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. 2021 Dec 8;27(1):55–70. doi: 10.1007/s12192-021-01248-8

Protective effect of hydrogen sulfide against stress-induced lung injury: involvement of Nrf2, NFκB/iNOS, and HIF-1α signaling pathways

Fatma F Ali 1,✉, Hanaa Hassanein Mohammed 2, Doaa M Elroby Ali 3
PMCID: PMC8821758  PMID: 34881408

Abstract

Stress is a common phenomenon that is attracting increasing attention. Hydrogen sulfide (H2S) is a gasotransmitter that plays an important role in many physiological and pathological events. Our study aimed to estimate the effect and the underlying mechanisms of the H2S donor, sodium hydrosulfide (NaHS), against immobilization stress (IS)–induced lung injury. Forty adult male rats were classified into control group, NaHS group, and IS groups with and without NaHS treatment. Serum was obtained to determine corticosterone (CORT), total antioxidant capacity (TAC), tumor necrosis factor‐α (TNF‐α), and interleukin-10 (IL-10) levels. Lung H2S, nitric oxide (NO), inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) levels were measured. Lung expressions of H2S synthesizing enzymes and Western blot analysis of nuclear factor erythroid 2–related factor 2 (Nrf2) and hypoxia-inducible factor 1 alpha (HIF 1α) were estimated. Histopathological changes and immunohistochemical assessment of nuclear factor kappa B (NF-κB) and caspase‐3 were also done. Pretreatment with NaHS led to marked histological protection from lung damage seen in IS rats. Furthermore, pretreatment with NaHS before IS protected lung H2S levels and expressions of H2S-synthesizing enzymes. Similarly, the levels of CORT, TNF-α, IL-10, MDA, TAC, NO, iNOS, HIF-1 α, and nuclear Nrf2 and expressions of NF-kB and caspase 3 were all maintained at near control levels in contrast to that in the IS rats. In conclusion, NaHS is protective against stress‐induced lung injury due to its antioxidant, anti-inflammatory, anti-fibrotic, and antiapoptotic effects. Thus, NaHS can be used to minimize stress complications on lung.

Keywords: Stress, Lung, Sodium hydrosulfide, Nuclear factor erythroid 2–related factor 2, Nuclear factor kappa B, Hypoxia-inducible factor 1 alpha

Introduction

Stress is a major threat to body homeostasis. It is considered a risk factor in 75–90% of all diseases, including those that lead to morbidity and mortality (Yisireyili et al. 2019). Immobilization stress is considered an easy method for inducing both psychological and physical stresses in mammals (Samad et al. 2020). These days, people experience psychological stress for days or even months. Therefore, it is necessary to use real animal models of long‐term psychological stress to understand the effects of exposure to chronic stress (Han et al. 2020).The production of reactive oxygen species (ROS) is as a key mechanism of stress that leads to oxidative stress in different tissues, including the lungs (Yisireyili et al. 2019). The mechanism by which ROS increases during stress remains unclear and is likely mediated in part by stress-related hormones such as cortisol and corticosterone that have been shown to accelerate glucose metabolism and ROS production (Chen et al. 2018).

Nuclear factor erythroid 2–related factor 2 (Nrf2) is the primary modulator of molecular systems regulating redox homeostasis that activates a network of antioxidants, anti-inflammatory molecules, and free radical scavengers. For that, downregulation or suppression of Nrf2 activity increases the cell susceptibility to the damaging effects of ROS and pro-inflammatory stimuli (Sivandzade et al. 2019). On the other side, nuclear factor kappa B (NF-κB) is a transcription factor that is activated and induced by various molecules such as cytokines and ROS and in turn it triggers the transcription of proinflammatory mediators such as tumor necrosis factor alpha (TNF-α) and iNOS (Ali et al. 2019; Zhang et al. 2018). Chronic stress also enhances the activation of NF-κB in response to inflammatory stimuli (Koo et al. 2010).

Hypoxia-inducible factor (HIF)-1 is composed of two subunits: HIF-1α and HIF-1β. HIF-1α acts as an adaptive and survival factor for cells exposed to hypoxia or undergoing stress. However, HIF-1α may be deleterious due to its ability to augment both apoptotic and inflammatory processes (Wu et al. 2019). On the other hand, fibrosis is a complex process that occurs in various organs including the lung and can cause structural damage, functional decline, and even failure that is seriously threatening to human health. During fibrosis, the tissue repair that occurs after injury is out of control and leads to excessive formation of fibrotic connective tissue. Fibrosis is induced by various factors such as inflammation that occurs in cases of stress exposure (Zhang et al. 2015).

