Abstract
Acute lung injury (ALI) is a major outcome of exposure to high levels of hydrogen sulfide (H2S). Dexamethasone (DXM) has been used to treat ALI. However, the mechanisms involved in H2S-induced ALI and the protective mechanisms of DXM in treating ALI are still nebulous. To explore the mechanisms involved, we evaluated the role of claudin-5 in the protective effect of DXM against H2S-induced ALI. Sprague-Dawley rats were exposed to H2S to establish the ALI model. In parallel with the animal model, a cell model was also established by incubating human umbilical vein endothelial cells (HUVECs) with NaHS. Lung hematoxylin–eosin staining, electron microscope assay, and wet/dry ratio were used to identify whether the ALI was successfully induced by H2S, and changes in claudin-5 expression were detected in both rats and HUVECs. Our results revealed that claudin-5 was markedly decreased after H2S exposure and that DXM significantly attenuated the H2S-induced downregulation of claudin-5 in both rats and HUVECs. In the animal experiment, p-Akt and p-FoxO1 presented a similar tendency as claudin-5, but their levels decreased 6 h prior to the levels of claudin-5. In a further investigation, the DXM-induced protective effect on ALI and rescue effect on downregulation of claudin-5 were both blocked by LY294002. The current study demonstrated that claudin-5 was involved in the development of H2S-induced ALI and that DXM exerted protective effects through increasing claudin-5 expression by activating the phosphatidylinositol 3-kinase pathway. Therefore, claudin-5 might represent a novel pharmacological target for treating ALI induced by H2S and other hazardous gases.
Introduction
Hydrogen sulfide (H2S) is a fatal and colorless suffocating gas that possesses a characteristic rotten-egg odor. 1 Exposure to H2S contributes to concentration-dependent toxicity in the human respiratory system. Levels of 500–1000 ppm H2S pose a risk of “sudden death” and levels of 250–500 ppm H2S result in acute lung injury (ALI), which is the main fatal outcome after exposure to H2S. 2 –4 However, the current mechanisms of H2S-evoked ALI are still insufficiently understood.
Endothelial cells (ECs) serve as a semipermeable barrier between vascular contents, and the pulmonary airspaces play a critical role in regulating tissue fluid homeostasis and the inflammatory response. 5 The adhesive structures between adjacent cells, including tight junctions (TJs), adherens junctions, and desmosomes, enable the establishment of cell polarity, differentiation, and survival and are critical to the maintenance of tissue integrity. EC TJs are mainly composed of three types of proteins, including claudins, occludins, and junctional adhesion molecules, which interact in a coordinated manner to form EC barriers. 6 Among them, the major functional transmembrane proteins that are known as claudins directly regulate the paracellular permeability of TJs. 7 To date, 27 human claudin proteins have been identified and display a tissue-restricted expression pattern. 8 In the claudin family, claudin-5 is the only member that is expressed in ECs. 9 It is a critical component to maintaining the blood–brain barrier 10 and can be upregulated by VE-cadherin via the phosphatidylinositol 3-kinase (PI3K) signaling pathway. 11 In a murine model of acrolein-induced ALI, reduced lung claudin-5 expression was considered to be associated with increased susceptibility to injury. 12 However, the role of claudin-5 in H2S-induced ALI is still unknown.
Glucocorticoids have been demonstrated to exert barrier-tightening effects on ECs both in vitro 13,14 and in vivo. 15 Dexamethasone (DXM), one of the glucocorticoids, has been used or proposed as a therapeutic approach to toxic gas–induced ALI. 16 We hypothesize that the therapeutic effects of DXM on ALI might be partially due to its significant increasing of claudin-5 in cases of ALI. Therefore, in the present study, our aim was to study whether claudin-5 is involved in H2S-induced ALI and to explore whether DXM exerts its protective effects by regulating claudin-5. Moreover, the effects of the signaling pathway PI3K, which might participate in claudin-5 regulation, were also discussed.
Materials and methods
NaHS (#161527) and DXM (#4902) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Gas cylinders containing 1% (10,000 ppm) H2S in nitrogen were purchased from Shangyuan GASES (Nanjing, China). A digital H2S gas analyzer was purchased from Lasting Star Safety Equipment Company (Nanjing, China). Claudin-5 antibodies were obtained from Sigma-Aldrich. All other antibodies were from Cell Signaling Technology (Danvers, Massachusetts, USA) unless otherwise stated.
