Protective effects of ulinastatin on rats with acute lung injury induced by lipopolysaccharide

ABSTRACT

We aimed to evaluate the protective effects of ulinastatin (UTI) on rats with acute lung injury induced by lipopolysaccharide (LPS) via the Toll like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. Forty-eight male Wistar rats were randomly divided into model, control, dexamethasone (DXM) and UTI groups. The body weight loss ratio and wet-to-dry weight ratio (W/D) of lung tissue were calculated at 10 h. The permeability of pulmonary vascular endothelium was detected by Evans blue method. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β levels in bronchial lavage fluid were detected by enzyme-linked immunosorbent assay. Total cells and neutrophils were counted by microscopy. TLR4, MyD88 and NF-κB expressions were detected by Western blotting. Compared with model group, DXM and UTI groups had significantly higher body weights and lower W/D values (P < 0.05). In DXM and UTI groups, the lung tissue structure was close to normal, inflammatory cell infiltration was alleviated, and hematoxylin-eosin staining scores were significantly lower than that of model group (P < 0.05). Compared with model group, the concentrations of Evans blue, IL-1β, IL-6 and TNF-α levels, and protein expressions of TLR4, MyD88 and NF-κB in DXM and UTI groups decreased significantly (P < 0.05). UTI inhibits LPS-induced activation of the TLR4/MyD88/NF-κB signaling pathway, thereby alleviating inflammatory response and protecting against lung injury.

KEYWORDS:

• Acute lung injury
• lipopolysaccharide
• Myd88
• NF-κB
• TLr4
• ulinastatin

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Publisher’s Note

Introduction

With the aggravation of air pollution and changes in lifestyles in recent years, the incidence rate of acute respiratory distress syndrome (ARDS) is increasing annually. ARDS occurs mostly upon diseases such as severe trauma, shock and infection [1,2]. Due to injury of pulmonary capillary and alveolar epithelial cells, patients may undergo diffuse pulmonary interstitial and alveolar edema, with the clinical manifestations of hypoxemia and respiratory distress. It is well-established that inflammatory response is the root cause for ARDS [3]. The onset and progression of acute lung injury (ALI) are also caused by uncontrollable inflammatory response, leading to mutual activation and interaction among a large number of inflammatory factors and effector cells [4]. Regardless of many studies on ALI/ARDS treatment, the mortality rate remains high, and the molecular mechanism is still unclear. Lung injury has been associated with the activation of Toll like receptor 4 (TLR4) [5], which then releases large amounts of inflammatory mediators via the downstream pathway myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB). Ulinastatin (UTI) is a broad-spectrum hydrolase inhibitor, and its anti-inflammatory effect has attracted widespread attention [6]. It can inhibit proteolytic activity and prevent endotoxin from stimulating macrophages to release inflammatory factors, thereby relieving the damage to tissues [7]. However, whether UTI can mitigate lung injury by suppressing lipopolysaccharide (LPS)-induced inflammatory cascade via the TLR4/MyD88/NF-κB signaling pathway remains elusive.

Therefore, we herein intended to explore whether UTI exerted protective effects on an ALI rat model established by LPS injection via the TLR4/MyD88/NF-κB signaling pathway. This study provided valuable experimental evidence for future clinical use.

Materials and methods

Animals and main reagents

Forty-eight SPF Wistar male rats aged 12–15 weeks and weighing 200 g-220 g were provided by the Animal Experimental Center of our hospital. All the experimental procedures involving animal study were performed in accordance with the guidelines for Animal Ethical Committee (Xuzhou Central Hospital, China).

LPS was purchased from Sigma (USA). UTI was bought from Merck & Co., Inc. (USA). Positive control drug dexamethasone (DXM) was obtained from the National Institute for Food and Drug Control (China). Evans blue was provided by Tianjin Baihaoxin Biotechnology Co., Ltd. (China). BCA protein concentration determination kit and DAB chemiluminescence kit were purchased from Beijing Huaxia Biotechnology Co., Ltd. (China). TLR4, MyD88 and NF-κB antibodies were bought from Santa Cruz Biotechnology (USA). Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Zhongda Hospital, China).

