Establishment of Acute Lung Injury Model Induced by Intraperitoneal Injection of Lipopolysaccharide in Rats

Zhang Hua-yun, Feng Xiu-jing, Yao Yu-jie, Ma Biao, Wang Chu-qiao, Zhao Yue, Bai Jing-chun, and Fan Honggang

Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University,Harbin 150030, China

Abstract: The model of acute lung injury (ALI) was established by intraperitoneal administration, but there was no time-point observation and comparison.ALI model was established by intraperitoneal injection of lipopolysaccharide (LPS) at the concentration of 10 mg · kg-1 (10 mg LPS dissolved in 1 mL normal saline to prepare 1 mL · kg-1 solution) in rats.The control group (CG) was intraperitoneally injected with saline of the same dose.In the LPS group, lung tissues were collected at 4, 6, 8, 12 and 24 h after administration.Then, the morphology changes, the ratio of wet-to-dry weight (W/D), the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) proteins, the levels of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH) were measured.To verify the success of the model, the degrees of lung injury via Western blot, RT-PCR, ELISA and other techniques were detected at different time points, and the severe time of the ALI model established was deterimined by intraperitoneal administration, which provided a stable model basis for the study of the pathogenesis of ALI in the future.The results showed that the lung injury occurred in LPS group.W/D and lung pathological changes at 12 and 24 h of LPS group were significantly different from those in the CG.Compared with the CG, the expression of IL-1β and TNF-α proteins and the content of MDA in lung tissues of LPS group increased and most significant difference was found at 12 and 24 h (p＜0.01).Compared with the CG, the activities of SOD and GSH in LPS 12 h group decreased significantly (p＜0.01).In conclusion, inflammation and oxidative damage were the main causes of the ALI in rats.Lung injury was most obvious 12 h after intraperitoneal injection of 10 mg · kg-1 LPS.

Key words: rat, lipopolysaccharide, intraperitoneal injection, acute lung injury model

Introduction

Acute lung injury (ALI) is an acute and severe inflammatory process in the lungs, caused by a variety of direct or indirect injuries, including sepsis,shock, pancreatitis, blood transfusion and inhalation of pathogenic bacteria (Kimet al., 2017).It is often accompanied by acute respiratory failure caused by pulmonary edema and has always maintained a high mortality rate (Lvet al., 2017).Due to its unique blood supply and tissue structure, the lung is one of the most vulnerable organs in an uncontrolled systemic inflammatory environment.Its pathological features include excessive activation of inflammatory response,excessive oxidative stress caused by abnormal release of reactive oxygen species, pulmonary edema caused by increased permeability of alveolar epithelial cellsand microvascular endothelial cells (Gonget al., 2018).

Lipopolysaccharide (LPS) is a major component of the Gram-negative bacterial wall and is a major inflammatory priming factor that activates the host immune response against pathogens, resulting in plenty of pro-inflammatory cytokines (such as TNF-αand IL-1β) produce.Proinflammatory cytokines appear in the early stage of the inflammation, which indicate the severity of the ALI in a certain sense(Giebelenet al., 2007).By binding to lipoid A and LPS binding proteins, LPS forms complexes and binds LPS monomer to CD14 receptors on the surface of the target cell, leading to activation of pathways,such as inflammation and oxidation (Jeyaseelanet al., 2005).LPS released into tissues or blood at the time of bacterial death or cell wall disintegration is an important causative factor causing sepsis and multiple organ failures, which not only causes dysfunction of the endothelial cell barrier, but also causes damage to the lung tissue structures and series of pathophysiological changes, such as necrosis.

At present, the establishment of the ALI model varies widely, and there is no detailed introduction of establishing the ALI model by intraperitoneal LPS.This study discussed the selection of the time of taking materials in the preparation of rat ALI model from the aspects of lung histomorphology,inflammatory reaction and oxidative stress and determined a more serious time point of lung injuries,which provided a stable model basis for the study of the pathogenesis of the ALI in the future.

Materials and Methods

Animals and drugs

Thirty-six healthy male SD rats, weighing (200±20) g, were provided by Harbin Medical University Experimental Animal Center.The rats were housed with a constant temperature of (22±1)℃ and a relative humidity of 55%±5%, a 12 h day/night cycle.The rats weread libitumaccessed to water and food.

LPS (Escherichia coli055: B5, Sigma, St.Louis,MO, USA), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH)commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China); IL-1βand TNF-α(Bioss,Beijing, China); GAPDH (Wanlei Biotechnology,Shenyang, China).

