JTE-013 Alleviates Inflammatory Injury and Endothelial Dysfunction Induced by Sepsis In Vivo and In Vitro

AbstractBackground

Nowadays, there is no approved targeted agent for lung injury induced by sepsis. S1PR2 is confirmed to be a promising diagnosis and treatment target. JTE-013 as S1PR2 antagonists may be an agent of great potential. In this research, we sought to determine the functional role of JTE-013 in lung injury induced by sepsis.

Materials and methods

Seventy-two rats were assigned into normal group, sepsis model group and JTE-013 group. The animal model of lung injury induced by sepsis was constructed by cecal ligation and puncture. The human pulmonary microvascular endothelial cells (HPMECs) were divided into control, LPS and LPS + JTE-013 group. HPMECs induced by LPS served as the cell model of lung injury induced by sepsis. HE staining assay was performed for assessment of the pathological condition and Evans blue was applied for assessment of pulmonary tissue permeability. Wet/dry ratio was measured as indicators of pulmonary edema degree and neutrophil count was measured as indicators of infection status. The levels of inflammatory factors were detected by corresponding kits, cell survival by CCK-8 assay and protein expression level by western blot.

Results

S1PR2 was highly expressed in vivo model of lung injury induced by sepsis. It was observed that JTE-013 as antagonist of S1PR2 alleviated the lung tissue injury, endothelial dysfunction and pulmonary edema induced by sepsis. In addition, JTE-013 reduced neutrophil count and levels of inflammatory factors. Moreover, results confirmed that JTE-013 enhanced cell viability and mitigated inflammatory response in cell model of sepsis.

Conclusions

Overall, JTE-013 as an antagonist of S1PR2 could relieve inflammatory injury and endothelial dysfunction induced by sepsis in vivo and vitro, resulting in attenuation of lung injury. These findings elucidated that JTE-013 may be a promising targeted agent for lung injury induced by sepsis.

1. IntroductionSepsis is defined as organ dysfunction caused by the inadequate anti-infection ability of host.Rello J Valenzuela-Sánchez F Ruiz-Rodriguez M et al.Sepsis: a review of advances in management. Sepsis caused by viruses, fungi or bacteria is closely related to inflammation response and is a life-threatening disease. Up to now, the etiology and mechanism of sepsis remain unclear and present researches have achieved very little success. Therefore, it is urgent to clarify the mechanism and explore the efficient therapy for sepsis. The sepsis is a kind of syndrome leading to the serious loss in function of kidney, lung, thyroid heart and so forth.Aslan A van den Heuvel MC Stegeman CA et al.Kidney histopathology in lethal human sepsis.Liu J Du J Cheng X et al.Effect of netrin-1 anti-inflammatory factor on acute lung injury in sepsis rats.Thyroid hormone disorders and sepsis.Complement and sepsis-induced heart dysfunction. Organ dysfunction weakens the response of body to infection, resulting in more severe sepsis. Hence, reducing the organ injury is one of the important parts in sepsis therapy.Among all the organs, lung is more vulnerable to sepsis. As reported, more than 50% patients are suffering from acute lung injury during the development of sepsis.Gu WJ Wan YD Tie HT et al.Risk of acute lung injury/acute respiratory distress syndrome in critically ill adult patients with pre-existing diabetes: a meta-analysis. Undoubtedly, acute lung injury caused by sepsis has become a crucial problem to be solved. Increasing evidence has confirmed that pulmonary microvascular endothelial cell function and inflammation play vital roles in acute lung injury. Pulmonary microvascular endothelial cells, the major component of the alveolar-capillary, are responsible for paracellular exchange of fluid and solutes, forming an alveolar-capillary barrier.Su VY Chiou SH Lin CS et al.Induced pluripotent stem cells attenuate endothelial leakage in acute lung injury via tissue inhibitor of metalloproteinases-1 to reduce focal adhesion kinase activity. An elevated colony count of endothelial progenitor cells was positively correlated with an increased patient survival.Burnham EL Taylor WR Quyyumi AA et al.Increased circulating endothelial progenitor cells are associated with survival in acute lung injury. Pulmonary microvascular endothelial cells are able to repair the endothelial injury and keep integrity of pulmonary alveolar-capillary barrier.Yang N Tian H Zhan E et al.Reverse-D-4F improves endothelial progenitor cell function and attenuates LPS-induced acute lung injury. Pulmonary microvascular endothelial cells dysfunction contributed to acute lung injury by increasing permeability as well as neutrophil adhesion. During the process of sepsis, inflammatory factors or pro-inflammatory mediators impair the endothelial cells, enhance the permeability, and lead to neutrophil migration from blood to inflammatory site to further promote permeability.The endothelium in acute lung injury/acute respiratory distress syndrome.