Modeling and grouping

All rats were fed for 7 days to adapt to the environment, numbered, and then randomly divided into 4 groups (n = 12), i.e. control group, model group, DXM intervention group and UTI intervention group. For the model group, 0.5 mL of LPS (5 mg/kg) was injected into the tail vein [8], and the control group was given an equal volume of normal saline. The DXM intervention group and UTI intervention group were intraperitoneally injected with 1 mL of DXM solution (2 mg/kg) and 1 mL of UTI solution (5 mg/kg) respectively 24 h and 10 min before LPS injection for pretreatment. The control and model groups were given equal volumes of normal saline. The rats were intraperitoneally injected with 10% chloral hydrate 10 h after modeling. Under anesthesia, they were intubated after tracheotomy, and sacrificed by opening the abdomen and bled through the abdominal aorta. Three rats from each group were used to measure the wet-to-dry weight ratio (W/D) of lung tissue and pulmonary vascular endothelial permeability, three were utilized for the collection of bronchial lavage fluid (BALF) sample and ELISA, three were used for the pathological observation of lung tissue, and the remaining three were employed for Western blotting.

Body weight measurement

Body weights were recorded before and 10 h after modeling to calculate the weight loss ratio: (Weight_{10 h after modeling} – weight_{before modeling})/weight_{before modeling}

Measurement of W/D of lung tissue

After the rats were sacrificed, the lower lobe of the left lung was taken after thoracotomy, and the blood on the surface of lung tissues was gently sucked dry. The tissues were weighed (wet weight W), dried in a 60°C oven for 48 h, and weighed (dry weight D) to obtain W/D [9].

Detection of pulmonary vascular endothelial permeability

The Evans blue method was used for detection [10]. Briefly, dried lung tissue was added formamide according to the ratio of 1 mL/100 mg wet lung weight, homogenized, and centrifuged at 4,000 rpm for 0.5 h after reaction in a 60°C water bath for 24 h. The resulting supernatant was added to a 96-well plate and measured at 620 nm. The Evans blue concentration was calculated according to a standard curve and converted to μg/g dry lung tissue.

Collection of BALF sample

After anesthesia, the rats were sacrificed by right ventricular blood collection. Tracheal separation was performed under aseptic conditions. The trachea cannula was inserted into the left main bronchus, and then ligated by silk thread. The left lung was washed with PBS three times, 5 mL each time. BALF was collected with the recovery of above 80%, and thereafter centrifuged at 2,000 rpm for 10 min to obtain the supernatant which was stored in a − 80°C refrigerator prior to use. The precipitate was resuspended, evenly spread on a glass slide, and stained with Wright Giemsa solution. The slide was observed under a microscope to count total cells and neutrophils [11].

Pathological observation of lung tissue

Tissue samples were fixed in 10% formaldehyde for 24 h, sectioned and observed after hematoxylin-eosin (HE) staining for pathological changes under a light microscope. Evaluation was conducted in terms of infiltration of alveolar cells, alveolar hemorrhage, alveolar septal edema and alveolar fibrin deposition. Four sections were taken for each rat and six different optical fields were selected for each section. The scores were finally averaged. Pathological findings were scored from 0 to 4 points: 0, without injury; 1 point, mild injury (with an area of about 25% of optical field); 2 points, moderate injury (with an area of about 50% of optical field); 3 points, severe injury (with an area of about 75% of optical field); 4 points, injury covers almost the entire optical field. The sections were observed and paragraphed by experienced pathologists in a blinded manner under a light microscope [12].

Detection of TLR4, MyD88 and NF-κB protein expressions by Western blotting

Lung tissue was homogenized by grinding, lysed on ice for 30 min and centrifuged to collect the supernatant. Protein samples were resolved by 5% SDS-PAGE, and the products were electronically transferred onto a PVDF membrane. Subsequently, the membrane was blocked overnight at 4°C, incubated with primary antibodies against TLR4, MyD88, NF-κB and β-actin (1:1500 diluted) for 1 h, washed, and incubated with HRP-labeled goat anti-rabbit IgG antibody (1:10,000 diluted). After being treated with ECL reagent for 30 min, the membrane was exposed to X-ray film, developed and air-dried at room temperature. Relative protein expression levels were represented by using β-actin expression as the internal reference [13].

Detection of TNF-α, IL-6 and IL-1β levels in BALF by ELISA

The TNF-α, IL-6 and IL-1β levels in BALF were measured strictly according to the instructions of ELISA kits using a multifunctional microplate reader [14].

Statistical analysis

All data were analyzed by SPSS16.0 software. The categorical data conforming to normal distribution were expressed as mean ± standard deviation. Multigroup comparisons were performed by one-way analysis of variance, and pairwise comparisons were conducted with the independent samples t test. P < 0.05 was considered statistically significant.