Animals and cells
Male Sprague-Dawley rats weighing (200 ± 20) g were approved by the Animal Center of Jiangsu Province, Nanjing, China (SCXK(Su) 2002-0031) and had free access to standard rat chow and tap water. All animals were housed in independent ventilation cages at an ambient temperature of 18.5–21.5°C and humidity of 40–70%. Human umbilical vein endothelial cells (HUVECs), purchased from ATCC (Manassas, Virginia, USA) with the passage number CRL-1730, were kindly provided by Dr. Xiaohang (Nanjing Medical University). The seeding density was 1 × 106 cells/ml. HUVECs were put in cell culture dishes with a size of 60 mm in diameter and 15 mm in height. HUVECs were cultured in endothelial cell medium (ECM, ScienCell Research Laboratories) supplemented with 5% fetal bovine serum (FBS), 1% EC growth supplement, and 1% penicillin/streptomycin solution (P/S) at 37°C in 5% CO2 and 95% air.
Exposure of rats to H2S
Rat model
Rats were acutely exposed to H2S gas (300 ppm) for 3 h, as described previously. 17 Briefly, the rats were placed inside a cylindrical glass chamber (Specialty Glass, Nanjing, China). Two mass flow controllers sealed with Kalrez and a digital H2S gas analyzer were used to control the flow rates of compressed air and H2S to reach the target H2S concentrations in the exposure chamber. After exposure to H2S (300 ppm) for 3 h, the rats were returned to room air and sacrificed 0, 6, 12, and 24 h later.
Cell model
NaHS, a H2S donor, was dissolved in PBS at 100 mM for the stocking concentration as in our usual way. 18 The HUVECs were kept in ECM (without FBS) prior to NaHS treatment. The diluted NaHS (500 μM) was used to incubate with HUVECs for 30 min, 1, 3, 6, 12, and 24 h in a thermostatic incubation box. Since H2S can escape as a gas from the solutions, the cell culture dishes were sealed for 30 min. NaHS was freshly prepared before each experiment.
Experimental design
The first aim of the present work was to evaluate the expression of claudin-5 in lung tissues and HUVECs following H2S exposure. In the in vitro study, HUVECs were incubated with NaHS for 30 min, 1, 3, 6, 12, and 24 h, but in the in vivo study, 30 rats were randomly divided into a control (unexposed) group and four time-point groups (n = 6 per group) using a computer-generated randomization schedule. Control rats were kept in room air, whereas the other 24 rats were exposed to 300 ppm H2S for 3 h, returned to the room air and then anesthetized by an intraperitoneal administration of pentobarbital sodium at 0, 6, 12, and 24 h after H2S exposure. Subsequently, the effects of H2S exposure on claudin-5 and/or p-AKT, t-AKT, p-FOXO1, t-FOXO1 expression were detected both in vivo and in vitro by Western blot.
The second objective was to confirm whether DXM could ameliorate H2S-induced ALI by increasing claudin-5 expression. Based on our pilot study, 18 rats were randomly divided into three groups (n = 6 per group), and DXM (2 mg/kg/day) was intraperitoneally injected for three consecutive days prior to H2S exposure. 18 On the third day, 12 rats were exposed to 300 ppm H2S for 3 h and were sacrificed 6 or 12 h later (n = 6 per group), whereas the other rats (n = 6) were sacrificed directly after returning to room air to obtain tissue specimens for further use.
We were also interested in determining whether the PI3K signaling pathway participated in claudin-5 regulation. To verify our hypothesis, HUVECs were kept in ECM without FBS and pretreated with DXM (100 nM) for 24 h 18 and/or LY294002 (10 µM) for 1 h. Then, NaHS (500 mM) was added to incubate HUVECs for 6 h before claudin-5 expression was detected.
Determination of lung wet/dry (W/D) weight ratio
The W/D weight ratio was measured as described previously. 18 The upper lobe of the right lung was removed and weighed. Then, specimens were dried in an oven (50°C) for 3 days and weighed again to determine the dry weight. The W/D weight ratio was calculated by dividing the wet weight by the dry weight.