Hydrogen sulfide (H2S) is the third gasotransmitter in mammals (Guan et al. 2020) and is produced by three enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). H2S possesses potent anti-oxidant, anti-inflammatory, and other physiological regulatory functions and acts as a critical biological factor involved in the response to injury in the lung, liver, heart, adrenal gland, and other organs (Zhang et al. 2016). Endogenous H2S participates in the regulation of important physiological functions in the respiratory tract such as airway tone, pulmonary circulation, cell proliferation, and apoptosis, and to modulate lung fibrosis, oxidative stress, and inflammation. An intact H2S-generating pathway appears to be an important protective mechanism against disease (Bazhanov et al. 2017). Therefore, our study aimed to estimate the potential effect and underlying mechanisms of the H2S donor, NaHS, against chronic stress–induced lung injury. This was done through estimation of H2S production and gene expressions of H2S synthesizing enzymes, CSE, CBS, and 3-MST in lung tissues, measuring the serum level of the stress hormone, CORT, the inflammatory markers, TNF-α and IL-10, and the determination of oxidative stress markers, iNOS, NO, malondialdehyde (MDA), and total anti-oxidant capacity (TAC). Moreover, lung tissue gene expressions of HIF-1 α and Nrf2 were determined. Furthermore, the lung histopathological changes and expressions of NF-κB and pro-apoptotic marker, caspase 3, were examined.

Materials and methods

In this study, forty adult male Sprague–Dawley rats weighing about 220–250 g were bought from the National Research Center, Giza, Egypt. They were harbored under standard laboratory conditions with natural day/night cycle with normal rat chow and water was available ad libitum. Animals were left to acclimatize to the environment for 1 week prior to inclusion in the experiment. The use of animals was in accordance with the guidelines of the “Animal Care and Use Committee” of the Faculty of Medicine, Minia University, Egypt (approval No. 676–9/2020), and in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (Health 1985).

Animals were randomly classified into the following groups (10 rats each):

Control group: rats were left undisturbed without exposure to stress. They were injected intraperitoneally (i.p.) with saline for 10 days.

Sodium hydrosulfide (NaHS) group: rats were injected i.p. with NaHS (100 µmol/kg/day) for 10 days (Han et al. 2020).

Immobilization stress (IS) group: Each rat was injected i.p. with saline 45 min before being exposed to IS. Then, rats were immobilized 2 h once a day for 10 days (Saber et al. 2019).

NaHS + IS group: rats were injected i.p with NaHS (100 µmol/kg/day) for 10 days (Han et al. 2020), 45 min before being subjected to IS (Elbassuoni and Nazmy 2018).

Induction of chronic immobilization stress:

Immobilization stress rats were bound in the supine position to a wooden board by taping the four limbs with surgical tapes to a specially prepared metal mounts but head motion was not limited (Stojkov et al. 2012). Animals were subjected to the stress protocol daily for 2 h from 10:00 a.m. to 12 p.m. for 10 days (Tripathi et al. 2017; Saber et al. 2019). Immobilization stress is considered an easy and convenient method for physiological and psychological stress in rodents (Gomaa et al. 2017). Immobilization is a common method of stress induction in animals and its effects are comparable to that of chronic stress in humans and this is in accordance with other previous studies (Stojkov et al. 2012; Saber et al. 2019). The 10-day stress duration was sufficient to result in marked decrease in H2S production and expression of H2S synthesizing enzymes in other previous studies done on different organs (Han et al. 2020).

Drug protocol

NaHS (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline. Doses were selected on the basis of our preliminary experiments and on the basis of previous reports which used the same or similar dose but on different organs (Zayachkivska et al. 2014; Sun et al. 2017; Han et al. 2020; Elbassuoni and Nazmy 2018). Multiple studies have demonstrated that H2S can play cytoprotective effects at micromolar concentrations. Some animal experiments have confirmed that infusion of NaHS or H2S inhalation has anti-oxidative and anti-inflammatory effects in animal models of various types of lung injury (Zhang et al. 2016).

Biochemical analysis

At the end of the experiment and stress period, the rats were decapitated and blood samples were obtained and left to clot. Then, centrifuged at 3,000 rpm for 15 min at 4 °C for separation of sera that were collected and stored at − 20 °C for analysis of corticosterone, TAC, TNF-α, and IL-10. Determination of corticosterone concentrations was done according to the method of Mattingly (1962) that is based on extraction of free 11-hydroxycorticosteroids, mainly cortisol and CORT, by methylene chloride followed by their condensation with an acidic fluorescence reagent. The induced fluorescence was measured using a spectrofluorometer. Measurement of TAC was carried out using commercially available kit (Biodiagnostic, Giza, Egypt). Determination of serum TNF-α and IL-10 was done by ELISA kits (My BioSource, USA) according to the manufacturer’s instructions.

Analysis of lung homogenates

The chest of each rat was opened to take out the lung tissues. Pieces of lung tissues were weighed and homogenized in cold phosphate-buffered saline (PBS) (pH 7.4). The homogenates were centrifuged at 4 °C at 4,000 rpm for 15 min and the supernatants were obtained and stored at − 80 °C for estimation of H2S content, NO, iNOS, and MDA levels.

Measurement of H2S content

Measurement of lung H2S content was done following the manufacturer’s instructions using H2S ELISA kit (AFG Bioscience, USA).