Based on the above theoretical researches, anti-inflammatory injury and endothelial dysfunction are two strategies to improve acute lung injury caused by sepsis. It is meaningful to find the effective endogenous therapy target that works on inflammatory injury and endothelial dysfunction in acute lung injury caused by sepsis.

S1PR2, a crucial regulatory factor in microvascular endothelial cells, has been confirmed to be highly expressed in microvascular endothelial cells.LPS and TNF-α induce expression of sphingosine-1-phosphate receptor-2 in human microvascular endothelial cells.,Zhang G Yang L Kim GS et al.Critical role of sphingosine-1-phosphate receptor 2 (S1PR2) in acute vascular inflammation. S1PR2 aggravates inflammatory lesions induced by IL-1β in sepsis and knockdown of S1PR2 can promote the cell survival induced by sepsis.Song F Hou J Chen Z et al.Sphingosine-1-phosphate receptor 2 signaling promotes caspase-11-dependent macrophage pyroptosis and worsens Escherichia coli sepsis outcome. S1PR2 contributes to sepsis via elevating inflammation and enhancing endothelial dysfunction.Camerer E Regard JB Cornelissen I et al.Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice.,Zhu B Luo GH Feng YH et al.Apolipoprotein M protects against lipopolysaccharide-induced acute lung injury via sphingosine-1-phosphate signaling. JTE-013 is the inhibitor of S1PR2 and may be a promising therapeutic agent for sepsis. Based on the evidence above, the present study aims to explore the effects of JTE-013 on inflammatory injury and endothelial dysfunction in acute lung injury induced by sepsis.2. Materials and methods2.1 Cell culture and treatment

Human Pulmonary microvascular endothelial cells (HPMECs) were purchased from CELLBIO (Shanghai, China). HPMECs at a density of 1 × 105 were cultured in Endothelial Cell Medium (CA, USA) containing 10% FBS and 2% endothelial cell growth supplement (ECGS) (CA, USA). Cell injury model induced by sepsis was established using LPS, which was used as the common methods. After 90% confluence, the cells were divided into control group, LPS and LPS + JTE-013. For LPS group, the cells were treated with LPS (1 µg/mL) for 4h. For LPS + JTE-013 group, the cells after LPS (1 µg/mL) pretreatment were treated with JTE-013 (10µm) for 4h.

2.2 Animals experimental model establishment and treatment

Male Sprague Dawley rats (6 weeks old, weighing 180-220 g) were obtained from Laboratory Animal Center and housed three days with free access to food and water for adaptability to the environment. 72 rats were randomly assigned into three groups (24/group): normal group, sepsis model group and JTE-013 group. For sepsis model group, the experimental model of acute lung injury induced by sepsis was established using cecal ligation and puncture. The rats after anesthesia with pentobarbital (40mg/kg) was shaved and disinfected. The incision was made in the middle abdomens of rat. The cecum was taken out and its root was ligated. The end of the cecum was perforated. For the normal group, the rats only received anesthesia with no surgeries. For JTE-013 group, the rats with acute lung injury induced by sepsis were treated with 30mg/kg JTE-013 via intraperitoneal injection. The research project is approved by the Ethics Committee of The fourth sanatorium area of Hangzhou special service sanatorium center of air force. The experimental procedure followed the Guidelines for the Care and Use of Laboratory Animals and the “3R” principle.

