The primary purpose of these research was to demonstrate the preventive effect of IMA on lipopolysaccharide (LPS)-induced inflammation in acute lung injury(ALI) mouse models and Human umbilical vascular endothelial cells (HUVECs). LPS stimulation for 24h to induce ALI and inflammation of cells. The pathological results of lungs was evaluated by W/D ratio, pulmonary vascular permeability measurement and immunohistochemistry of myeloperoxidase(MPO). Cell viability was measured using CCK8 assay. Expression of pro-inflammatory mediators was analyzed by RT-PCR and ELISA. The protein level was analyzed by Western blot. The structure of cell junction was detected by immunofluorescence. IMA dose-dependently improved pulmonary pathological damage induced by LPS, reduced the lung wet/dry weight ratio and MPO expression in lung tissue. IMA decreased bronchoalveolar lavage fluid (BALF) inflammatory cell count, as well as the release of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) in blood. Pretreatment with IMA in HUVECs significantly attenuated LPS-induced actin stress fiber formation and VE-cadherin disruption. In addition, IMA down-regulated the mRNA abundances of Vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), interleukin-1B (IL-1β), IL-6 and TNF-α expression, the phosphorylation of protein p-p65, p-IκBα, p-p38, p-ERK and p-JNK induced by LPS were attenuated after IMA treatment in vivo and in vitro. In summary, IMA modulate of NF-κB and MAPK signaling pathways as well as the production of pro-inflammatory cytokines to prevent cellular damage due to LPS infection. These results indicate that in the treatment of LPS-induced acute lung injury, IMA could be a potential modulator.
Research Article
Effect of imatinib on lipopolysaccharide‑induced acute lung injury and endothelial dysfunction through P38 MAPK and NF-κB signaling pathway in vivo and in vitro
https://doi.org/10.21203/rs.3.rs-3130073/v1
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Acute lung injury
Imatinib
NF-κB
MAPK
Endothelial dysfunction
Acute lung injury (ALI) is a common adverse clinical consequence of sepsis and a key contributory factor to the incidence rate and mortality in intensive care [1], characterized by diffuse alveolar damage and increased permeability of alveolar capillary [2]. So far, few effective treatments for ALI have been available, including preventative antibiotic therapy and changing the patient's position. And these therapies lack efficacy and may have severe side effects.[3]. Thus, there remains a pressing need to develop novel targets for clinical treatment of ALI.
Lipopolysaccharide (LPS) as an endotoxin of gram-negative bacteria, can cause oxidative stress and inflammation response to induce ALI through the TLR4 pathway [4], and also is the most common way to establish experimental model of ALI [5]. LPS can stimulate the release of various cytokines, including tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β)[6]. Furthermore, the stimulation of LPS can cause cell apoptosis and activate the NF-κB signaling pathway mediated by TLR-4, ultimately leading to septic shock and death [7]. Some researchs have shown that activation of NF-κB is linked to the activation of the p38 MAPK pathway, which regulates various downstream transcription factors including NF-κB [8, 9]. There is sufficient literature to indicate that p38MAPK/NF-κB signaling pathway phosphorylation was closely bound up with the progression of LPS-induced ALI [10].
Endothelial cells are found in the innermost layer of blood vessels and they are one of the earliest cells to react to different inflammation stimulation such as LPS [11]. During the process of inflammation, EC responds to LPS by activating of pattern recognition receptors (PRRs), producing pro-inflammatory chemokines, cytokines and adhesion molecules [12], leading to the increase of mortality [13]. Therefore, there is an urgent need for the development of new treatment strategies for the treatment of endothelial dysfunction and the amelioration of inflammatory damage in ALI.