Histological analysis
Lung histology was performed using light microscopy and transmission electron microscopy. The right lower lobe from each rat was harvested and fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, cut into 4-mm-thickness slices and stained with hematoxylin–eosin. Pathologic changes were evaluated by two independent observers who had no knowledge of the H2S exposure. As characteristics of ALI, the following four parameters were assessed: inflammatory cells, congestion and edema, hemorrhage, and septal thickening. The samples were then graded on a 4-point scale (0 = absent; 1 = mild; 2 = moderate; 3 = severe), and an overall histological score was calculated by totaling the scores, as previously described. 18 The same fixation procedure was used for electron microscopy, except paraformaldehyde was replaced with 2.5% glutaraldehyde. Small blocks of lung tissue were postfixed in 1% osmium tetroxide in PBS, dehydrated in a graded series of ethanol washes, and embedded. Ultrathin sections stained with uranyl acetate and lead citrate were examined using a transmission electron microscope (JEM 1010; JEOL, Tokyo, Japan).
Western blot analysis
Sample proteins were extracted using the RIPA (Sigma, USA) buffer with Protease Inhibitor Cocktail (Sigma, USA), centrifuged at 12,000 g and incubated for 30 min at 4°C. The protein concentration was measured with the BCA (bicinchoninic acid, BCA) method (Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. An equal amount of protein (20 µg) was loaded onto tris-glycine sodium dodecylsulphate polyacrylamide gel (10%) for electrophoresis and subsequently blotted onto a PVDF (polyvinylidene fluoride, PVDF) (Millipore, Billerica, Massachusetts, USA) membrane. The membranes were blocked with 5% nonfat milk in TBST (Tris-Buffered Saline and Tween 20, TBST) for 2 h and then incubated with anti-claudin-5 (dilution 1:1000), anti-p-AKT/t-AKT (1:1000), anti-p-FOXO1/t-FOXO1 (1:1000), or mouse anti-β-actin (1:5000) for at least 8 h. After incubation with (horseradish peroxidase, HRP) HRP-conjugated anti-rabbit or anti-goat secondary antibody (1:50,000; Jackson Immuno Research Laboratories, Baltimore, Maryland, USA) for 1 h at room temperature, labeled proteins were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, USA). Band density was normalized to β-actin in each sample.
Statistical analysis
Data are expressed as the mean ± standard error of the mean. Statistical analyses were performed with one-way analysis of variance or an independent student’s t-test using a software package (Graph Pad Prism5) (Version 5.03 (Graph Pad Software, Inc., La Jolla, CA)). p-Values < 0.05 were considered statistically significant.
Results
W/D weight ratio in lung tissues after H2S exposure
As the lung tissue W/D weight ratio is an important index for assessing the ALI, 19 we sought to examine the effects of H2S on the ratio. As shown in Figure 1, the W/D weight ratio of rat lung tissues was significantly elevated at 6, 12, and 24 h after 300 ppm H2S exposure compared with the control group. Moreover, the W/D weight ratio reached the maximum value at 12 h after H2S exposure, implying that the most severe lung injury might occur at this time point.
Effects of H2S on the W/D ratio of rat lungs. The W/D ratio was detected 0, 6, 12, and 24 h after rats’ exposure to 300 ppm H2S. The results are from six rats (each group) and are presented as the means (±SE). Statistically significant differences between the treatment groups and the control group were determined by one-way ANOVA or an independent student’s t-test. *p < 0.05 indicates a significant difference when the values were compared with the control. SE: standard error; ANOVA: analysis of variance.
Histopathological abnormalities in lung tissues induced by H2S
To further confirm H2S-induced lung injury, we investigated the histopathological changes in lung tissue after H2S exposure. Under light microscopy, within 6, 12, and 24 h after H2S exposure (Figure 2(c) to (e)), the lung specimens displayed evident histopathological abnormalities, including infiltration of inflammatory cells, capillary congestion extravasation, edema, hemorrhage, alveolar wall thickening, fracture, and alveolar fusion. Furthermore, the histological score based on these abnormalities accompanied the ALI scores (Figure 2(f)). Using an electron microscopy assay, some ultrastructure changes, such as mitochondrial swelling and shrinking, empty lamellar bodies and nucleus collapse, were observed in type II alveolar epithelial cells (Figure 3(b) to (d)).
