry

The lung tissues were fixed in 10% formalin for 24 h at 4˚C. Sections of paraffin-embedded specimens were sectioned into 5 µm thick slices and prepared as above. The sections were then rinsed with PBS after being incubated in 3 percent H2O2 for 10 min. Following DAB chromogen incubation at room temperature for 5 min, the sections were counterstained with hematoxylin. Images were taken using a Nikon Eclipse 80i microscope (magnification, x400; Nikon Corporation).

Statistical analysis

Statistical analysis was performed using SPSS version 19.0 (IBM Corp.). Data are presented as the mean ± SD of three independent repeats. A one-way ANOVA followed by a Tukey's post hoc test or an unpaired Student's t-test were used for comparisons between groups. P<0.05 was considered to indicate a statistically significant difference.

Results

SFN attenuates I/R-induced lung lesions in rats

To determine the effects of SFN on lung injury after I/R, hematoxylin-eosin staining for lung histology was evaluated (Fig. 1C). Notably, a disordered alveolar structure was observed in the I/R group, with significant pulmonary interstitial edema, and a large number of inflammatory cells in the alveolar cavity. However, pretreatment with SFN significantly attenuated I/R-induced lung injury histopathologically. All of these changes were corroborated by the histological scoring. Pretreatment with SFN significantly decreased the lung injury score. (Fig. 1D).

An increase in lung permeability will promote the occurrence of pulmonary edema (27). Here, the extent of pulmonary edema based on the W/D ratio and PPI was assessed. As shown in Fig. 1E-F, the W/D ratio and PPI were markedly increased after I/R compared with the Sham group. SFN pretreatment significantly decreased the W/D ratio and PPI in the I/R rats. MPO activity was next assessed to evaluate neutrophil accumulation in the lung tissues. As shown in Fig. 1G, I/R treatment significantly increased MPO activity, whereas MPO activity was significantly reduced by SFN pretreatment.

Rat arterial blood gas analysis was performed to further assess lung injury. As shown in Table I, the arterial blood pH value was significantly decreased after I/R compared with the Sham group, and the I/R-induced acidosis was significantly prevented by SFN pretreatment. In addition, pretreatment with SFN also significantly prevented the increase in PaCO2 and decrease in PaO2 values in the I/R-induced rats. These results indicate that SFN pretreatment attenuated I/R-induced lung lesions in rats.

Table I Arterial blood gases at the end of reperfusion.

Table I

Arterial blood gases at the end of reperfusion.

Group	pH	PaO2 (mmHg)	PaCO2 (mmHg)
Sham	7.35±0.11	262.21±18.12	55.22±6.41
Ischemia-reperfusion	7.12±0.08a	167.35±21.21a	76.61±6.71a
SFN	7.22±0.09b	214.10±23.54b	62.24±7.43b

[i] ^aP<0.05, Sham vs. I/R group;

[ii] ^bP<0.05, I/R vs. SFN group. All data are presented as the mean ± SD. SFN, Sulforaphane; PaO₂, arterial partial pressure of oxygen; PaCO₂, arterial partial pressure of carbon dioxide.

SFN alleviates I/R-induced oxidative stress and inflammation in the lungs of rats

The effect of SFN pretreatment on the redox metabolism and inflammatory response in the lungs of I/R rats was next evaluated. As shown in Fig. 2A and B, compared with the Sham group, the levels of MDA and 8-OH-dG were significantly increased in the I/R group. However, SFN pretreatment significantly downregulated the levels of MDA and 8-OH-dG in the I/R-induced rats. In addition, pretreatment with SFN markedly reversed the decrease in the antioxidant enzyme activities of CAT, SOD, and GSH-Px in the lung of I/R treated rats (Fig. 2C-E). Furthermore, serum IL-6, IL-1β, and TNF-α levels in the I/R group were significantly increased compared with those in the Sham group, and a significant decrease was observed in the SFN pretreatment group (Fig. 2F-H). These results show that SFN pretreatment alleviated the I/R-induced imbalance of redox metabolism and the aggravation of inflammatory reactions in the lungs of rats.

Figure 2 SFN alleviates I/R-induced lung oxidative stress and inflammation in rats. The levels of (A) MDA and (B) 8-OH-dG oxidative stress parameters. The antioxidant enzyme activities of (C) CAT, (D) SOD, and (E) GSH-Px. The levels of serum inflammatory cytokines (F) IL-6, (G) IL-1β, and (H) TNF-α. Data are presented as the mean ± SD. **P<0.01, Sham vs. I/R group; #P<0.05, ##P<0.01, I/R vs. SFN group. SFN, sulforaphane; I/R, ischemia/reperfusion; MDA, malondialdehyde; CAT, catalase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; prot, protein.