Imatinib is an tyrosine kinase inhibitor approved by FDA, was originally designed to treat chronic myelogenous leukemia by inhibits the BCR-Abl fusion protein, it also acts on a number of other kinases including c-Abl, Abl-related gene (Arg), c-kit, and platelet-derived growth factor receptor (PDGFR), indicating the potential for pleiotropic effects in vascular function [14]. According to reports, it also has beneficial pharmacological effects in humans [15] and in experimental animals [16], such as anti-inflammatory and anti-fibrotic effects. Thus, in the treatment of gram-negative sepsis and sepsis-induced acute respiratory distress syndrome (ARDS), IMA is considered as an unique medicine [17]. Recent works indicate that IMA also attenuates vascular permeability [18, 19, 20]. As these researchs have demonstrated that IMA may be a valid candidate for the treatment of LPS-induced lung injury, we further explore the specific anti-inflammatory mechanism of IMA. We found that IMA ameliorated pulmonary inflammation through the inhibition of the p38MAPK/NF-κB signaling pathway in vivo and in vitro.
Animals
C57BL/6 mice (Male, 8 to 12-week-old)were purchased from the SPF Biotechnology Co., Ltd.(Beijing, China). Before the experiment, mice were acclimated for 7 days. Sixty mice were randomly divided into control, LPS challenged, IMA (IMA, 60 mg/kg) and LPS + IMA (20, 40, 60 mg/kg) groups. The chemical structure of IMA is shown in Fig. 1. LPS was intraperitoneally administered at a dose of 15 mg/kg to induce the ALI mouse model and IMA (20, 40 or 60 mg/kg) was intravenous injection thirty minutes before intraperitoneal injection of LPS. The control group received equal amounts of saline. All mice were sacrificed by cervical dislocation after twenty-four hours, serum and lung tissues were obtained for subsequent experiments.The Laboratory Animal Management and Use Committee of Tianjin Medical University approved all experimental procedures.
Cell culture and treatment
Human umbilical vein endothelial cells (HUVEC, CELLBIO, Shanghai, China) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 0.5% penicillin/streptomycin in a 37℃, 5% CO2 incubator. All tests used cells with a fusion degree of 80–90%.
HUVECs were plated in 6-well culture plates and divided into six groups: Control, IMA10µM, LPS1µg/ml, LPS + IMA(5µM, 10µM, 20µM). Cells in the control group were left untreated.Cells were cultured with IMA at a dosage of 10 µM in the IMA 10µM group. In the LPS group, HUVECs were cultured with 1µg/ml LPS. As fot the three different concentrations of IMA + LPS treatment group, cells were pretreated with IMA for 30mins ,and then incubated with LPS for a duration of 24 h.
Chemicals and reagents
LPS(from Escherichia coli 055: B5) and IMA were purchased from Sigma-Aldrich China Branch (Shanghai, China). The chemical structure of IMA is shown in Fig. 1. All the primary antibodies used in western blotting, immunofluorescence and immunohistochemistry were purchased from Cell signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated (HRP) secondary antibodies and donkey anti-rabbit IgG H&L (Alexa Fluor® 488) preadsorbed were purchased from Abcam (Cambridge, UK). Mouse IL-6, TNF-α and MCP-1 ELISA kits were purchased from Biolegend (CA, USA). All other chemicals were of reagent grade.
Survival analysis
Another 48 mice were split into six groups for the survival curve and subjected to the same experimental procedure as described above for survival analysis. Monitored and recorded survival every 12 h for up to 72 h after LPS injection.
Lung W/D Ratio
Excised the wet left lung of mice, weighed and oven-dried at 60°C for 48 hours. Took it out and weighed again, calculated the ratio of wet to dry mass.
Histological analysis
Fixed the lungs with 4% paraformaldehyde for 48h. After paraffin embedding, cut the lung tissues into sections (4 µm) then fixed on slides. Stained the slides with hematoxylin and eosin. The Mikawa method was used to score lung injury based on the follows four indicators which imcluding alveolar congestion, haemorrhage, neutrophils infiltration and alveolar wall thickening or hyaline membrane formation. [21]. The cumulative increase in the total number of points is the score for ARDS..
Increase of the Protein and Cells in BALF
Flushing the lung (0.5 ml ×3 times) to procure the BALF liquid, then centrifuged at 3000 rpm for 15 min at 4°C. The collected supernatants were used to determine the total protein concentration using the Solarbio® BCA kit (Cat# PC0020) according to the manufacturer’s instructions, counting the number of cells in the sediment by Wright–Giemsa staining.