Histopathological changes in lungs after H2S exposure. (a) Control group; (b) 0 h after H2S exposure, low-grade inflammatory cell infiltration (white arrow); (c) 6 h after H2S exposure, inflammatory cell infiltration (white arrow); (d) 12 h after H2S exposure, inflammatory cell infiltration (white arrow) and interlobular septal thickening (black arrow); (e) 24 h after H2S exposure, alveolar wall fracture, alveolar fusion, alveolar hemorrhage, and pulmonary capillary edema; (f) the ALI scores. The ALI scores were significantly elevated compared with the control group at 6, 12 and 24 h after H2S exposure. Magnification ×200. ALI, acute lung injury. p-Values < 0.05 were considered statistically significant.
Ultrastructure abnormalities induced by H2S in type II alveolar epithelial cells. (a) Control group; (b) 6 h after H2S exposure, mitochondria were slightly swelling; (c) 12 h after H2S exposure, mitochondria clearly swelled (black arrow); (d) 24 h after H2S exposure, with mitochondrial shrinking (white arrow), the nuclear structure was not clear and disaggregated, and the multilamellar body decreased with significant cavitation (black arrowheads). Magnification ×15,000.
Effects of H2S on claudin-5 protein expression in rats and HUVECs
To assess whether claudin-5 was involved in H2S-induced ALI, we detected the expression changes of claudin-5 protein induced by H2S. In the in vivo study, compared with the control group, claudin-5 protein expression was significantly decreased 12 h (0.42 vs. 1.12) and 24 h post-H2S exposure. However, no obvious changes in claudin-5 were observed 0 and 6 h post-H2S exposure (Figure 4(b)). In the in vitro study, the protein expression of claudin-5 in HUVECs was markedly alleviated when incubating HUVECs with NaHS for 6 h. Compared with the control group, a decrement of approximately 30% was observed (0.70 vs. 1.02); subsequently, the claudin-5 expression gradually returned to the normal levels (Figure 6(a) and (b)). These data indicated that H2S significantly downregulated claudin-5 levels after exposure to H2S.
Effects of H2S on claudin-5, p-AKT, and p-FOXO1 in lung tissues from H2S-induced rats. The protein levels of claudin-5, p-AKT and t-AKT, p-FOXO1 and t-FOXO1 in lung tissue from six rats (each group) were detected by Western blot 0, 6, 12, and 24 h after exposure to H2S. (a) Representative immunoblots of claudin-5, p-AKT and t-AKT, p-FOXO1 and t-FOXO1 protein in lung tissues induced by H2S. (b) Semiquantitation of blot densities for the specific claudin-5 bands (23 kD). (c) Semiquantitation of blot densities for the specific p-AKT and t-AKT bands. (d) Semiquantitation of blot densities for the specific p-FOXO1 and t-FOXO1 bands. Means (±SE) are representative of five independent experiments. Statistically significant differences between the treatment groups and the control group were determined by one-way ANOVA or student’s t-test. *p < 0.05 indicates a significant difference when the values were compared with those of the control. SE: standard error; ANOVA: analysis of variance.
Effects of DXM on the expression of claudin-5 protein
To explore whether claudin-5 expression is regulated by DXM, we investigated the effects of DXM on claudin-5 protein expression at 12 h in the rat model and at 6 h in HUVECs. When DXM was applied, compared with the H2S-exposed group, the H2S-induced downregulation of claudin-5 protein expression was substantially rescued in both the in vivo (0.78 vs. 0.37; Figure 5(a) and (b)) and the in vitro studies (1.04 vs. 0.70; Figure 6(c) and (d)).
Effects of DXM on claudin-5, p-AKT, and p-FOXO1 in lung tissues from H2S-induced rats. The protein levels of claudin-5, p-AKT and t-AKT, p-FOXO1 and t-FOXO1 in lung tissue from six rats (each group) were detected by Western blot 6 or 12 h after exposure to H2S. (a) Representative immunoblots of claudin-5 (post-6 h), p-AKT and t-AKT, p-FOXO1 and t-FOXO1 protein (post-12 h) in lung tissues induced by H2S. (b) Semiquantitation of blot densities for the specific claudin-5 bands (23 kD). (c) Semiquantitation of blot densities for the specific p-AKT and t-AKT bands. (d) Semiquantitation of blot densities for the specific p-FOXO1 and t-FOXO1 bands. Means (±SE) are representative of four independent experiments. Statistically significant differences between the treatment groups and the control group were determined by one-way ANOVA or the student’s t-test. *p < 0.05 indicates a significant difference when the values were compared with those of the control. # p < 0.05 indicates a significant difference when the values were compared with those of the H2S-treated group. DXM: dexamethasone; SE: standard error; ANOVA: analysis of variance.
