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Inhibition of RHO Kinase by Fasudil Attenuates Ischemic Lung Injury After Cardiac Arrest in Rats

Wei, Jian; Wang, Peng†,‡; Li, Yi§; Dou, Qingli; Lin, Jiali†,‡; Tao, Wuyuan; Lin, Jinle†,‡; Fu, Xuan; Huang, Zitong†,‡; Zhang, Wenwu

Author Information
doi: 10.1097/SHK.0000000000001097

Abstract

INTRODUCTION

Effective cardiopulmonary resuscitation (CPR) can help patients with cardiac arrest (CA) attain a return of spontaneous circulation (ROSC); however, CA mortality is higher than 55% of those afflicted (1, 2). Multiple organ dysfunction syndrome following resuscitation caused by ischemia-reperfusion injury results in high mortality (2, 3). Acute lung injury and the ensuing acute respiratory distress syndrome are the key factors responsible for this process. Routinely, respiratory function recovery is an important index for assessing the prognosis of patients following CPR (4). The pathogenesis mechanisms underlying CA-induced lung injury involve in the production of inflammatory mediators, which may promote the breakdown of the lung endothelial barrier (2, 3, 5, 6). Emerging evidence has suggested that post-CA care is associated with the survival rate of patients (7). It is well recognized that the heart and brain should be immediately protected following ROSC (2, 3). In addition, lung tissue is susceptible to injury following CPR due to ischemia-reperfusion or precordial compression (2, 8). However, there are currently no effective therapies for lung injury induced by CA. Therefore, it is necessary to find novel therapeutic agents for CA-induced lung injury.

Rho kinase (Rho-associated coiled-coil-containing protein kinase, ROCK), a serine/threonine protein kinase, is a major downstream effector of the small GTPase RhoA. The two isoforms of ROCK, ROCK1 and ROCK2, are encoded by two different genes (9). The active form of RhoA binds to ROCK, which remodels the cell cytoskeleton by increasing the phosphorylation of myosin light chain (MLC) (10). The Rho/ROCK signaling pathway not only regulates cell contraction, adhesion, and motility but also affects other cellular processes, including transcriptional regulation, proliferation, differentiation, and apoptosis (9, 11). Vascular endothelial (VE)-cadherin is specifically expressed in endothelial cells, and functions as a key mediator in maintaining endothelial junction stability and vascular integrity (12). Previous studies have reported that VE-cadherin expression in endothelial cells was dependent on Rho/ROCK signaling (13). In addition, intercellular adhesive molecule (ICAM)-1 is the primary endothelial cell adhesion molecule that regulates leukocyte adhesion and transmigration (14). A previous study revealed that ICAM-1 expression is regulated through a ROCK-dependent mechanism, which mediates various cellular functions related to cardiovascular disease (15).

ROCK inhibition has been shown to be effective against ischemia-reperfusion injury (16, 17). Moreover, inhibition of ROCK in vivo prevents neutrophil recruitment and edema formation in endotoxin-induced acute lung injury (5, 18). Fasudil is the only Rho kinase inhibitor used clinically for the treatment of cerebral vasospasms following subarachnoid hemorrhage (19). Fasudil has since been tested in various clinical trials and showed remarkable safety profiles including angina pectoris, hypertension, pulmonary hypertension, stroke, and heart failure (20). As the Rho/ROCK signaling pathway plays an important role in lung injury (10, 18, 21, 22), we hypothesized that fasudil, a widely used ROCK inhibitor, may inhibit the development of lung injury induced by CA and CPR.

In this study, we established a rat CA model through asphyxia and investigated if treatment with fasudil could improve the outcome of CA-induced lung injury in rats by blocking the Rho/ROCK signaling pathway. Lung injury was detected by performing a histopathological examination. Moreover, lung edema, myeloperoxidase (MPO) activity, proinflammatory cytokines, and oxidative stress associated with lung injury were detected. To elucidate the underlying protective mechanism of fasudil, ROCK1/2, VE-cadherin, and ICAM-1 protein expressions in the lung tissue were also evaluated.