Measurement of MDA and NO

Measurement of lung MDA and NO levels was performed using colorimetric assay kits (Biodiagnostic, Giza, Egypt).

Measurement of iNOS

Measurement of lung iNOS levels was done using ELISA kit (Elabscience, USA) following the manufacturer’s instructions.

Western blot analysis

Proteins from lung tissue were extracted on ice for 30 min on a shaker using lysis buffer (radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor). 16,000 g centrifugation for 30 min at 4 °C eliminated cell debris. The supernatant was then transferred to a separate tube to be analyzed for protein concentration. BIO BASIC INC donated the Bradford Protein Assay Kit (SK3041) that was used to conduct quantitative protein analysis (Markham, Ontario, Canada). Each sample was loaded with a 20 µg protein concentration of Laemmli sample buffer in an equal amount. To ensure that proteins were denatured, an aliquot of 7.5.µg protein from each sample was boiled in Laemmli buffer at 95 °C for 5 min, then put into an individual lane in sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene (PVDF) membranes The membranes were blocked for 1 h at room temperature with a blocking solution made up of Tris-buffered saline with Tween 20 (TBST) buffer and 3% bovine serum albumin (BSA), then incubated overnight at 4 °C with a primary antibody (rabbit HIF-1 NB100-479,1:1000, Nrf2 NBP1-32,822,1:500, Histone-3 NB500-171,1:100, and β-actin NB600-503,1:500) (rabbit IgG-Novus Biologicals USA) diluted in TBST against the blotted target TBST was applied to the blot three to five times for a total of 5 min. Following that, the blotted target protein was incubated for 1 h at room temperature in a peroxidase conjugated secondary antibody solution (goat antirabbit IgG-HRP-1 mg goat mab—Novus Biologicals). TBST was applied to the blot three to five times for a total of 5 min. An enhanced chemiluminescence (ECL) system (Clarity Western ECLyTM substrate—BIO-RAD, USA) was used to visualize protein bands. A CCD camera-based imager was used to capture the chemiluminescent signals. On the Chemi Doc MP imager, image analysis software was utilized to compare the band intensity of the target proteins to the control sample by normalizing to β-actin and histone-3 (BIO RAD, USA).

Real-time PCR

Jena Bioscience (Munich, Germany) provided a total RNA purification kit, which was used to isolate total RNA from homogenized tissue and preserve it at 80 °C. The cDNA archive kit was used to convert RNA into its complementary DNA (Applied Biosystems, Foster City, California, USA). The GoTaq PCR master mix was used for qPCR (Promega Co., Madison, USA). On a Phase One Real-Time PCR System, a protocol that included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 15 s, annealing and extension at 60 °C for 1 min, then 60 °C for 30 s was used (Applied Biosystems, Foster City, California, USA). The oligonucleotide primers listed below were used.:

5′-GAACCAGACGGAGCAAACAG-3′ (sense).

5′-GGCGAAGGAATCGTCATCA-3′ (antisense) for CBS.

5′-GGCCTGAAGTGTGAGCTCTT-3′ (sense).

5′-TTGGGGATTTCGTTCTTCAG-3′ (antisense) for CSE.

5′-GACCCCGCCTTCATCAAG-3′ (sense).

5′-CATGTACCACTCCACCCA-3′ (antisense) for 3-MST.

5′-GACGAGGCCCAGAGCAAGAGAGG-3′ (sense).

5′-GATCCACATCTGCTGGAAGGTGGAC-3′ (antisense) for β-actin as an internal control.

Histological examination

Samples of lung tissues were taken. The tissues were directly fixed in neutral buffered 10% formalin solution, and managed into 5-µm-thick paraffin sections. Then, stained with hematoxylin and eosin (H&E) and Masson’s trichrome stain (Suvarna et al. 2018). The stained sections of the lung tissues were studied under the light microscope.

Staining technique for immunohistochemical studies

Immunocytochemical staining was done using polyclonal rabbit antibodies against cleaved caspase 3 (PA1-26,426) and nuclear factor kappa beta (NF-κB) (PA04206) that were obtained from Sigma-Aldrich. Paraffin sections of the lung tissues of different groups were cut into 5-μm thickness and incubated at 42 °C in an oven for 24 h. The sections were deparaffinized in xylene (1 h), hydrated in descending grades of alcohol, incubated in hydrogen peroxide (5 min), and then washed twice in PBS (5 min each). The primary antibody (diluted 1:100) was applied to the sections that were then incubated for 1.5 h. Next, the sections were washed twice in PBS for 5 min each. The secondary antibody (1–1000) was applied and the sections were again incubated for 20 min, following which they were washed three times in PBS for 5 min each. Diaminobenzidine tetra hydrochloride solution was then applied to the sections and they were again incubated for 10 min. The sections were then washed in distilled water and counterstained with Mayer’s hematoxylin (2 min), following which they were washed in tap water, dehydrated, cleared, and mounted by DPX.

Photography

Olympus light microscopy (Olympus, Japan) was used for examining and capturing images for the histological and immunohistochemical sections. Slides were photographed using Olympus digital camera (U.TV0.5XC-3).