Lung vascular permeability measurements.
To assay lung vascular permeability, 100 µL 0.5% Evans blue was injected into anesthetized mice for 30 minutes. Harvested and weighed 100 mg of lung tissues then homogenized in 1 mL formamide, incubated the homogenate in 37°C for 24 hours and then centrifuged at 4000g for 10 minutes, the determination of the absorbance of supernatants were using spectrophotometry at a wavelength of 630 nm [22].
Immunohistochemistry
For immunocytochemistry, after the slices were deparaffinized, rehydrated, retrieved antigen and nonspecific protein docking, the slices were incubated with anti-MPO primary antibody and appropriate biotin-labelled secondary antibody. Stained the slices with DAB under the microscope, washed it and counterstained with hematoxylin, dehydrated, cleared in xylene, then fixed. Randomly chose five different microscopic fields under 400× magnification. The counting of MPO-positive cells in random fields was by a blinded evaluator.
Cytokine measurement
ELISA assay kits (Invitrogen, Carlsbad, CA, USA) were used to measure the expressions of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in serum according to the manufacturer’s instructions. The OD value was measured using a microplate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm.
CCK8 assays of cell viability
To analyze cell viability of drug‑treated cells compared with un-treated cells by the CCK8 kit (Sigma‑Aldrich; Merck KGaA), cells were seeded at a density of 1x105 cells/well into 96-well culture plates and allowed to adhere for 12h. After washed twice with PBS, cells were subsequently received with different concentrations of IMA ranging from 0.1 to 100µM for 24 hours.The absorbance was determined at 450 nm by using microplate reader (Bio-Rad, Hercules, CA, USA) at end.
Immunofluorescence staining and image analysis
Cells were seeded on 12-well plates where placed cell slides (Biosharp; China), stimulated for 24h. After the challenge, HUVECs were fixed with 4% formaldehyde, washed with PBST buffer, permeabilized with 0.1% Triton X-100 for 5 minutes, then blocked with 5% bovine serum albumin in PBS in 37℃ incubator. Subsequently immunostained cells with VE-cadherin antibody at 4℃ overnight, after wash 3 times, the cells were stained with Alexa Fluor 488 conjugated secondary antibodies (Abcam; England). Actin filaments were stained at RT with Texas red phalloidin (UElandy; Suzhou, China) for 1 h. Capture images using a fluorescence microscope.
RNA extraction and quantitative RT-PCR
To evalute the mRNA levels of inflammatory factors and focal adhesion factor, total cellular RNA was isolated by using Eastep® Super Total RNA Extraction Kit (Promega, American; LS1040) and cDNA synthesis was performed by TransScript All-in-One First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China; AT341-01). The SYBR Green PCR Kit (TransGen, Beijing, China; AQ602) and the ABI 7500 System (Thermo Fisher Scientific, Beijing, China) were used to perform PCR amplification with the primers listed in Table 1. PCR parameters were set for an initial cycle at 95 ℃ for 2 min and 40 cycles at 95 ℃ for 30 s, 57 ℃ for 45 s, and 72 ℃ for 45 s. With GAPDH for normalization, the relative levels of target genes were analysed by the 2−ΔΔCt method. The expression of the genes in the control group was standardized as 1 and then calculated other groups.
Immunoblotting
The lung tissues were homogenized and HUVECs was lysed in cold RIPA buffer which containing with protein inhibitor cocktail (Sigma-Aldrich, 1:100) and phosphatase inhibitor cocktail (Sigma-Aldrich, 1:100). Measured protein concentration by the BCA protein estimation kit (Solarbio®) after centrifugation. The same quantity of protein samples was separated by SDS-PAGE and then transferred onto PVDF membrane. At the end of the transfer, the membrane was transferred to 5% non-fat dry milk in TBST to block for 1 h, then incubated with ICAM-1 (1:1000), VCAM-1 (1:1000), VE-Cadherin (1:5000), p65(1:1000), p-p65(1:1000), p-IκBα(1:1000), IκB-α (1:1000), p-p38(1:1000), p38(1:1000), p-JNK (1:1000), JNK(1:1000), p-ERK (1:1000), ERK(1:1000), β-actin (1:5000) primary antibodies respectively at 4°C to react overnight. After washed three times, the membranes were treated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:5000) in TBST to conjugate with horseradish peroxidase. Also after three times washed by TBST, the membranes were followed by ECL detection reagent (Beyotime). These proteins were visualised using Tanon 5200 Multi Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China). β-actin protein levels were quantified to normalize protein levels by Image J software (NIH Image, Bethesda, MD, USA).