MATERIALS AND METHODS

Animals and drugs

All animals were cared for humanely in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (8th edition; Washington DC, National Academic Press, 2011). The protocol was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

Male Sprague-Dawley rats (body weight, 310–340 g) were purchased from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Animals were maintained on laboratory chow and housed in a specific pathogen-free room at a constant temperature (20–22°C) with 12 equal hours of light and dark exposure.

Fasudil was purchased from AdooQ Bioscience (Irvine, Calif) and was dissolved in 0.9% saline as a stock solution. Pentobarbital sodium was purchased from Sigma Aldrich (St. Louis, Mo).

Experimental procedures

The rats were randomized into three groups: Sham, Fasudil, and Control groups. The animals in the Fasudil group were intraperitoneally injected with 10 mg/kg of the drug 1 h prior to asphyxial cardiac arrest (ACA) and then were given daily for 2 days. The dose of fasudil was based on previous dose-dependent effects data (Fig. S1, https://links.lww.com/SHK/A692) and previous reports (5, 18). The rats in the Control group received an equal volume of placebo (0.9% saline) intraperitoneally 1 h prior to ACA. The animals in the Sham group underwent the same treatment as the Control group, except that they were not subjected to ACA induction.

ACA was established by endotracheal tube clamping in both the Control and Fasudil groups. CA was defined as the mean arterial pressure (MAP) ≤ 20 mm Hg, which occurred approximately four and a half minutes after endotracheal tube clamping. After 6 min of untreated CA, CPR was started by reconnecting the ventilator, performing precordial compression, and injecting epinephrine as previously described (23). If rats had ventricular fibrillation, defibrillation was attempted with up to three 2-J counter shocks after 4 min of precordial compression (PC). PC was stopped when there was no ROSC after 5 min. ROSC was defined as the return of supraventricular rhythm with a MAP ≥ 60 mm Hg and duration of at least 5 min.

Arterial blood gas analysis

A 23-gauge polyethylene 50 catheter (Abbocath-T, North Chicago, Ill) was advanced through the left femoral artery for the measurement of blood pH, partial pressure of oxygen (PaO2), and partial pressure of carbon dioxide (PaCO2) using a blood gas analyzer (Abbott, Chicago, Ill) at baseline, and 3, 6, 12, 24, and 48 h following CA.

Histopathological examination

The lung tissues were harvested and immediately fixed in 4% formaldehyde in 0.1 M phosphate buffer. The tissues were then embedded in paraffin and sectioned. Paraffin sections were subjected to hematoxylin and eosin (HE) staining and analyzed by an experienced pathologist in a blinded fashion. The severity of lung injury was scored (0–4, with 0 = minimal injury, and 4 = severe injury) by evaluating the degree of congestion in the alveoli, degree of hemorrhage, infiltrated, and/or aggregated neutrophils, and thickness of the alveolar wall or hyaline membrane formation.

Wet/dry (W/D) lung weight ratio measurements

The severity of pulmonary edema was assessed by calculating the ratio of W/D lung weight. The left lower lobes of the lungs were harvested and were weighed (wet), then dried at 70°C for 48 h and weighed again (dry).

Measurement of malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity

The fresh lung tissue was harvested and homogenized immediately. The homogenate was centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was used for measurements. MDA level in lung tissue was determined using a lipid peroxidation assay kit (Beyotime, Nantong, China) according to the instructions of the manufacturer. SOD activity in lung tissue was evaluated by a water-soluble tetrazolium-8 (WST-8) assay kit (Beyotime, Nantong, China) according to the instructions of the manufacturer.

Measurement of lung cytokine levels and MPO activity

The fresh lung tissue was harvested and homogenized immediately. The homogenate was centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant was used for measurements. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in tissue were measured by an ELISA kit (Ray Biotech, Norcross, Ga) according to the instructions of the manufacturer. MPO activity in lung tissue was measured by a MPO colorimetric activity assay kit (Biovision, Milpitas, Calif) instructions of the manufacturer.