H&E scoring

The four aspects, i.e., alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in alveoli or vessel wall, and thickness of alveolar wall/hyaline membrane formation, were graded with 5-point scales: 0 = minimal damage, 1 = mild damage, 2 = moderate damage, 3 = severe damage, and 4 = maximal damage. A total lung injury score was designed as the sum of the four items (Tan et al. 2014).

Masson scoring

Each lung section was assessed histologically and scored as follows: 0—absence of alveolar fibrosis; 1—mild fibrosis; 2—moderate fibrosis; and 3—marked fibrosis (Stocker 1986).

Measuring area fraction (morphometric analysis) of NF-κB and caspase 3 immunoreactivity

NF-kB and caspase 3 immunoreactivity were examined under 400 × objective and assessed as area fraction by image analysis software Image J (Schneider et al. 2012).

Statistical analysis

The study data were represented as means ± standard deviation (SD). GraphPad Prism 6 software (La Jolla, CA) was used for statistical analysis. Significant difference between groups was done by one‐way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test and p value < 0.05 was considered statistically significant.

Results

Changes in the measured serum parameters

The current results showed significant increase in both CORT and TNF-α levels accompanied with significant decrease in both TAC and IL-10 levels in chronic immobilization stress group as compared to the control and NaHS groups. However, treatment of rats with NaHS before IS suppressed the increase in serum CORT and TNF-α seen in IS rats. Treatment of rats with NaHS before IS also attenuated the decrease in serum TAC and IL-10 levels in comparison with IS group (Table 1).

Table 1.

Serum parameters in different experimental groups

Parameters Groups
Control NaHS IS NaHS + IS
CORT (ng/ml) 84.51 ± 4.68 86.50 ± 5.63 137.50 ± 6.05ab 107.50 ± 5.97abc
TNF-α (pg/ml) 90.87 ± 4.60 95.19 ± 4.93 165.90 ± 5.37ab 127.40 ± 4.72abc
IL-10 (pg/ml) 66.32 ± 5.21 69.40 ± 3.75 33.39 ± 4.57ab 49.66 ± 4.63abc
TAC (mmol/L) 1.78 ± 0.36 1.80 ± 0.38 0.63 ± 0.24ab 1.19 ± 0.20abc
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NaHS sodium hydrosulfide, IS immobilization stress, CORT corticosterone, TNF-α tumor necrosis factor-alpha, IL-10 interleukin 10, TAC total antioxidant capacity

aSignificant difference from the control group

bSignificant difference from the NaHS group

cSignificant difference from the IS group, p < 0.05. Values are expressed as mean ± SD of 10 rats in each group

Oxidative stress parameters

In regard to lung tissue NO, iNOS, and MDA, there was significant increase in their levels in the chronic immobilization stress group as compared to the control and NaHS groups. Interestingly, treatment of rats with NaHS before IS suppressed the increase in NO, iNOS, and MDA levels in comparison with IS group (Table 2).

Table 2.

Oxidative stress parameters in different experimental groups

Parameters Groups
Control NaHS IS NaHS + IS
NO (µmol/gm) 9.48 ± 0.53 9.93 ± 0.69 16.53 ± 0.70ab 12.90 ± 0.79abc
MDA (nmol/gm) 59.16 ± 6.38 63.27 ± 6.46 107.50 ± 6.18ab 87.96 ± 5.03abc
iNOS (ng/mg) 2.87 ± 0.44 3.16 ± 0.53 6.92 ± 0.53ab 4.96 ± 0.48abc
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NaHS sodium hydrosulfide, IS immobilization stress, NO nitric oxide, MDA malondialdehyde, iNOS inducible nitric oxide synthase

aSignificant difference from the control group

bSignificant difference from the NaHS group

cSignificant difference from the IS group, p < 0.05. Values are expressed as mean ± SD of 10 rats in each group

Lung H2S content and expression of H2S-synthesizing enzymes

The results showed that both lung tissue H2S levels and expression of H2S-synthesizing enzymes were significantly decreased in chronic IS rats compared to control and NaHS rats. Treatment of rats with NaHS before IS effectively protected them from the decline seen in both lung tissue H2S levels and expression of H2S-synthesizing enzymes when compared to the IS group (Fig. 1).

Fig. 1.

Fig. 1

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Lung tissue H2S content and relative expression of H2S synthesizing enzymes in different experimental groups. NaHS: sodium hydrosulfide; IS: immobilization stress; H2S: hydrogen sulfide; CBS: cystathionine-β-synthase; CSE: cystathionine-γ-lyase; 3-MST: 3-mercaptopyruvate sulfurtransferase. aSignificant difference from the control group and bsignificant difference from the NaHS group and csignificant difference from the IS group, p < 0.05. Values are expressed as mean ± SD of 10 rats in each group

Lung Nrf2 and HIF-1 α protein levels

In lung tissue, there were significant increases in both cytoplasmic Nrf2 and HIF-1 α levels and a decrease in nuclear Nrf2 levels in rats exposed to chronic immobilization stress compared with the control and NaHS rats. Treatment of rats with NaHS before IS effectively suppressed both cytoplasmic Nrf2 and HIF-1 α and attenuated the decrease in nuclear Nrf2 levels as seen in the IS rats (Fig. 2).