Effects of the PI3K pathway in DXM-induced alterations of claudin-5 expression in HUVECs. The HUVECs were co-incubated with DXM (100 nM) for 24 h, and/or LY294002 (10 μM) for 1 h, prior to NaHS (500 mM) treatment for 6 h, and then claudin-5 expression was detected by Western blot. (a) Representative immunoblots of claudin-5 expression in HUVECs. HUVECs were incubated with NaHS for 0.5, 1, 3, 6, 12, and 24 h. (b) Semiquantitation of blot densities for the specific claudin-5 bands (23 kD). (c) Representative immunoblots of claudin-5 expression with DXM and/or LY294002 treated. (d) Semiquantitation of blot densities for the specific claudin-5 bands. Means (±SE) are representative of seven independent experiments (a) and four independent experiments (c). *p < 0.05 indicates a significant difference when the values were compared with those of the control. # p < 0.05 indicates a significant difference when the values were compared with those of the post-6 h treated group. & p < 0.05 indicates a significant difference between the post-6 h + DXM and the post-6 h + DXM + LY treated group. DXM, dexamethasone; HUVEC, human umbilical vein endothelial cell; PI3K, phosphatidylinositol3-kinase.
Role of PI3K/AKT/FOXO1 pathway in H2S-induced downregulation of claudin-5
It was reported that the regulation of claudin-5 expression was mainly mediated by the extracellular regulated protein PI3K/AKT/FOXO1 pathway. Therefore, we next examined the role of the PI3K/AKT/FOXO1 pathway in the H2S-induced downregulation of claudin-5. 20,21 We first assessed the effects of H2S on AKT and FOXO1 phosphorylation. As shown in Figure 4(a), (c), and (d), after 300 ppm H2S stimulation for 3 h, p-AKT displayed a transient increase and then a significant decrease 6 h after H2S exposure, after which it was gradually restored to normal levels. A similar tendency was also observed in p-FOXO1 expression. There was no significant change in the total AKT and FOXO1 protein expression. Overall, there was a significant reduction in the phosphorylated/total AKT and FOXO1 ratio post-6 h in H2S-treated rats: The change is not synchronized with the change in claudin-5 but is 6 h ahead of schedule. We further examined the effects of DXM on AKT and FOXO1 phosphorylation. As shown in Figure 5(a), (c), and (d), DXM treatment significantly prevented the H2S-induced reduction of p-AKT and p-FOXO1 proteins, but no significant changes were detected in the total AKT and FOXO1 expression. To explore whether the PI3K/AKT/FOXO1 pathway was implicated in the DXM-induced rescue effects on claudin-5 downregulation, LY294002, a PI3K inhibitor, was applied to block the PI3K/AKT/FOXO1 pathway. In the H2S-exposured rats with LY294002 treatment, the DXM-induced upregulation of claudin-5 was markedly attenuated by LY294002 (1.04 vs. 0.72; Figure 6(c) to (d)). Such data revealed the essential role of the PI3K/AKT/FOXO1 pathway in regulating claudin-5 expression
Discussion
It is known that severe pulmonary edema and respiratory failure are major adverse events due to ALI after H2S inhalation. In the present study, pathological changes in the lung tissues exposed to H2S displayed aggravation of diffuse alveolar damage, such as alveolar edema and hemorrhage. As reported by other studies, we also observed ultrastructure abnormalities such as mitochondrial swelling and shrinking, empty lamellar bodies, and nucleus collapse. 17,18 In addition, a markedly increased W/D weight ratio indicated that serious pulmonary edema occurred after H2S exposure. Combined, these results provided definite evidence for H2S-induced ALI.
Recently, several new mechanisms involved in H2S-induced ALI, including mitochondrial damage, 22 epithelial sodium channel (α-ENaC) downregulation, 17 and matrix metalloproteinase-2,9 (MMP-2,9) up-regulation, 18 as well as reactive oxygen species and inflammatory reaction were reported. 23,24 However, the mechanisms are still not fully illustrated.