Experimental groups

Thirty-six rats were randomly divided into two groups:the control group (CG,n=6) and the LPS group (LPS,n=30).Rats in LPS group at each time point (4, 6,8, 12 and 24 h) were intraperitoneally injected with 10 mg · kg-1LPS (Fujiwaraet al., 2015) (10 mg LPS dissolved in 1 mL saline solution) and the CG was intraperitoneally injected with the same volume of normal saline.Then, the rats were euthanized and the lung tissues were removed quickly and stored at -80℃or fixed with 10% buffered formalin.

Determination of histopathological lung

The lung right upper lobe was fixed with 10% formalin fixative for 24 h, rinsed with running water for 24 h,and embedded in paraffin after being transparent and dipped in wax.The paraffin section was prepared,according to the routine procedure, and the slice thickness was 4-5 μm.Sections were stained with standard histological techniques of hematoxylin and eosin, and histopathological changes were observed under a microscope.The lung injury score criteria were based on the following items (Wanget al., 2014):edema, hemorrhage, neutrophil infiltration and alveolar wall thickness, which were scaled from 0, no injury;1, slight injury (25%); 2, moderate injury (50%);3, severe injury; 4, very injury (almost 100%).

Determination of lung wet-to-dry (W/D) ratio

The right middle lobe lung tissue was placed in a dry glass dish and weighed to obtain a wet weight (W).Then, the lung dry weight (D) was obtained by drying at 80℃ for 24 h to the constant weight in a vacuum drying box.The degree of edema of the lung tissue was evaluated by calculating W/D ratio.

Determination of expression of inflammatory indexes in lung tissues

Western blot was used to detect the expression of IL-1βand TNF-αprotein in lung tissues.The 100 mg of lung tissue was homogenized by adding 1 mL of RIPA lysate in an ice bath and centrifuged at 12 000×g for 15 min at 4℃.The supernatant was taken and the protein concentration was determined by the BCA method and added 5×loading buffer and boil for 10 min.The protein was separated by SDS-PAGE,electrophoresed onto a PVDF membrane, which was blocked in 5% skim milk for 2 h at room temperature.Then, they were incubated overnight at 4℃ using IL-1β(1 : 1 000), TNF-α(1 : 1 000) and GAPDH(1 : 2 000) antibodies, respectively.After washing with TBST for five times of 5 min, the membranes were incubated with goat anti-rabbit horseradish peroxidaseconjugated antibody for 2 h at room temperature, then washed with TBST.

Determination of oxidative indexes in lung tissues

The lower lobe of the left lung was made into 10%homogenate.The content of MDA, the activity of SOD and the activity of GSH in lung tissues of rats were determined according to the instructions of the kit.

Statistical analysis

The statistical analyses of all the data were performed using GraphPad Prism (version 7.0, GraphPad Software Inc., San Diego, CA, USA).The significant values were obtained using at-test.All the data were expressed as mean±SD, displayed a normal distribution and passed the test for the equal variance.

Results

Effects of LPS on pulmonary histopathological changes

As shown in Fig.1, there were obvious pathological changes in the lungs of each LPS group compared with CG.Significant lung damage had been seen at 4 h of LPS, including inflammatory cell infiltration and alveolar septal thickening.After 12 h of LPS,obvious congestion and edema of the lungs were observed.The lung tissue of the rats in the CG was normal and no obvious damage was observed.The W/D of LPS at 8, 12 and 24 h was significantly different from that of CG (p＜0.05), and the 12 h W/D reached the maximum, and the degree of pulmonary edema was the most severe at 8, 12 and 24 h.

Fig.1 Pathological changes of lung tissue induced by LPS

Expression of inflammatory indexes in lung tissues

The results of Western blot showed that the expression of IL-1βand TNF-αprotein in LPS group was significantly different from that in the CG (p＜0.01).The expression of IL-1βand TNF-αproteins varied with time and the trend was the same.The protein results are shown in Fig.2.

Fig.2 Relative expression of IL-1β and TNF-α protein in lung tissues

Results of determination of oxidation indexes in lung tissues

As shown in Fig.3, MDA content of each LPS group was significantly higher than that of the CG and gradually increased with time, and was most significant at 12 and 24 h.After 6 h, SOD activity and GSH activity of each LPS group were significantly lower than those of the CG.SOD activity of the 4 h LPS group was significantly lower than that of the CG.SOD activity of the 24 h LPS group was significantly different from that of the 12 h LPS group.