2.3 Hematoxylin-eosin staining assay

The lung tissues from different group were fixed with 4% formaldehyde overnight. After dehydration, the samples of different groups were embedded with paraffin and then made into slices. Subsequently, samples were dewaxed with dimethylbenzene and hematoxylin-eosin was used to stain the samples for hematoxylin-eosin staining assay.

2.4 Western blot

The total proteins from lung tissues and cells were extracted by corresponding kits. Equal amount of protein were separated on 10% SDS-PAGE and then transferred to PVDF membranes. After blocking in 5% skimmed milk, membranes were incubated with the primary antibodies against ICAM-1(#ab7815), VCAM-1 (#ab134047), VE-cadherin (#ab166715), ZO-1 (#ab221547) as well as occluding (#ab240150). The secondary antibody was then incubated with the membranes for 1h. MultiImager was used to capture the blots and the results were analyzed by Quantity-One software.

2.5 Evans blue assay

Pulmonary tissue permeability was evaluated by Evans blue assay. The rats of different groups after treatment were injected with 2% Evans blue (2 mg/kg) via cauda vein. 1 h later, the rats were anesthetized, and the chest of rats were opened. Left atrium was sheared to irrigate the vessel. The blood in the lung was removed using cold normal saline. The lungs of rats were isolated, finally.

2.6 Neutrophil count

Chloral hydrate was used for anesthetization of rat via intraperitoneal injection. The right main bronchus was clamped with vascular forceps following opening the chests of rats. A tracheal catheter was used to extract the liquid after the left lung was treated with precooled sterile saline. After this step was repeated three times and the bronchoalveolar lavage fluid was collected. After that, the bronchoalveolar lavage fluid was submitted to centrifugation and cell precipitation was taken. The cells were classified, and neutrophil count was counted after resuspension.

2.7 Assessment of TNF-a, IL-1β, and IL-6

The levels of TNF-a, IL-1β and IL-6 in the serum were detected by the corresponding kits. The detection of TNF-a, IL-1β and IL-6 was performed according to the corresponding protocols of manufacturer.

2.8 Assessment of wet/dry ratio

Pulmonary edema was evaluated by detection of wet/dry ratio. The right lower lungs from different groups after isolation were weighed and then subjected to dehydration at 80 °C in an oven overnight. The lungs after dehydration were weighed and the wet/dry ratio was calculated by the weight before/after dehydration.

2.9 Cell counting kit-8 (CCK-8) assay

CCK-8 assay was performed for assessment of cell viability. The cells (1 × 105) were cultured in a 96-well plate. After treatment, each well of the cells in different groups were treated with 10 µL CCK-8 agent. The absorbance at 450nm which represented the cell viability was obtained using a microplate reader (Thermo Scientific, CA, USA).

2.10 Statistical analysis

All statistical analysis was performed using GraphPad Prism version 6.00. One-way ANOVA analysis followed by Tukey's post hoc test were used to test the differences among different treatment groups. p< 0.05 indicated a difference with statistical significance.