Statistical analyses
Experimental data were presented as mean ± S.E.M. The data were analyzed by one-way ANOVA by Tukey-Kramer multiple comparisons.
IMA significantly improves the survival of LPS-induced ALI mice
In Fig. 2, eight mice for each group were followed for survival rates. The rate in the LPS group was only 12.5% at 48h, low dose (20mg/kg) IMA administered to mice did not prevent LPS-induced death, and medium dose (40mg/kg) IMA treatment increased mouse survival to 37.5% and delayed the rate of death. The high dose of IMA (60mg/kg) raised the survival rate from 37.5–62.5%, according to the Kaplan–Meier survival analysis (P < 0.01), confirmed the protective effect of IMA.
IMA improve lung injury and Alleviates LPS-Induced Alveolar Permeability
All sections of structures of the lung in the Control and IMA groups were morphologically normal. In contrast, the significant alterations in the LPS group were manifested as thickening of peribronchial and alveolar walls, alveolar hemorrhage, vascular congestion, pulmonary edema and numerous infiltrating inflammatory cells. Compared with the control group and the IMA group, the lung injury score was considerably higher in the LPS group(P < 0.001). However, pretreatment with IMA attenuated the bleeding and inflammatory cells numbers, alleviating lung injury in Fig. 3A, B. As shown in Fig. 3C, the lung W/D ratio was also significantly lower in IMA group, especially at the high dose, compared to the LPS group(P < 0.001). Furthermore, to observe pulmonary vascular leakage by measured the extravasation of EBD, pulmonary tissue permeability in ALI mice was higher in contrast to that in normal mice and different doses of IMA partially reversed this effect (Fig. 3D). To further confirm the effect of IMA on ALI, we detected the total protein content in BALF, which also is a characteristic of capillary permeability. As shown in Fig. 3E, protein levels was markedly higher in the LPS group, with IMA treatment resulting in the decline of leaking protein content. The datas above showed that IMA protects against acute lung injury and high vascular permeability.
Cells in BALF of LPS-induced ALI mice
As shown in Fig. 3F, exposure to LPS trigger the overall number of cells in BALF significantly elevated(P < 0.001), whereas treatment with IMA (20 mg/kg, 40 mg/kg and 60mg/kg) (P < 0.05, P < 0.01 and P < 0.001), the numbers of cells was markedly decreased. The Wright Giemsa staining method was used to count the amount of neutrophils and macrophages in BALF. From Fig. 3G, H, stimulation with LPS resulted in a significant increase in the number of both neutrophils and macrophages in the BALF(P < 0.001). IMA (40 mg/kg, 60mg/kg) treatment prevented the injury by reduced neutrophil and macrophage cell counts.(P < 0.01).
IMA decrease the neutrophil infiltration during sepsis
As the exposure to LPS causes infiltration of neutrophils, immunohistochemical analysis was used to quantify the numbers of neutrophils. MPO-Positive cells were brown in colour in the interstitium of the lung tissues (Fig. 4A, B). In the LPS-induced mice lungs were detected MPO with strong staining density, demonstrated upregulated MPO activity(P < 0.001). IMA treatment improved lung injury by decreased neutrophil infiltration and migration significantly.
Impact of IMA on LPS-induced inflammatory response in HUVECs
In vitro, compared with viability in the Control groups, HUVECs treated with IMA at a concentrations in the range of 0.1 to 10 µM for 24 h had no significant differences in viability measured by CCK8 assay (Fig. 5A). When the concentration is 100µM, the drug shows certain cytotoxicity. Thus select 5, 10 and 20µm as appropriate concentration gradients for subsequent experiments.