SFN improves I/R-induced lung apoptosis in rats

TUNEL staining was used to determine whether SFN attenuated lung tissue apoptosis following I/R in rats. As shown in Fig. 3A and B, the results of the TUNEL assays showed a significant increase in the AI in the I/R group compared with the Sham group, and SFN pretreatment significantly decreased the AI in the lung tissues of the I/R rats.

Figure 3 SFN improves I/R-induced lung apoptosis in rats. (A) Representative images of TUNEL-stained lung sections. (B) Quantification of the apoptosis index. (C) The protein expression levels of the Bax/Bcl-2 pathway members were evaluated by western blotting. Densitometry analysis of (D) Bcl-2, (E) Bax, and (F) C-casp3 protein expression levels. Data are presented as the mean ± SD. *P<0.05, **P<0.01, Sham vs. I/R group; #P<0.05, ##P<0.01, I/R vs. SFN group. SFN group. SFN, sulforaphane; I/R, ischemia/reperfusion; C-casp3, cleaved-caspase 3.

To determine the potential molecular mechanism of SFN in reducing lung apoptosis in I/R rats, the expression of several apoptosis-associated proteins in the lung tissues was measured (Fig. 3C). As shown in Fig. 3D-F, the expression of the pro-apoptotic proteins Bax and C-casp-3 were significantly increased, whereas the expression of the anti-apoptotic protein Bcl-2 was markedly decreased in the I/R group compared with the Sham group. SFN pretreatment notably attenuated the decrease in Bcl-2 expression and the increase in Bax and C-casp-3 expression compared with the I/R group. These data showed that SFN improved I/R-induced lung apoptosis in rats by suppressing the Bax/Bcl-2 pathway.

SFN protects against I/R-induced lung lesions via activation of the Nrf2/HO-1 pathway

It has been shown that the Nrf2/HO-1 pathway plays an important role in maintaining redox metabolism and inhibiting apoptosis in lung tissues (28). Thus, the expression of Nrf2 and its downstream antioxidant genes in the lung tissues of I/R rats with or without SFN pretreatment was assessed (Fig. 4A). As shown in Fig. 4B, the nuclear transfer of Nrf2 (n-Nrf2) was markedly increased in the I/R group compared with the Sham group. Accordingly, the expression levels of Nrf2 target antioxidant genes HO-1, NQO1, and CAT were also markedly increased in the I/R group compared with the Sham group (Fig. 4C-E). Interestingly, SFN pretreatment further increased the expression of n-Nrf2 and its downstream antioxidant genes compared with the I/R group (Fig. 4B-E). These results suggested that SFN protected against I/R-induced lung lesions in rats via activation of the Nrf2/HO-1 pathway.

Figure 4 SFN protects against I/R-induced lung lesions via activation of the Nrf2/HO-1 pathway. (A) The protein expression levels of the Nrf2/HO-1 pathway members were evaluated by western blotting. Densitometry analysis of (B) nuclear Nrf2, (C) HO-1, (D) NQO1, and (E) CAT protein expression levels. Data are presented as the mean ± SD. *P<0.05, **P<0.01, Sham vs. I/R group; ##P<0.01, I/R vs. SFN group. SFN group. SFN, sulforaphane; I/R, ischemia/reperfusion; CAT, catalase.

Discussion

As lung I/R injury is a recognized fatal complication following lung transplantation, there is an urgent need to identify novel therapeutic targets for alleviating lung I/R injury. I/R is directly related to the formation of ROS, increased vascular permeability, the activation of neutrophils, and cytokine release (29). The findings of the present study demonstrate that SFN can protect the lung against I/R-induced oxidative stress and inflammation, as evidenced by reducing ROS production and the release of pro-inflammatory cytokines. The beneficial effects of SFN on lung I/R injury involved activation of the Nrf2-related antioxidant pathway.