Results

Effects of UTI on body weight and lung tissue W/D of LPS-induced rats

Lung tissue W/D reflects the abnormality of vascular permeability and the severity of pulmonary edema [15]. Compared with the control group, the body weights of model, DXM and UTI groups decreased significantly (P < 0.05). Compared with the model group, the body weights of DXM and UTI groups increased significantly (P < 0.05). Compared with the control group, W/D of the model group significantly increased after LPS stimulation (P < 0.05). Compared with the model group, the W/D values of DXM and UTI groups decreased significantly (P < 0.05) (Figure 1).

Figure 1. Effects of UTI on body weight and lung tissue W/D of LPS-induced rats. *Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Effects of UTI on numbers of neutrophils and total cells in BALF of LPS-induced rats

The numbers of neutrophils and total cells in BALF indirectly reflect the infiltration of neutrophils in lung tissue and the severity of inflammation [16]. Compared with the control group, the numbers of neutrophils and total cells in BALF of the model group increased significantly (P < 0.05). Compared with the model group, the numbers of DXM and UTI groups significantly decreased (P < 0.05) (Figure 2). Therefore, UTI significantly inhibited LPS-induced inflammatory cell infiltration.

Figure 2. Effects of UTI on numbers of neutrophils and total cells in BALF of LPS-induced rats. *Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Effects of UTI on pathological injury of lung tissue in LPS-induced rats

The lung tissue of the control group was intact, without congestion, and the alveolar space was clear. No edema or inflammatory cell infiltration was found in the septum. Compared with the control group, the alveolar space of the model group was significantly smaller. The septum was thickened, the alveolar wall showed congestion and edema, a large amount of inflammatory cell infiltration appeared, and the HE staining score was significantly higher (P < 0.05). In DXM and UTI groups, the lung tissue structure was close to normal, inflammatory cell infiltration was alleviated, and the HE staining scores were significantly lower than those of the model group (P < 0.05) (Figure 3 and Table 1).

Figure 3. Effects of UTI on pathological injury of lung tissue in LPS-induced rats. Magnification: 100 × . A: Control group, normal alveoli (asterisk) and alveolar septa (black arrow) with few neutrophils are shown; B: model group, with thickened septum (asterisk), alveolar bleeding (black arrow) as well as considerable cell infiltration and alveolar fibrin deposition (yellow arrow); C: DXM group, with significantly alleviated cell infiltration and alveolar fibrin deposition; D: UTI group, with significantly alleviated cell infiltration and alveolar fibrin deposition.

Table 1. HE staining scores.

Group	Alveolar cell infiltration	Alveolar bleeding	Alveolar septal edema	Alveolar fibrin deposition
Control	0.56 ± 0.09	0.43 ± 0.04	0.51 ± 0.06	0.59 ± 0.07
Model	3.04 ± 0.16*	2.46 ± 0.11*	2.74 ± 0.12*	2.85 ± 0.13*
DXM	2.18 ± 0.15#	1.58 ± 0.13#	1.69 ± 0.11#	1.88 ± 0.16#
UTI	2.25 ± 0.18#	1.65 ± 0.12#	1.71 ± 0.11#	1.92 ± 0.14#

*Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Effects of UTI on pulmonary vascular endothelial permeability of LPS-induced rats

After being stimulated by inflammatory factors, multiple signaling pathways in endothelial cells are activated, eventually leading to cell contraction, morphological changes and increase in vascular permeability [17]. Evans blue concentration was used to reflect the pulmonary vascular endothelial permeability. Compared with the control group, the concentration of Evans blue in the model group was significantly elevated (P < 0.05). Compared with the model group, the concentrations of DXM and UTI groups decreased significantly (P < 0.05) (Figure 4).

Figure 4. Effects of UTI on pulmonary vascular endothelial permeability of LPS-induced rats. A: Control group; B: model group; C: DXM group; D: UTI group. *Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Effects of UTI on levels of IL-1β, IL-6 and TNF-α in BALF of LPS-induced rats

The lung injury model established by administration of bacterial endotoxin LPS is in line with the clinical course of ALI. LPS leads to the massive production and release of pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β. Meanwhile, it induces oxidation/antioxidation imbalance, lipid peroxidation, and production of considerable oxygen free radicals causing damage to tissues and cells [18]. Compared with the control group, the levels of IL-1β, IL-6 and TNF-α in the model group were significantly raised (P < 0.05). Compared with the model group, the levels of DXM and UTI groups significantly dropped (P < 0.05) (Figure 5).