Western blotting analysis

The lung tissues were homogenized and cleared by centrifugation at 4°C for 30 min. Total protein concentrations in supernatants were measured using bicinchoninic acid protein assay kits (Pierce Biotechnology, Rockford, Ill). Approximately 40 μg protein sample each was run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. For immunoblotting, ROCK1, ROCK2, ICAM-1 (diluted 1:1,000; Abcam, Cambridge, UK), VE-cadherin (diluted 1:1,000; LifeSpan Biosciences, Seattle, Wash) and β-actin (diluted 1:2,000; Cell Signaling Technologies, Danvers, Mass) antibodies were used. Protein bands were detected using an enhanced chemiluminescence system (Cell Signaling Technologies, Danvers, Mass). The densities of protein blots were quantified by using Image J (National Institutes of Health, Bethesda, Md) and normalized to β-actin levels.

Statistical analysis

All the values were shown as mean ± standard deviation (SD) and the statistical analyses were performed using SPSS version 19.0 for Windows (SPSS, Chicago, Ill). Comparison of the same parameters among three groups was done using one-way analysis of variance, and the difference between pairs of means was tested post hoc with Tukey test. A difference of P < 0.05 was considered statistically significant.

RESULTS

Baseline characteristics of the rats

A total of 130 rats were prepared for the study. Among them, 100 underwent CPR; however, 15 rats failed to achieve ROSC, and the remaining 30 rats received sham operations. Of the 85 rats that achieved sustained ROSC, 25 failed to survive in the expected time after ROSC and were excluded from the group. Finally, 90 rats were used in this study to assess the potential effects of fasudil on CA-induced lung injury. There were no significant differences in baseline physiologies, hemodynamic and blood analytical measurements among the three groups (Table 1).

T1
Table 1:
Baseline characteristics of all groups

Effect of fasudil on blood gas

The pH, PaO2, and PaCO2 levels of arterial blood in the Sham group showed no significant differences over time (P > 0.05, Fig. 1). In comparison with the Sham group, the pH and PaO2 of arterial blood in the Control group was significantly decreased from 3 to 24 h after ROSC; however, it was restored to normal levels at 48 h after ROSC (P < 0.05, Fig. 1, A and B). Moreover, while the PaCO2 of arterial blood in the Control group was significantly increased from 3 to 24 h after ROSC, it decreased to normal levels at 48 h after ROSC (Fig. 1C). However, treatment with fasudil significantly increased the pH and PaO2 of arterial blood within 24 and 6 h after ROSC respectively (P < 0.05, Fig. 1, A and B), and it also significantly decreased the PaCO2 of arterial blood at 3 h after ROSC (P < 0.05, Fig. 1C).

F1
Fig. 1:
Effect of fasudil on arterial blood gas after return of spontaneous circulation (ROSC).

Effect of fasudil on lung histological injury

HE staining was used to assess the potential effects of fasudil on lung injury caused by CA. As shown in Figure 2, the results showed that pulmonary histological damage was observed from 3 to 48 h after ROSC, as evidenced by an increase in inflammatory cell infiltrate, interstitial edema, vascular congestion, as well as lung hemorrhaging. However, fasudil treatment significantly reversed CA-induced histological alternations in rat lung tissue from 3 to 48 h after ROSC.

F2
Fig. 2:
Fasudil significantly decreased the lung histopathological injury after resuscitation.

Fasudil reduced lung tissue edema after CA

The increase in lung tissue W/D weight ratio is a key indicator for evaluating lung edema. Therefore, we calculated the lung tissue W/D weight ratio from 3 to 48 h after ROSC. We found that CA led to a significant increase in the W/D weight ratio at all time points; however, fasudil treatment significantly decreased the W/D weight ratio (P < 0.05, Fig. 3).

F3
Fig. 3:
Fasudil significantly reduced the pulmonary wet/dry (W/D) weight ratio at 3, 6, 12, 24, and 48 h after resuscitation.