Fig.3 Detection of MDA, SOD and GSH in lung tissues

Discussion

LPS, also known as endotoxin, is a key component of the cell wall of Gram-negative bacteria.It was cytotoxic and could cause extensive and intense inflammatory reactions and oxidative stress (Leiet al.,2018).LPS could cause damages to various organs ofthe body and obstacles to systemic function, and the lung was one of the first target organs to be damaged.LPS was dissociated by LPS-binding protein into a potent activated form of LPS monomer, which fully exposed the internal binding sites.Then, after binding to toll-like receptor 4 and myeloid differentiation protein-2, it activated downstream transcription factors, such as NF-κB, which promoted the explosive growth of inflammatory factors and reactive oxygen species, causing a series of damages (Qiuet al., 2018).Therefore, LPS was considered as an effective inducer for acute lung injury (Wanet al., 2018).

There are many ways to establish the ALI in LPS.The time points for establishing the ALI by intraperitoneal administration of LPS are different,including 4, 8, 12 and 24 h.It had been reported that the ALI model established by tail vein injection of LPS was most severe in 6 h of pulmonary edema and neutrophil infiltration and the expression of TNF-αand IL-1βin lung tissues was the highest (Luoet al.,2011).Therefore, this experiment was performed by adding a 6 h time point to the intraperitoneal injection of LPS model to observe lung changes.Although the effect of tail vein administration was better and the repeatability was stable, the surgical technique was highly demanded, and the drug was easily injected into the blood vessel at the time of administration,so that the effective dose of the drug was inaccurate.Therefore, it was not suitable for plenty ofin vivoexperiments.Kastiset al.(2013) established the ALI model by intratracheal instillation of LPS and detected the changes of pulmonary inflammation at different time points, and their results showed that the lung injury score reached the maximum at 6 h after tracheal drip of low dose LPS.Tracheal perfusion of LPS could directly damage epithelial cells to cause inflammation in the lungs, leading to significant lung tissue damage and stable methods.However, this method was relatively complicated to operate and was prone to cause suffocation in animals, and had limitations for studying acute lung injury caused by systemic inflammation.Intraperitoneal injection of LPS established the ALI modeling stability, high reproducibility and simple operation (Abdelmageedet al., 2016).However, there was no intraperitoneal injection of LPS to establish a screening for the ALI time points.Therefore, in this study, the ALI model was established by intraperitoneal injection of 10 mg · kg-1LPS.The lung tissues were judged at 4,6, 8, 12 and 24 h to determine whether the model was successful and the degree of lung injury was observed.

In this experiment, the ALI model was established by intraperitoneal injection of LPS.From pathological observation, it was found that the pathological changes of lung tissues in the model group were obvious, the alveolar septum thickened, and more inflammatory cells could be seen in the alveolar cavity.The damage was aggravated with the prolongation of LPS time.This was consistent with lung pathological injury caused by intravenous administration and intratracheal instillation of LPS and acid in trachea (Kogaet al.,2010; Puiget al., 2016).The W/D ratio of lung tissues in 12 h LPS group significantly increased and decreased slightly after 12 h of LPS.The results suggested that pulmonary dysfunction caused by acute lung injury caused by LPS might be due to edema in the lungs.The expression of IL-1βand TNF-αproteins in lung tissues of LPS group increased gradually, and reached the peak at 12 h.Therefore, the expression of inflammatory factors in lung tissues was the highest at 12 and 24 h after LPS administered, which proved that the inflammatory injury was most significant at 12 and 24 h.The index of oxidation factor showed that the contents of MDA increased, the activities of SOD and GSH decreased in LPS group.In LPS group, the activities of GSH increased at 24 h compared with those of 12 h, but the contents of MDA decreased slightly and the activities of SOD increased significantly, which indicated that the ability of antioxidant injury increased at 24 h.This could be caused that LPS was constantly consumed in the body, the stimulation to the body decreased gradually and the body stimulated the mechanism of antioxidant protection, which was mainly played by SOD.

In summary, in the ALI model of intraperitoneal injection of LPS, inflammatory factors and oxidative factors participated in the process of lung injury and were time-dependent.The pathological changes of the lungs were observed 4 h after LPS administration.The antioxidation factor increased continuously after 12 h,but it still showed inflammatory damage and oxidative damage, due to the time required for the body to metabolize LPS.

Conclusions

The results showed that inflammatory reaction and oxidative damage were the main causes of lung injuries in rat ALI model and lung injuries were most obvious 12 h after intraperitoneal injection of 10 mg · kg-1LPS.