3.3. JTE-013 alleviated endothelial dysfunction and inflammation induced by sepsisEndothelial dysfunction and inflammation play crucial role in lung injury induced by sepsis. Based on this point, the effects of JTE-013 on endothelial dysfunction and inflammation induced by sepsis were evaluated herein. ZO-1, VE-cadherin and occludin as junction proteins play vital roles in maintenance of endothelial barrier integrity as well as permeability of endothelium.Adherens and tight junctions: structure, function and connections to the actin cytoskeleton.Kim D Eom S Park SM et al.A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function.Singh GB Zhang Y Boini KM et al.High mobility group box 1 mediates TMAO-induced endothelial dysfunction. In comparison with normal rats, the levels of ZO-1, VE-cadherin and occludin in sepsis rats were greatly decreased, indicating that the endothelial function was destroyed by sepsis (Fig. 4). JTE-013 treatment partly restored ZO-1, VE-cadherin and occludin expression, suggesting that JTE-013 protected the endothelial cells against injury induced by sepsis. Intercellular adhesion molecule-1 (ICAM-1) and cell adhesion molecule-1 (VCAM-1) play a crucial role in mediating the adhesion of leukocytes on endothelial cells.Burne MJ Elghandour A Haq M et al.IL-1 and TNF independent pathways mediate ICAM-1/VCAM-1 up-regulation in ischemia reperfusion injury.,Zittermann SI Issekutz AC. Basic fibroblast growth factor (bFGF, FGF-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression. The interaction between endothelial cells and leukocytes is an essential step in inflammatory response. Therefore, ICAM-1, VCAM-1 and inflammation factors are all measured herein. Sepsis rats appeared an obvious increase in the levels of ICAM-1, VCAM-1 and inflammation factors and upregulation of ICAM-1, VCAM-1, TNF-α, IL-1β and IL-6 expression were visibly suppressed by JTE-013, confirming that JTE-013 was able to reduce the inflammation injury induced by sepsis (Fig. 5 A, B).Fig 4

Fig. 4JTE-013 alleviated endothelial dysfunction induced by sepsis. The expression levels of ZO-1, VE-cadherin and occludin. ***P < 0.001 versus Normal group; ##P < 0.01 versus sepsis group.

Fig 5

Fig. 5JTE-013 alleviated inflammation induced by sepsis. (A) The levels of adhesion molecules (ICAM-1 and VCAM-1); (B) The levels of inflammation factors (TNF-a, IL-1β and IL-6). ***P < 0.001 versus Normal group; ###P < 0.001 and ##P < 0.01versus sepsis group.

3.4. JTE-013 enhanced cell viability, reduced the inflammation level and mitigated endothelial dysfunction in cell model of sepsisMoreover, we evaluated the effects of JTE-013 on cell injury by exploiting the cell model of sepsis. The cell model of sepsis was established using the LPS, which is the common method. LPS significantly decreased the cell viability when compared with the control, indicating the successful establishment of cell model of sepsis. Then, JTE-013 restored the decreased cell viability induced by LPS, demonstrating that JTE-013 could protect against LPS-mediated cell injury (Fig. 6). In addition, we investigated the effects of JTE-013 on inflammation in vitro. The levels of ICAM-1, VCAM-1, TNF-α, IL-1β and IL-6 were all increased in LPS group in contrast to control group. Furthermore, the upregulated expression of ICAM-1, VCAM-1, TNF-α, IL-1β and IL-6 induced by LPS were suppressed following treatment withJTE-013, confirming that JTE-013 possessed protective effects on cell model of sepsis via decreasing ICAM-1, VCAM-1 and inflammation levels (Fig. 7 A, B). We also assessed the effects of JTE-013 on endothelial dysfunction in vitro. The levels of junction proteins including VE-cadherin, ZO-1 and occludin were all evaluated in cells of different groups. In comparison with that in control group, the levels of VE-cadherin, ZO-1 and occludin were all decreased in cell model of sepsis. As expected, the suppressive effects of LPS on the levels of junction proteins were abolished by JTE-013. The findings herein confirmed that JTE-013 attenuated endothelial dysfunction induced by LPS (Fig. 8).Fig 6

Fig. 6JTE-013 enhanced cell viability in cell model of sepsis. Cell viability in HPMEC, LPS, LPS+JTE-013 group. ***P < 0.001 versus HPMEC group; ##P < 0.01 versus LPS group.

Fig 7

Fig. 7JTE-013 reduced the inflammation level in cell model of sepsis. (A) The levels of adhesion molecules (ICAM-1 and VCAM-1); (B) The levels of inflammation factors (TNF-a, IL-1β and IL-6). ***P < 0.001 versus HPMEC group; ###P < 0.001 and #P < 0.05 versus LPS group.