Fig. 2.

Fig. 2

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Lung Nrf2 and HIF-1 α relative protein levels in different experimental groups. NaHS: sodium hydrosulfide; IS: immobilization stress; Nrf2: nuclear factor erythroid 2-related factor 2; HIF-1α: hypoxia-inducible factor -1 alpha. aSignificant difference from the control group and bsignificant difference from the NaHS group and csignificant difference from the IS group, p < 0.05. Values are expressed as mean ± SD of 10 rats in each group

Histological changes

Study of lung sections stained with H&E demonstrated that both the control (A1 and A2) and NaHS groups (B1 and B2) had normal architecture. Bronchioles of different sizes appeared regular with intact folded mucosa and surrounded by regularly arranged smooth muscle fibers and outer adventitia. Lung tissues consisted of alveolar ducts, alveolar sacs, and different-sized alveoli with thin inter alveolar septa. The alveoli were lined by simple squamous epithelium that has two types of cells: squamous, thin cells with flat nuclei (pneumocyte type I) and cuboidal cells with rounded nuclei (pneumocyte type II) bulging into the alveolar lumen. Blood capillaries were seen within the inter alveolar septa (Fig. 3, Table 3).

Fig. 3.

Fig. 3

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A representative photomicrograph of a section in the lung of control and NaHS groups. A1 and B1 showing normal structure of a bronchiole (B), the alveolar duct (AD), alveolar sac (AS), and alveolus (A) which is lined by simple squamous epithelium within a thin interalveolar septum (arrow heads) and has a wide lumen. An intervening blood vessel could be seen (BV) H&E, × 100. A2 and B2 showing normal alveoli (A) which is lined by a squamous pneumocyte type I (arrows) and a pneumocyte type II bulging into the alveolar lumen (elbow arrows). A blood capillary (C) can be seen in the interalveolar septa. H&E, × 400

Table 3.

Histological scores and immunohistochemical mean area fraction in different experimental groups

Groups
Control NaHS IS NaHS + IS
H&E score 0.2 ± 0.42 0.3 ± 0.48 2.9 ± 0.31ab 1.1 ± 0.56abc
Fibrosis score 0.2 ± 0.42 0.1 ± 0.31 2.5 ± 0.52ab 1 ± 0.81abc
NF-κB 0.4 ± 0.51 0.7 ± 0.48 26.8 ± 3.99ab 10.20 ± 2.15abc
Caspase 3 1.8 ± 0.63 2.4 ± 0.69 33 ± 1.94ab 9.8 ± 1.55abc
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NaHS sodium hydrosulfide, IS immobilization stress, H&E hematoxylin and eosin, NFκB nuclear factor kappa B

aSignificant difference from the control group

bSignificant difference from the NaHS group

cSignificant difference from the IS group, p < 0.05. Values are expressed as mean ± SD of 10 rats in each group

The lung tissues exposed to chronic immobilization stress showed destruction and denudation of the epithelial lining of the bronchioles. Inflammatory mass infiltrating the wall of bronchioles with intra bronchiolar cellular debris collapsed alveoli with thick prominent inter alveolar septa studded with inflammatory cells and RBCs. Macrophages with hemosiderin granules and dilated congested blood vessel could be seen. Hemorrhagic areas and dilated congested blood vessels with margination of inflammatory cells mainly neutrophils also were noticed. Multiple macrophages with highly acidophilic cytoplasm and irregular outline, inflammatory cell infiltration mainly neutrophils in the highly thickened inter alveolar septum, and Langhans giant cells in which macrophages fuse together, the nuclei forming a cute horse-shoe shape around the periphery of the cell (Fig. 4, Table 3).

Fig. 4.

Fig. 4

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A representative photomicrograph of section in the lung of the chronic immobilization stress group. C1 showing inflammatory mass (arrows) infiltrating (circle) the wall of a bronchiole with intra bronchiolar cellular debris (rectangle), collapsed alveoli (A) with thick prominent inter alveolar septa (double arrow) studded with inflammatory cells and RBCs (elbow arrows). A macrophage with hemosiderin granules (red arrow) and dilated congested blood vessel (BV) could be seen (H&E, × 100). C2 is a larger magnification (H&E, × 400) of C1 showing the intra bronchiolar cellular debris (oval), destructed bronchiolar epithelium (thick arrows), inflammatory cell infiltration (arrows) both in the inter alveolar septa and invading the bronchial wall. Hemorrhagic areas (red arrows) and macrophage with hemosiderin granules (circle) are present. C3 and C4 showing dilated congested blood vessels (BV) with margination of inflammatory cells mainly neutrophils (arrows). Inflammatory cell infiltration in the thickened inter alveolar septa (circles) (C3, H&E, × 400–C4 H&E, × 1000). C5 showing multiple macrophages with highly acidophilic cytoplasm and irregular outline (arrows) (H&E, × 1000). C6 showing inflammatory cell infiltration mainly neutrophils (arrows) in the highly thickened inter alveolar septum (double arrow) (H&E, × 1000). C7 shows Langhans giant cells (oval) in which macrophages fuse together, the nuclei forming a cute horse-shoe shape around the periphery of the cell. (H&E, × 1000)