ICAM-1 and VCAM-1 expression was determined at mRNA and protein levels. The mRNA expressions of ICAM-1 and VCAM-1 were notably increased by LPS challenge compared to the Control and IMA groups (P < 0.001), which were significantly reversed by IMA treatment (P < 0.001). The results of western blot analysis further suggested IMA exerted suppressive effects on LPS-induced excessive inflammation in HUVECs (Fig. 5B). The results in RT-PCR are consistent with the protein levels(Fig. 5C). The levels of ICAM-1, VCAM-1, TNF-α, IL-1β and IL-6 were all increased in LPS group in comparison to the Control group and IMA group. Furthermore, the elevated mRNA expression of these proinflammatory factors evoked by LPS were suppressed by pre-treatment with IMA( Fig. 5C), especially since the concentration was 10µM, confirming that IMA possessed protective effects on the LPS-induced inflammation of cells via decreasing the levels of ICAM-1, VCAM-1 and other inflammation factors ( Fig. 5D).
Impact of IMA on LPS-induced inflammatory response in lung tissue of ALI mice
Administration of LPS alone elevated the levels of inflammation cytokines and ICAM-1 in the lungs of mice(Fig. 6A). The up-regulation of TNF-α, IL-6 and MCP-1 expression were visibly decreased by IMA in a dose-based manner(Fig. 6B), confirming that IMA was able to reduce the inflammation injury induced by LPS.
Effects of IMA on LPS-induced endothelial Adherens Junctions
After exposure to LPS, the expression levels of VE-cadherin in HUVECs were depressed (Fig. 7A, B). LPS leads to a significant loss of this critical junctional protein in the extracellular domain and interferes with the localization of VE-cadherin and F-actin between cells. The changes detected by immunofluorescence indicated that compared with LPS, 10µM imatinib increased VE-cadherin expression. Individual exposure to imatinib (10µM, 24 h) had no influence on the distribution of VE-cadherin in static HUVECs. IMA eliminated the suppressive effect of LPS on junction proteins levels as we expected.
Impact of IMA on NF-κB, JNK, and p38 MAPK pathway
As inhibition of NF-κB and MAPKs signaling activation are considered important target in inflammation. To further reveal which potential mechanism IMA through to relieves LPS-induced ALI, the immunoblotting was used to determine the effects of IMA on IκBα, p65, JNK, Erk1/2 and p38 MAPK signaling proteins .
Western blot analysis showed in Fig. 8A, C, the protein expression level of p-IκBα, p-p65 in the LPS group was higher as compared to the Control and IMA groups. With IMA treatment, these changes were mitigated. As shown in Fig. 8B, D, p-p38, p-JNK and p-ERK levels in the LPS group were higher than those in the Control and IMA groups. With IMA treatment, these phosphorylated proteins were significantly affected. The results in vivo and in vitro were consistent. IMA effects was significant from 40mg/kg dose, the effect was maximum at 60 mg/kg.
The high permeability of blood vessels means fluid and protein excessively infiltrate into the interstitium from the intravascular space, resulting in tissue edema and life-threatening complications. To improve the prognosis of ALI, it is important to find ways on protecting endothelial function [23], preventing endothelial damage and inflammation in ALI has profound implication. This study examined that during sepsis, Imatinib was associated with vascular endothelial cell damage and decreased inflammation and endothelial injury via the NF-κB and MAPK pathways. Our data suggested the potential role and mechanism of IMA and it should be considered as an potential effective drug in the treatment of patients with ALI in the future.
Cell adhesion molecules such as VCAM-1 and ICAM-1 can promate interaction between endothelial cells and leukocytes, leading to the recruitment of leukocytes to inflammatory sites, resulting in subsequent impairment of organ function [24]. MPO present in the neutrophils is one of the most abundant pro-inflammatory factors [25]. Our data demonstrate that IMA quells ICAM-1, VCAM-1 and other pro-inflammatory cytokines mRNA expression in HUVECs. In vivo, IMA attenuates ICAM-1, MPO and pro-inflammatory cytokines expression but has no effect on VCAM-1 of the lung tissue, which is same as previous researches [26]. In addition, our novel data indicate that higher concentrations of IMA have exacerbated inflammatory injury on endothelial cells. Why the lower dose of IMA seemed more effective than the higher dose in this study in HUVECs is unclear, however, there has been one study that suggested the potential mechanisms of DNA instability and impaired angiogenesis in PAH are related to the absence or inhibition of c-Abl in maintaining the integrity of normal pulmonary endothelial cells [27], this could be related to the over inhibition of c-abl.