Fig 8

Fig. 8JTE-013 mitigated endothelial dysfunction in cell model of sepsis. The expression levels of ZO-1, VE-cadherin and occludin. ***P < 0.001 versus HPMEC group; ###P < 0.001 and ##P < 0.01 versus LPS group.

4. Discussion

Sepsis as the leading cause of mortality from infection remains unsolved. Sepsis could cause a series of organ dysfunction and make the condition worse. As the susceptible organ, lung was more frequently injured by sepsis. It is beneficial to control illness through reducing the lung injury caused by sepsis. Therefore, exploring the effective agent for lung injury caused by sepsis is pressing. In the current study, we found that JTE-013 alleviated inflammatory injury and endothelial dysfunction induced by sepsis.

JTE-013 was an antagonist of S1PR2 which was highly expressed in endothelial cells and confirmed to contribute to sepsis via pro-inflammatory roles and suppressing macrophage phagocytosis.Song F Hou J Chen Z et al.Sphingosine-1-phosphate receptor 2 signaling promotes caspase-11-dependent macrophage pyroptosis and worsens Escherichia coli sepsis outcome.Camerer E Regard JB Cornelissen I et al.Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice.Zhu B Luo GH Feng YH et al.Apolipoprotein M protects against lipopolysaccharide-induced acute lung injury via sphingosine-1-phosphate signaling.Adherens and tight junctions: structure, function and connections to the actin cytoskeleton.Kim D Eom S Park SM et al.A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function.Singh GB Zhang Y Boini KM et al.High mobility group box 1 mediates TMAO-induced endothelial dysfunction.Burne MJ Elghandour A Haq M et al.IL-1 and TNF independent pathways mediate ICAM-1/VCAM-1 up-regulation in ischemia reperfusion injury.Zittermann SI Issekutz AC. Basic fibroblast growth factor (bFGF, FGF-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression.Hou J Chen Q Zhang K et al.Sphingosine 1-phosphate receptor 2 signaling suppresses macrophage phagocytosis and impairs host defense against sepsis. Currently, there is no approved effective therapeutic agents for the targeted treatment of sepsis. JTE-013 as an antagonist of S1PR2 is expected to become a new agent for the targeted therapy of sepsis. Sepsis was an inflammatory disease. In addition, both inflammation and endothelial dysfunction play important roles in lung injury.Recent advances in understanding and managing sepsis.Leonard A Su PY Yule DI et al.Critical role of mortalin/GRP75 in endothelial cell dysfunction associated with acute lung injury.Du XK Ge WY Jing R et al.Necroptosis in pulmonary macrophages mediates lipopolysaccharide-induced lung inflammatory injury by activating ZBP-1. Thus, the current study was designed with emphasis on the effects of JTE-013 on inflammation and endothelial dysfunction in the acute lung injury induced by sepsis.Male animals are usually applied to establish the sepsis-induced lung injury model in vivo. Male animals are more prone to immunosuppression in sepsis. In comparison with male animals, female animals are more tolerant to sepsis. Besides, the sex hormone levels of female animals in estrus may change a lot and estrogen could partly inhibit excessive inflammatory response following sepsis by suppressing signal transduction. For the above reasons, 6-week-old male Sprague Dawley rats were selected for establishment of sepsis model in vivo in our present study. Previous study has reported that S1PR2 was elevated in peripheral blood of sepsis patients.Hou J Chen Q Zhang K et al.Sphingosine 1-phosphate receptor 2 signaling suppresses macrophage phagocytosis and impairs host defense against sepsis. In the present research, S1PR2 was also found to be upregulated in the lung tissues of sepsis rats. In addition, in line with the previous studies, sepsis rats appeared visibly severe lung injury.Liu J Du J Cheng X et al.Effect of netrin-1 anti-inflammatory factor on acute lung injury in sepsis rats.,Efficacy of pulmonary transplantation of engineered macrophages secreting IL-4 on acute lung injury in C57BL/6J mice. Furthermore, in this research, the pulmonary tissue permeability, neutrophil count and wet/dry ratio were also greatly increased. All these results