Examination of lung sections of the immobilization stress group pretreated with NaHS revealed evident improvement of all alveolar changes. Nearly normal structure of alveolar ducts, alveolar sacs, and alveoli which are lined by simple squamous epithelium (pneumocytes type I) and pneumocytes type II was noticed. The inter alveolar septa was thin except in a few areas (Fig. 5, Table 3).

Fig. 5.

Fig. 5

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A representative photomicrograph of section in the lung of the NaHS + IS group. D1 showing more or less normal structure of the alveolar duct (AD), alveolar sac (AS), and alveoli (A). The inter alveolar septa appear relatively thin (arrows) H&E, × 100. D2 showing normal alveoli (A) which is lined by a squamous pneumocyte type I (arrows) and a pneumocyte type II bulging into the alveolar lumen (thick arrow). A blood capillary (C) can be seen in the interalveolar septa. H&E, × 400

Immunohistochemical results

NF-κB expression

The lung tissues of both control and NaHS groups showed negative immune expression of NF-κB. However, the IS group showed strong positive immune expression (brown color) in comparison with both control and NaHS groups. On the other hand, treatment of rats with NaHS before IS markedly suppressed the expression of NF-κB as seen in the IS group (Fig. 6, Table 3).

Fig. 6.

Fig. 6

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Representative photomicrographs of rat lung tissue of different groups immunohistochemically stained for NF-κB. (A) Control group and (B) NaHS group both showing negative immune expression (arrows). C1 and C2: the IS group showing strong positive immune expression in the epithelial cells lining the bronchioles (yellow arrow), cells lining the alveoli (blue arrows) and the cells scattered in the interalveolar septa (black arrows). D1 and D2: The NaHS + IS group showing marked decrease in expression except for scattered cells (arrows). (IHC by NF-κB antibody × 100 and × 400)

Caspase 3 expression

Concerning caspase 3 expression, there was faint immune expression in both control and NaHS groups. The chronic IS group showed strong positive immune expression as compared to both control and NaHS groups. On the other hand, in the NaHS + IS group, there was markedly less caspase 3 expression compared to the IS group except for scattered cells (Fig. 7, Table 3).

Fig. 7.

Fig. 7

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Representative Photomicrographs of rat lung tissue of different groups immunohistochemically stained for caspase 3. (A) Control group and (B) NaHS group both showing faint immune expression (arrow). C1 and C2: the IS group showing strong positive immune expression in the epithelial cells lining the alveoli (blue arrows) and the cells scattered in the inter alveolar septa (black arrows). D1 and D2: the NaHS + IS group showing marked decrease in expression except for scattered cells (arrow). (IHC by caspase 3 antibody × 100 and × 400)

Masson’s trichrome stain results

The obtained results demonstrated the presence of traces of collagen fibers in both the control and NaHS groups. The IS group showed significant increased deposition of collagen fibers compared to both the control and NaHS groups. Interestingly, treatment of rats with NaHS before IS showed more or less normal distribution of collagen around bronchiole and in the inter-alveolar septa in contrast with the IS group (Fig. 8, Table 3).

Fig. 8.

Fig. 8

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A representative photomicrograph of Masson’s trichrome stain in the lung tissues of different groups. The control and NaHS groups (A, B) showing traces of collagen fibers around blood vessel, bronchiole and in the inter-alveolar septa (arrows). The chronic immobilization stress group (C) showing increase deposition of collagen fibers around the wall of the bronchiole, surrounding blood vessels, around alveoli and in the inter-alveolar septa (arrows). The NaHS + IS group (D) showing more or less normal existence of collagen around bronchiole and in the inter-alveolar septa (arrows). (Masson trichrome × 100)

Discussion

The current study confirmed that H2S had a pathophysiological role in the stress-induced lung injury in the rat model. Our results show the association between the significant lung damage revealed by histological examination and the significant decrease in both lung H2S content and expression of H2S-synthesizing enzymes, CSE, CBS, and 3-MST in the IS group compared to the control and NaHS groups. Our findings are in agreement with previous studies that demonstrated decreased expression of H2S-synthesizing enzymes in other animal models of stress- and non-stress-induced injury (Magierowski et al. 2016; Han et al. 2020; Guan et al. 2020).

The obtained results showed that chronic exposure of rats to IS led to a significant increase in serum CORT as compared to both control and NaHS groups that is in line with (Saber et al. 2019; Palumbo et al. 2020). Stress activates the hypothalamic pituitary adrenocortical (HPA) axis and elevates level of glucocorticoids (cortisol in humans; corticosterone in rodents) that are part of the humoral adaptive response to the stressor (Elbassuoni and Nazmy 2018).