Figure 5. Effects of UTI on levels of IL-1β, IL-6 and TNF-α in BALF of LPS-induced rats. *Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Effects of UTI on protein expressions of TLR4, MyD88 and NF-κB in lung tissue of LPS-induced rats

HMGB1, TLR4 and NF-κB play key roles in the onset and progression of sepsis. TLR-4 is a downstream factor of the HMGB1/TLR-4/NF-κB signaling pathway [19]. When HMGB1 binds TLR-4, it activates the NF-κB pathway, thereby promoting the release of inflammatory factors [20]. Compared with the control group, the protein expressions of TLR4, MyD88 and NF-κB in the model group significantly increased (P < 0.05). Compared with the model group, the expressions of DXM and UTI groups significantly reduced (P < 0.05) (Figure 6). Thus, the protective effects of UTI on rat lung injury were associated with proteins of the TLR4/MyD88/NF-κB signaling pathway.

Figure 6. Effects of UTI on protein expressions of TLR4, MyD88 and NF-κB in lung tissue of LPS-induced rats. *Compared with control group, P < 0.05; #compared with model group, P < 0.05.

Discussion

ALI, as a common clinical respiratory critical disease, is pulmonary parenchyma injury mainly caused by the interaction of inflammatory mediators and effector cells [21]. At present, effective therapies are still lacking in clinical practice, so it is imperative to seek prevention and treatment measures. UTI can inhibit fibrin decomposition and stabilize lysosomal membranes [22], which is often used to treat shock and acute pancreatitis. However, the influence of UTI on ALI has rarely been reported. Therefore, this study used LPS to construct an ALI model to explore the mechanism for protecting lung injury.

The pathological characteristics of ALI mainly include lung epithelial cell damage, osmotic pulmonary edema, as well as migration and infiltration of a large number of inflammatory cells [23]. Alveolar epithelial or endothelial cells are damaged mainly because the regulatory network composed of inflammatory mediators is uncontrollable. Neutrophils are recruited when stimulated by the external environment. After being activated, they can release a large amount of superoxide oxidase or free radicals which in turn damage lung epithelial and endothelial cells, and indirectly causing pulmonary edema [24]. UTI can inhibit the activity of proteolytic enzymes, thereby attenuating further activation of macrophages by inflammatory stimuli produced by tissue cell destruction, and the release of inflammatory factors by endotoxin-stimulated macrophages [25]. In this study, LPS significantly reduced body weight and lung tissue W/D, which was relieved by UTI. HE staining results showed that UTI protected lung tissue structure, as well as alleviated lung hemorrhage, edema and inflammatory infiltration. The concentration of Evans blue was measured to reflect the permeability of rat pulmonary vascular endothelium. The results showed that the concentration of Evans blue in the model group was significantly higher than that in the control group. Compared with the model group, the concentration of Evans blue decreased significantly in DXM and UTI groups, indicating that UTI not only protected against LPS-induced ALI, but also decreased the pulmonary vascular permeability.

Upon ALI, a large number of inflammatory cells are infiltrated to produce cytokines and inflammatory mediators, and inflammatory signals are amplified step by step, which result in microcirculatory disorders, enhanced permeability of alveolar epithelial cells and injury of pulmonary capillary endothelium, eventually causing alveolar hemorrhage and pulmonary edema [26]. As key factors involved in the inflammatory cascade, IL-1β, IL-6 and TNF-α play crucial roles in inducing cell infiltration and accelerating the progression of respiratory system diseases. To investigate the role of UTI in pulmonary gas exchange, this study examined the changes of these factors in BALF, and showed that UTI significantly reduced IL-1β, IL-6 and TNF-α levels. When TLR4 is released outside cells, it not only promotes inflammatory reaction, but also interacts with the inflammatory mediators to form an immune-stimulating complex [27]. Besides, TLR4 is a key receptor for endotoxin transmembrane signaling, which can activate MyD88-dependent pathway, promote TκB phosphorylation, and then activate NF-κB [28]. This pathway is associated with cellular oxidation, apoptosis and inflammatory response [29–31]. Herein, the protein levels of TLR4, MyD88 and NF-κB significantly increased after modeling, while those of the UTI group decreased, suggesting that the protective effects of UTI on LPS-induced ALI in rats were associated with the TLR4-mediated MyD88/NF-κB pathway.

Conclusion

In summary, UTI can inhibit LPS-induced activation of the TLR4/MyD88/NF-κB signaling pathway, thereby alleviating inflammatory response and protecting against lung injury. Nevertheless, further studies are in need to clarify whether UTI can affect LPS-induced lung injury by regulating other pathways.

Highlights

• Lipopolysaccharide induces activation of the TLR4/MyD88/NF-κB signaling pathway.

• Ulinastatin inhibits the pathway activation.

• Ulinastatin relieves inflammatory response and protects against lung injury.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.