Endothelial cell junctions are consists of tight junctions and adhesion junctions, which are closely related to vascular permeability [28]. As the main component of intercellular adhesion connections, cadherin is a characteristic calcium-dependent transmembrane adhesion protein in endothelial cells[29]. The cytoskeleton of actin is a highly dynamic structure related to barrier integrity, undergoes polymerization and depolymerization according to the demands of cells. Previous investigations indicate that the increase expression of pro-inflammatory factors such as IL-1β and TNF-α are capable of disrupting the expression of the tight junction protein and cytoskeleton protein such as vascular endothelial cadherin (VE-cadherin) and actin-myosin interaction [30]. IMA protects the integrity of HUVECs monolayer after LPS exposure by preventing the lost of VE-cadherin from cell junctions, regulating the rearrangement of actin cytoskeleton and maintaining VE-cadherin at cell-cell junctions. Our datas also suggest that IMA can alleviate lung permeability (BAL protein, EBD extravasation) in LPS-induced ALI.
Playing vital roles in regulating certain cellular properties, mitogen-activated protein kinases (MAPKs) is a family of serine-threonine protein kinases which is composed of stress-activated JNK, p38 and growth factor-regulated ERK1/2 [31]. During ALI process, MAPKs modulates the upstream pathway of LPS-induced NF-kB activation by regulating the phosphorylation of IkB-a and p65 [32]. Phosphorylated NF-κB pathway activates adhesion molecules (ICAM-1, VCAM-1), cytokines (TNF-α, IL1β, IL-6), and chemokines (IL-8 and MCP-1), converting the “anti-adhesive” pulmonary vascular endothelium to a “pro-adhesive” one, thereby promoting the adhesion of inflammatory cells and subsequent transendothelial migration [33].
As previous studies only investigated p38 in IMA-treated sepsis rats [34], we further explored the rest proteins expressed in MAPK pathway in mouses and HUVECs. In the present study, IMA treatment inhibited the phosphorylation of P-38, Erk1/2, JNK of MAPKs in HUVECs and lung tissues.
In conclusion, our data demonstrate that imatinib protects barrier function and inflammatory responses from acute lung injury induced by LPS. The mechanism likely involves suppression of NF-κB and MAPK pathway activation. The most recent research suggests that, in the context of systemic inflammatory response and endothelial dysfunction, only patients classified as alveolar injured due to elevated SP-D levels can benefit from imatinib therapy [35]. The mechanism and clinical treatment of IMA awaits further explored.
The present study demonstrates the effectiveness of IMA in preventing LPS-induced ALI by inhibit the NF-κB and MAPK pathway, reduce the release of systemic inflammatory factors and neutrophil infiltration, protect the pulmonary capillary barrier and endothelial barrier function. This topic is expected to provide evidence for a novel drug candidate for the treatment of endothelial inflammation-related diseases.
Fundings
This work was funded by Scientific Research Project of Integrated Traditional Chinese and Western Medicine of Tianjin Health Committee (2021207) and “New medical prominent youth program” of Tianjin high-level selection and training project [2018(19)].
Author contributions
Liu yaru conceived and designed the study, wrote the original draft preparation. Li duanyang in charge of data curation andd performed experimental operations. Zhang tianyi and Wang keruo were involved in vivo LPS-induced lung injury mice models. Liang xue and Zong xiaolong contributed to the figures. Yang hong provided technical supports and project generation. Li zhenyu is responsible for the overall concept and supervision.
Disclosure
The authors declare that there is no conflict of interests regarding the publication of this paper.
Ethics
The study was approved by the Laboratory Animal Management and Use Committee of Tianjin Medical University.
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No competing interests reported.