The imbalance between pro-oxidants and antioxidants causes various diseases (Samad et al. 2020). The present study indicates that chronic immobilization stress induced oxidative stress in rats’ lung tissues as evidenced by the significant increase in lung MDA and decrease in serum TAC in the IS group in comparison with control and NaHS rats. MDA is a marker for oxidative stress–induced lipid peroxidation (Marrocco et al. 2017). Lipid peroxidation changes membrane integrity and then leads to tissue damage (Samarghandian et al. 2016). Furthermore, glucocorticoids play an essential role in chronic stress. Induced oxidative injury enhances tissue MDA levels in stressed rats. In addition, the raised glucocorticoids levels during restraint stress may affect the animal antioxidant content (Gonchar et al. 2018) similar to what we see in our results.

Stress-induced secretion of glucocorticoids is able to modulate the immune cells and cytokine production such as interleukin‐6 (IL‐6) and TNF‐α (Yildirim and Yurekli 2010). This is in accordance with our study results where there was a significant increase in serum TNF‐α accompanied with significant decrease in serum IL-10 levels in IS group compared to both control and NaHS groups. A stress situation provokes the synthesis of inflammatory cytokines (Samad et al. 2020).

Nitric oxide (NO) is a free radical gas that is synthesized by two constitutive enzyme isoforms, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). The inducible nitric oxide synthase (iNOS) is scarcely present normally but can be expressed during immunological challenge and stress (Elbassuoni and Nazmy 2018). Additionally, iNOS activity is induced by a diversity of proinflammatory cytokines including TNF-α (Liu et al. 2016). The significant increase in lung tissues’ NO levels with IS exposure observed in our study can be explained by the significant increase of iNOS level that in turn increased NO production in rats exposed to chronic IS in comparison with control and NaHS groups and this result is in agreement with previous studies (Saber et al. 2019; Guo et al. 2017). The overexpression of NO with stress results in formation of reactive nitrogen species that is considered to be one of the leading causes of lung injury with stress exposure (Elbassuoni and Nazmy 2018) and as found in the present study.

Oxidative stress stimulates transcription factors, including NF-κB, and in turn, NF-κB regulates genes, such as iNOS (Guo et al. 2017). NF-κB is implicated in cellular responses to diverse stimuli like stress. NF-κB plays a role in the regulation of proliferation, cell death, immune, and inflammatory responses. In the present study, the expression of NF-κB was markedly elevated in the lung tissues of stressed rats that was associated with the significant decrease in the anti-inflammatory marker, IL-10, and increased levels of TNF-α, lung tissue NO and iNOS, and the lung expression of caspase 3 that is in agreement with a previous study (Guo et al. 2017). In fact, NF-κB activation is implicated in stress-induced lung injury and inflammation (Zhou et al. 2014).

HIF-1α is a critical oxygen sensor that correlates with inflammation (Guan et al. 2018). HIF-1α plays an important role in a variety of pathophysiologies. HIF-1α protein synthesis can be mediated by a variety of inflammation mediators (Guan et al. 2020). ROS generation caused the upregulation of NF-kB, which in turn led to HIF-1a mRNA induction (Chen et al. 2018) in agreement with our present study.

In the current study, exposure of rats to chronic IS resulted in significant decrease in the nuclear Nrf2 protein levels in the lung tissues with concomitant increase in the cytosol fraction compared to control and NaHS groups that is in line with other studies (Gonchar et al. 2018; Guo et al. 2017). Nrf2 plays a significant role in preventing the development of oxidative stress (Guo et al. 2017). The increased level of the NF-κB could be one of the reasons for the low level of nuclear Nrf2 (Djordjevic et al. 2015). Also similar to our results, under stress conditions, glucocorticoids can impair Nrf2-dependent antioxidant response (Gonchar et al. 2018).

In our study, there was significant increase in fibrosis score in chronic IS rats compared to both control and NaHS groups. Interestingly, long-term exposure to oxidative stress induces chronic inflammation and fibrosis which leads to disease progression (Yisireyili et al. 2019). ROS leads to fibrosis by increasing the expression of TGF-β1 that enhances the synthesis of collagen and suppresses its degradation. In our present study, there was a significant increase in lung immunohistochemical expression of caspase 3 in IS rats compared to control and NaHS groups. This increase in caspase 3 expression may be implicated in the development of pulmonary fibrosis via caspase‐3 activation in lung epithelial cell apoptosis (Predescu et al. 2017).

H2S has received attention as the third endogenous gaseous signaling transmitter in mammals, including humans. Some animal experiments have confirmed that the H2S donor, NaHS, has anti-oxidative and anti-inflammatory effects in other models of lung injury (Zhang et al. 2016). Although other H2S synthesis pathways have been described, the majority of endogenous H2S production occurs by desulfhydration of the amino acid cysteine by CSE and CBS or 3-MST enzymes (Osmond and Kanagy 2014).