suggested the successful establishment of acute lung injury induced by sepsis model.It is well known that pulmonary endothelial cells are the functional barrier in fluid balance.Ohmura T Tian Y Sarich N et al.Regulation of lung endothelial permeability and inflammatory responses by prostaglandin A2: role of EP4 receptor. Enhanced pulmonary tissue permeability will result in pulmonary edema, meaning severe endothelial cell damage. In this study, the pulmonary edema as indicated by the wet/dry ratio occurred in sepsis rats, which represented the injury of endothelial cells.It was observed that the lung injury was significantly alleviated following JTE-013 treatment. The obvious upregulation of wet/dry ratio, pulmonary tissue permeability and neutrophil count induced by sepsis were all distinctly suppressed by JTE-013. In addition, it was known that endothelial cells play a key role in preventing leakage of proteins and the cells influx in lung tissue. Junction proteins including VE-cadherin, ZO-1, and occludin together form the endothelial cell barrier, and the expression levels of VE-cadherin, ZO-1, and occludin represents the endothelial function situation. Maintenance of endothelial function lies in the expression of junction proteins.Zhu N Zhang GX Yi B et al.Nur77 limits endothelial barrier disruption to LPS in the mouse lung. In this study, JTE-013 elevated the levels of VE-cadherin, ZO-1, and occludin in vivo model of sepsis.Inflammatory level is an important parameter in both acute lung injury and sepsis, and a lot of researches reported that inflammatory level was up-regulated in acute lung injury and sepsis.Liu J Du J Cheng X et al.Effect of netrin-1 anti-inflammatory factor on acute lung injury in sepsis rats.,Deng G He H Chen Z et al.Lianqinjiedu decoction attenuates LPS-induced inflammation and acute lung injury in rats via TLR4/NF-κB pathway.,Radovic M Ristic L Krtinic D et al.Melatonin treatment prevents carbon tetrachloride-induced acute lung injury in rats by mitigating tissue antioxidant capacity and inflammatory response. In the current study, the inflammation level was also obviously upregulated in lung injury induced by sepsis, which is in line with previous studies. Furthermore, JTE-013 significantly inhibited the inflammatory level induced by sepsis in vivo.We also explored the effects of JTE-013 at cell level. LPS-induced cells have been reported as the common cell model of lung injury induced by sepsis.Wang YM Ji R Chen WW et al.Paclitaxel alleviated sepsis-induced acute lung injury by activating MUC1 and suppressing TLR-4/NF-κB pathway.,Li X Jamal M Guo P et al.Irisin alleviates pulmonary epithelial barrier dysfunction in sepsis-induced acute lung injury via activation of AMPK/SIRT1 pathways. JTE-013 treatment restored cell viability, inhibited inflammatory injury and endothelial dysfunction. The experiments in vivo and vitro were consistent, strongly supporting the conclusion that JTE-013 possessed alleviative effects on inflammatory injury and endothelial dysfunction induced by sepsis.5. Conclusion

The acute lung injury induced by sepsis commonly occurs and remains unsolved. So far, no effective targeted therapy was approved in human. In this study, JTE-013 was found to possess inhibitory effects on inflammatory injury and endothelial dysfunction induced by sepsis in vivo and vitro. To conclude, these findings revealed the protective effects of JTE-013 on the pathology of sepsis for the first time and may provide new insights for the development of novel therapeutic strategies for acute lung injury induced by sepsis. In spite of above achievement, this current study lacks the research referring to the underlying mechanism by which JTE-013 protected against acute lung injury induced by sepsis.

In summary, JTE-013 is a promising agent, providing a new strategy for sepsis therapy in clinic. More importantly, in depth mechanism by which JTE-013 alleviated inflammatory injury and endothelial dysfunction induced by sepsis should be further investigated. Still, clinical analysis should be studied in the future to confirm our results and excavate the predictive values of JTE-013.

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