This is the first study, to our knowledge, to demonstrate the involvement of claudin-5 in H2S-induced ALI. Claudin-5, a 23-kDa protein, is expressed only in the endothelium, where it is the dominant claudin and is required for the formation of confluent EC monolayers, 11 and it plays a critical role in the permeability of the EC barrier. Newborn claudin-5 KO mice die within 10 h after birth, possibly due to the altered permeability of the blood–brain barrier. 10 Additionally, inducing claudin-5 expression in leaky rat lung ECs can enhance paracellular barrier function against large molecules. 25 The downregulation of claudin-5 is believed to be responsible for the destruction of the blood–air barrier by increasing permeability during the pathogenesis of ALI. 26,27 In this work, both in vivo and in vitro studies showed that the data indicated a significant downregulation of claudin-5 expression after exposure to H2S compared with control subjects, which suggested that H2S-induced injury of the pulmonary endothelium and claudin-5 downregulation might contribute to the pathogenesis of ALI in H2S-induced individuals.
Glucocorticoids have been demonstrated to have barrier-tightening effects on cerebral ECs both in vitro 28,29 and in vivo. 15 DXM, which is a powerful and widely used glucocorticoid, was reported to exert protective effects in various pulmonary conditions, including H2S inhalation poisoning. Therefore, in the present work, we speculated that DXM partially exerts its protective effects by rescuing the H2S-induced downregulation of claudin-5. As we speculated, our results revealed that DXM significantly attenuated the H2S-mediated downregulation of claudin-5 expression and may have had a direct effect on the pulmonary vascular endothelium reducing the permeability of blood–air barrier, which manifested in phosgene- and lipopolysaccharide-induced ALI. 30
Several pathways participated in the regulation of claudin-5 expression, such as JAK-STAT6-FOXO1, 21 SOX18, 31 p38 MAPK/NF-κ B, 22 and PI3K-AKT-FOXO1. 12 By integrating various cell signals, FOXO1 makes a critical contribution to EC function at the transcriptional level. 32 Claudin-5 is expressed in the absence of the nuclear accumulation of FOXO1 transcription factor. This is controlled by the PI3K-mediated phosphorylation of AKT, which in turn mediates the downstream phosphorylation of FOXO1. p-FOXO1 can be retained in the cytoplasm or degraded rapidly after ubiquitination. It has been reported that DXM may prevent muscle protein loss via maintenance of the AKT/FOXO pathway. 33 Therefore, it is speculated that H2S might restrain the PI3K-AKT-FOXO1 pathway and downregulate claudin-5 expression, whereas DXM plays the opposite role. As we hypothesized, H2S significantly reduced the level of p-AKT and p-FOXO1 at the 6 h point, but DXM obviously retarded this process. These findings suggested that DXM administration protected against ALI, at least in part, by preventing the H2S-induced inhibition of AKT-FOXO1 signaling. This conclusion was further validated by the protective effects of DXM, which could be partially blocked by LY294002, the PI3K inhibitor, further proving that PI3K was also involved in the pathogenesis of ALI. 12
Intriguingly, the reduction of p-AKT/p-FOXO1 occurred 6 h prior to the change in claudin-5; therefore, claudin-5 might be the downstream location in the p-AKT/p-FOXO1 signaling pathway. Furthermore, during the early 300 ppm H2S exposure, the expression of p-AKT and p-FOXO1 did not decline, but there was a transient increase associated with the individual moderate stress reaction. With mounting stress, FOXO1 proteins accumulate in the cell, translocate to the nucleus, and partner to regulate target genes that promote stress resistance, cell cycle arrest, or apoptosis, 34 which is consistent with more severe outcomes, including ALI.
In summary, we suggest for the first time that H2S reduces claudin-5 expression, which might aggravate the development of ALI. DXM increases claudin-5 expression through the upregulation of p-AKT and p-FOXO1 by activating the PI3K pathway. Therefore, claudin-5 might represent a novel pharmacological target for treating ALI induced by H2S and other hazardous gases, and further research will focus on the endothelial barrier function of claudin-5.
Footnotes
Authors’ contribution
Ping Geng and Tianlong Ma have contributed equally to this study.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Plan Project of Jiangsu Province (BL2014088) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).