Oxidative stress is the outcome of an imbalance between the generation of ROS and the antioxidant defense systems, which is characterized by increases in ROS and other free radicals, leading to cellular injury (30). Oxidative stress is closely associated with the initiation and progression of lung I/R injury (31). The Nrf2-related pathway is considered a defense system aimed to counteract oxidative stress and preserve cellular homeostasis (32-34). Under physiological conditions, Nrf2 binds to its negative regulator Keap1 and is maintained in an inactive state. In the presence of ROS, Nrf2 dissociates from Keap1 and translocates to the nucleus, and binds to Maf (35,36). The Nrf2-Maf heterodimers then bind to antioxidant response elements in the promoters of key antioxidant genes (such as HO-1, NQO1, and CAT) and activates their transcription (37,38). HO-1 catalyzes the breakdown of heme to produce biliverdin, ferrous ions, and carbon monoxide, all of which are essential components of the inflammatory process (39). The homodimeric luteinase NQO1 promotes the elimination of hydrazine, which can produce harmful semihydroquinone radicals through the redox cycle, by catalyzing the reduction of hydrazine to hydroquinone (26,39). CAT promotes the synthesis of intracellular catalase, which catalyzes the decomposition of H2O2 into H2O and O2 (40). By scavenging excessive ROS levels and restoring redox homeostasis, Nrf2 can prevent I/R-related disorders (41). As previously described, SFN is a bioactive molecule present in broccoli, which exerts its cytoprotective effect by activating an Nrf2-related pathway (42). In this study, the expression of Nrf2 and its downstream target genes HO-1 and NQO1 were significantly decreased in the I/R rats, and SFN treatment significantly suppressed ROS generation and activated the Nrf2 antioxidant pathway, thus exerting therapeutic effects on IRI-induced injury by restoring cellular ROS homeostasis.

It is well established that the over-production of pro-inflammatory cytokines is another crucial trigger of cellular damage (43,44). Oxidative stress is closely correlated with the inflammatory response, especially during the I/R injury process. High levels of ROS produced during oxidative stress stimulate the release of pro-inflammatory mediators and increase inflammation, which may further aggravate I/R injury. In addition, activation of Nrf2 not only inhibits oxidative stress response but also contributes to the anti-inflammatory process by regulating cytokine secretion (45). SFN is a natural product that exerts its beneficial effects via the activation of the antioxidant systems and suppression of pro-inflammatory responses through the activation of Nrf2-related pathways (46,47). In the present study, elevated levels of pro-inflammatory cytokines were observed in the I/R rats, which were also effectively attenuated by SFN treatment. In line with the results of the present study, SFN was reported to ameliorate LPS-induced ROS, reactive nitrogen species, pro-inflammatory cytokine production, and cell death via Nrf2 activation (48).

Oxidative stress and inflammation are two major factors involved in the pathogenesis of lung I/R injury, which jointly contribute to the apoptosis of lung cells. Apoptosis is closely associated with the pathological process of lung I/R injury, according to earlier studies (49,50). Bcl-2, an anti-apoptotic protein, can inhibit the production of free radicals and endoplasmic reticulum Ca2+ as well as prevent the formation of lipid peroxides (51). Bax is an endogenous antagonist of Bcl-2; by physically attaching to the related protein homologs, it inhibits Bcl-2, thus inducing apoptosis (52). Bcl-2 and Bax expression levels are typically balanced in a healthy state (53). In the present study, it was found that lung cell apoptosis was activated in rats with I/R lung injury and that Bax protein expression increased while Bcl-2 protein expression was decreased. An important biochemical aspect of apoptosis is the activation of caspases. The beginning and completion of mammalian apoptotic processes are regulated by the caspase family of cysteine proteases (54). Caspase-3 is created from a 32 kDa zymogen and cleaves to a 17 kDa active subunit via mitochondrial and death ligand mechanisms (55). This zymogen is a crucial caspase effector that starts the cell's disintegration during the final stages of apoptosis (56). In the present study, it was found that SFN effectively inhibited the expression level of the pro-apoptotic proteins Bax and C-casp-3 in the I/R group rats, thus reducing apoptosis. A previous study showed that Nrf2/HO-1 activation counteracts the inflammatory response and apoptosis in contrast-induced renal injury (57). Herein, the beneficial effects of SFN on I/R injury via antioxidative stress, anti-inflammation, and anti-apoptosis were dependent on the activation of the Nrf2/HO-1 pathway. Conversely, as mitochondria are the primary source of ROS, the destruction of mitochondria leads to the accumulation of excessive ROS levels, resulting in increased mitochondrial dysfunction (58). MDA, SOD, and GSH are important indicators for detecting oxidative stress. Through a number of signaling mechanisms, the generation of ROS can lead to oxidative stress in cells and cause cells to undergo apoptosis (34,59). When mitochondria undergo apoptosis, the expression of Bcl-2 is inhibited (60). However, at present, whether SFN alleviates I/R-induced oxidative stress in lung tissue by alleviating mitochondrial damage remains to be further studied.

In summary, the present study provided evidence that SFN protected against lung I/R injury-induced oxidative injury, inflammation, and apoptosis via activation of the Nrf2/HO-1 pathway. These findings may provide novel insights into the development of therapeutic applications of SFN for lung I/R injury.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LZ and CY conceived and designed the experiments. SW and YZ performed the experiments. FL analyzed the data. LZ wrote the manuscript. All authors read and approved the final manuscript. LZ and CY confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Yantai Mountain Hospital, Yantai, China (approval no. 2021-12.).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References