Administration of NaHS before IS attenuated the effects of IS on lung injury as evidenced by the normal (control) levels of H2S and expression of H2S-synthesizing enzymes, CSE, CBS, and 3-MST in lung tissues. In addition, NaHS before IS protected the near normal histological appearance of the lung, and protected the levels of COR, oxidative stress markers (iNOS, NO and MDA), the proinflammatory cytokine TNF-α, and the anti-inflammatory cytokine IL-10, and TAC from the changes induced by IS. Similarly, administration of NaHS before IS protected the near-normal (control) protein levels of nuclear Nrf2 and HIF-1α and the expression levels of NF-κB and caspase 3.

In our study, there was association between the lesser amount of serum CORT and the greater amount of H2S-synthesizing enzymes and H2S content in lung tissue in NaHS + IS rats as compared to IS group. H2S has a role in neuroendocrine function through controlling the HPA axis (Mancuso et al. 2010). Also, exposure to H2S significantly improved the stress-induced increases in plasma corticosterone levels in a water immersion and restraint stress rat model. Furthermore, H2S inhibits activity of the HPA axis in vivo and in vitro (Lou et al. 2008).

Our results on the protective effects of NaHS on IS oxidative parameters, H2S content, and H2S-synthesizing enzyme expression, nuclear Nrf2 levels, serum TAC levels, and lung MDA and NO contents are similar to the results in other studies (Zhang et al. 2016; Aziz et al. 2020). Collectively, these results suggest that the anti-oxidant effect of NaHS in our model of stress-induced lung injury may be due to increasing the lung levels of nuclear Nrf2 (Aziz et al. 2020).

Our study demonstrated that administration of NaHS to rats before exposure to chronic IS resulted in significant attenuation of lung NFκB expression in addition to HIF-1α and iNOS levels and serum levels of TNF-α while suppressing the attenuation of serum IL-10 levels as compared to IS rats. This can be attributed to the levels of both lung tissue H2S contents and H2S-synthesizing enzyme expression. These results suggest that H2S had anti-inflammatory effect in the current study and is in accordance with previous studies (Magierowski et al. 2016; Lei et al. 2018). Similarly, H2S has anti-inflammatory effect that was secondary to suppression of NF-κB activation. Excess ROS activate NF-κB, resulting in enhancement of its expression and activity (Li et al. 2013). In accordance with our study, the anti-inflammatory effect of H2S might be secondary to its anti-oxidative effect. In addition, H2S inhalation inhibited the activation of the NF-κB signaling pathway and reduced iNOS expression and NO synthesis (Han et al. 2015).

Our finding that the pretreatment with NaHS before IS maintained both lung H2S content and expression of H2S-synthesizing enzymes at normal levels and suppressed pulmonary fibrosis compared to IS rats. This suggests an anti-fibrotic effect of H2S donor in the present study. This can be attributed to the significant decrease in oxidative stress and inflammation as indicated in our study findings that is in line with a previous study (Zhang et al. 2015).

The caspase family plays a substantial role in mediating the process of apoptosis (Li et al. 2016). According to our results, there was significantly less immunohistochemical expression of caspase 3 in lungs of NaHS + IS rats as compared to IS rats. This can be due to the demonstrated significant greater content of lung tissue H2S content and expression of H2S-synthesizing enzymes that confirm the anti-apoptotic action of H2S in this study that is in line with another study (Mendes et al. 2019). Similarly, NaHS conferred protection against lung injury in rats and inhibited lung apoptosis through attenuating the inflammatory response and suppressing oxidative stress (Xu et al. 2013).

Conclusion

In conclusion, the present study confirmed that administration of H2S donor, NaHS, had a protective effect against a rat model of chronic stress–induced lung injury. The detrimental effect of IS on lung H2S-synthesizing enzymes, CSE, CBS, and 3-MST expressions and on lung H2S production was reversed by the pretreatment of NaHS. This was confirmed by the marked attenuation of lung tissue damage through histological examination that was accompanied by less oxidative stress (iNOS, NO, and MDA), less serum CORT, TNF-α and lung HIF-1 α levels, and expressions of NF-kB and the apoptotic marker, caspase-3, compared to the IS rats. The NaHS pretreatment also maintained the higher levels of serum TAC and the anti-inflammatory marker, IL-10, and lung levels of nuclear Nrf2 compared to the IS rats. This protective effect of NaHS can be attributed to its antioxidant, anti-inflammatory, anti-apoptotic, and anti-fibrotic effects in lungs.

Acknowledgements

Not applicable

Author contribution

Dr. Fatma: conceptualization; methodology; formal analysis and investigation; writing—original draft; writing—review and editing and supervision.

Dr. Hanaa: investigation, writing—review and editing.

Dr. Doaa: investigation, writing—review and editing.

Declarations

Ethics approval

All procedures about animal use here were proved by the Institutional Committee of Animal Care in Faculty of Medicine, Minia University, Egypt (approval No. 676–9/2020).

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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