Fasudil decreased inflammatory response in lung tissues after CA

The levels of inflammatory cytokines were measured in rat lung tissues. Our data showed no significant differences in TNF-α or IL-6 levels in the Sham group over time (Fig. 4). However, the levels of TNF-α (Fig. 4A) and IL-6 (Fig. 4B) were markedly elevated at 3, 6, 12, 24, and 48 h after ROSC, and these increases were significantly attenuated by fasudil (P < 0.05). To evaluate total neutrophil infiltration in lungs, we performed the MPO activity assay. The results showed that fasudil treatment significantly decreased MPO activity in lung tissue at 3, 6, 12, 24, and 48 h after ROSC (P < 0.05, Fig. 4C).

F4
Fig. 4:
Fasudil significantly decreased the inflammatory response in lung tissue after cardiac arrest.

Fasudil reduced oxidative stress of lung tissues after CA

The level of MDA was measured in rat lung tissues. Compared with the Sham group, the MDA level was significantly increased at 3, 6, 12, 24, and 48 h after ROSC (P < 0.05, Fig. 5A). However, the MDA level was decreased by fasudil at 3, 6, 12, 24, and 48 h after ROSC (P < 0.05, Fig. 5A). Furthermore, the effect of fasudil on SOD activity in lung tissue was detected. The results showed that the level of SOD activity was markedly decreased at 3, 6, 12, 24, and 48 h following ROSC; however, fasudil treatment significantly increased SOD activity at 3, 6, 12, 24, and 48 h after ROSC (P < 0.05, Fig. 5B).

F5
Fig. 5:
Fasudil significantly reduced the oxidative stress in lung tissue after cardiac arrest.

Effect of fasudil on protein expression in the ROCK signaling pathway

We found that the protein expression of ROCK1 and ROCK2 was significantly upregulated in lung tissues at 3, 6, 12, 24, and 48 h after ROSC. However, fasudil treatment markedly attenuated the upregulation of both ROCK1 and ROCK2 after ROSC (P < 0.05, Fig. 6, A–C). Moreover, ICAM-1 expression was significantly increased, while VE-cadherin expression was significantly decreased at 3, 6, 12, 24, and 48 h after ROSC. However, fasudil treatment reduced the expression of ICAM-1, while restoring the expression of VE-cadherin at 3, 6, 12, 24, and 48 h after ROSC (P < 0.05, Fig. 6, A, D, and E).

F6
Fig. 6:
Fasudil significantly reduced the ROCK-1/2 and ICAM-1 protein expression, as well as increased VE-cadherin protein expression in lung tissue after cardiac arrest.

DISCUSSION

In this study, we demonstrated that pretreatment of fasudil (10 mg/kg) could attenuate CA-induced lung injury in rats. The protective effects of fasudil included a decrease in lung edema, oxidative stress, and inflammatory response after CA. The molecular mechanism underlying the protective effects of fasudil involves the inhibition of the Rho/ROCK signaling pathway.

It is well established that lung ischemia-reperfusion injury is related to endothelial dysfunction, increased vascular permeability, as well as the activation of neutrophils and production of cytokines (24–26). Furthermore, formation of reactive oxygen species is also involved in lung ischemia-reperfusion injury (25, 26). As the CA-induced lung injury is mainly caused by lung ischemia-reperfusion injury, it is possible that lung endothelial dysfunction, inflammation, and oxidative stress occur after CA. Therefore, the animal model of CA-induced lung injury is of immense value to assess therapeutic modalities for lung ischemia-reperfusion injury. Furthermore, this animal model could help in studying the underlying mechanism of lung injury pathogenesis.

Edema is a crucial feature of the lung injury after CA (27, 28). Our study showed that fasudil ameliorated lung edema after CA, as evidenced by the decreased lung W/D weight ratio. Arterial blood gas analysis demonstrated a decrease in PaO2 and pH, as well as an increase in PaCO2 after CA. Although the PaO2 and pH gradually increased, while PaCO2 gradually decreased after CA, these values remained abnormal even 24 h after CA compared with the values observed in the Sham group. These results might be associated with an increase in water content of lung tissue, thereby affecting gas exchange. The infiltration of neutrophils and ensuing transmigration are important symptoms of pulmonary injury clearly related to the endothelial vascular permeability and endothelial barrier integrity (29, 30). MPO is a peroxidase enzyme primarily abundant in neutrophil granulocytes. As evidenced by the reduced MPO activity in our study, fasudil could potently inhibit the infiltration of neutrophils in lung tissue. Moreover, histopathological analysis confirmed the protective effect of fasudil on CA-induced lung injury.

Inflammation occurs in the early phase of inflammatory diseases and persistent inflammation could potentiate lung dysfunction (25). IL-6 and TNF-α are key mediators of the systemic inflammatory response. IL-6 is related to acute inflammatory stimulation, whereas TNF-α is responsible for the transduction of pro-inflammatory signaling cascades (31). Previous studies have shown that ROCK is involved in NF-κB activation, and the effect of ROCK inhibition on NF-κB signaling is a marked decrease in the expression of proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8 (16, 32). We found that fasudil significantly decreased IL-6 and TNF-α expression in lung tissue, indicating its anti-inflammatory effects in our CA-induced lung injury model. Consistent with other studies, our data suggested that fasudil reduced TNF-α and IL-6 expression induced by lipopolysaccharides, which suggests its potential therapeutic utility in treating acute lung injury (18).

Previous studies have demonstrated that oxidative stress plays an important role in lung ischemia-reperfusion injury (25, 26). A high level of oxidative stress can be produced either by generating free radicals or by inhibiting antioxidant systems, leading to damage of cells and tissues. Therefore, reduction of oxidative stress could benefit in attenuating lung ischemia-reperfusion injury after CA. MDA is the end-product of lipid peroxidation by free radicals. However, SOD is the primary defense system for tissues and organs against free radicals. In the present study, our data showed that fasudil could significantly decrease lung MDA levels while restoring lung SOD activity after CA, indicating that the therapeutic effect of fasudil on lung injury may be partially attributed to its anti-oxidative ability.

The Rho/ROCK signaling pathway regulates several aspects of cellular function (9, 20). In the present study, we found that the protein expression of two ROCK isoforms, ROCK1 and ROCK2, was increased following CA in rats. Previous studies have proved the link between lung injury and the activation of the Rho/ROCK signaling pathway (10, 18, 21). ROCK activation can phosphorylate and inactivate MLC phosphatase, leading to increase vessel contraction and vascular hyperpermeability (33). By inhibiting the Rho/ROCK pathway, fasudil may increase MLC phosphatase activity, which facilitates the dephosphorylation of MLC, thereby resulting in attenuated lung ischemic-reperfusion injury following CA. In the present study, we also confirmed that fasudil restored the expression of VE-cadherin and decreased the expression of ICAM-1 in lung tissue after CA. Both ICAM-1 and VE-cadherin are closely related to the maintenance and regulation of the endothelial barrier integrity through cell–cell adhesion (12, 14). The endothelial barrier regulates the extravasation of fluids, plasma proteins, and leukocytes between blood and the interstitium. A significant decrease in VE-cadherin and increase in ICAM-1 at endothelial cell junctions results in an increase in vessel permeability, leading to edema and extravasation of leukocytes. Supporting our results, it has been reported that inhibition of ROCK attenuated endothelial barrier dysfunction through regulation of VE-cadherin and ICAM-1 (34, 35).

Nonetheless, there were some limitations in the current study. First, we only administered one dose of fasudil to study its effects on lung injury after CA and CPR. Additional studies should be performed to find the most suitable dose of intervention for lung injury after ROSC. In addition, fasudil was administered before CA in this study, which may be different from clinical practice with respect to drug delivery time. The results will be further confirmed when fasudil is administered after ROSC. Finally, the upstream components of the Rho/ROCK signaling pathway were not examined in this study.

CONCLUSIONS

Our study demonstrated the critical role of the Rho/ROCK signaling pathway in CA-induced lung injury in rats and found that fasudil could markedly improve the functional outcome of the lung following CA. The protective mechanism of fasudil may involve the inhibition of Rho/ROCK signaling pathway. Our study suggested that ROCK could be explored as a promising therapeutic target for improving lung function following CA and CPR.

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Keywords:

Cardiac arrest; Fasudil; lung injury; Rho/ROCK

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