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Original Article Protective Effects of Neurotensins on Lipopolysaccaride-Induced Acute Lung Injury by Blocking Tachykinin Mediated Pathway

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Original Article Protective Effects of Neurotensins on Lipopolysaccaride-Induced Acute Lung Injury by Blocking Tachykinin Mediated Pathway Int J Clin Exp Pathol 2017;10(7):7292-7302 www.ijcep.com /ISSN:1936-2625/IJCEP0038369 Original Article Protective effects of neurotensins on lipopolysaccaride-induced acute lung injury by blocking tachykinin mediated pathway Shuiqiao Fu, Xiaoqian Luo, Wenqiao Yu, Yun Ji, Weina Lu School of Medicine, The Second Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang, China Received August 21, 2016; Accepted August 28, 2016; Epub July 1, 2017; Published July 15, 2017 =Abstract: Neurotensin, a bioactive tridecapeptide, has been shown to regulate inflammatory process in lung tis- sues. However, the effect of neurotensin on LPS-induced lung injury and underlying detailed molecular mechanisms has not been studied. The aim of present study is to investigate the effect of neurotensin on LPS-induced acute lung injury in mice. Mice were treated with LPS intratracheally to induce acute lung injury. 1 hour after ALI induction, and then mice were treated with neurotensins (NTs) (20 mg/kg, 40 mg/kg, and 80 mg/kg) via tail vein injection. Next, the severity of lung injury, MPO activity, neutrophils infiltration, lung edema, protein and pro-inflammatory cytokines concentration in BALF were determined to evaluate the effect of Nts on ALI. Additionally, the expression of tachykinins receptors, including NK1, NK2, and NK3 and the production of IL-8, COX-2, and PGE2 mediated by tachykinins-tachykinins receptors pathway were determined to investigate the blocking effect of Nts on tachykinins and its receptors pathway. Neurotensins treatment significantly decreased the lung edema and the infiltration of inflammatory cells into lung tissue caused by LPS induction. Meanwhile, the elevation of pro-inflammatory cytokines and chemokine in BALF was dramatically reduced by neurotensins treatment. Furthermore, neurotensins could interact with tachykinins receptors and block the inflammatory responses activated by tachykinins pathways. In summary, neurotensins has a potentially protective effect on LPS-induced acute lung injury through the interaction with tachykinins receptors and subsequently blocking the inflammatory responses induced by activation of tachy- kinins pathway. Keywords: Neurotensin, inflammation, acute lung injury, tachykinins, COX-2, PGE2 Introduction rial infection. LPS was the one of mainly pro- inflammatory reaction factor in lung injury, Acute lung injury (ALI) and acute respiratory leading to neutrophil recruitment and pulmo- distress syndrome (ARDS) are devastating and nary edema [4]. Following the infection, a cas- life-threatening syndromes and a major cause cade of cytokines that are expressed and re- of death in critically ill patients [1]. ALI is char- leased, such as tumor necrosis factor-α (TNF- acterized by hypoxemia and respiratory failure α), interleukin-1β (IL-1β), and IL-6, was induced due to exudation to alveolar spaces which im- by LPS [5, 6]. Therefore, the endotoxin LPS- pairs gas exchange. Histologically, ALI in hu- induced model of ALI has been widely used mans is characterized by a severe acute in- to investigate the mechanisms of ALI. flammatory response in the lungs and neu- trophilic alveolitis [2, 3]. The risk factors for Tachykinins [7], particularly substance P (SP) developing this syndrome include pneumonia, and neurokinin A (NKA), are members of the gastric aspiration, sepsis, shock, and acute tachykinin family of peptides and are widely pancreatitis. A major cause for development distributed in airway sensory nerves of the of ALI is sepsis, wherein Gran-negative bacte- upper and lower airways and immune cells [8]. ria are a prominent cause. Lipopolysacchari- Previous studies [9, 10] have demonstrated des (LPSs) are the main components of the that substance P and neurokinin A have various outer membrane of gram-negative bacteria and effects that could contribute to the changes act as basic mediators that host the inflam- observed in asthmatic airways, such as submu- matory sequelae after a gram-negative bacte- cosal gland secretion, increasing of the vascu- Neurotensins protects acute lung injury lar permeability, stimulation of mast cells, B Materials and methods cells, T-lymphocytes, and macrophages, addi- tionally, they also could induce the infiltration Mice of neutrophils. Meanwhile, the release of SP and NKA also induces microvascular reactions 100 specific-pathogen free C57BL/6 mice (20- such as vasodilatation and plasma extravasa- 25 g, 6-8 weeks, male =50, female =50) were tion which contribute to the edema formation obtained from the Shanghai Laboratory Animal of lung tissue [11, 12]. Center (SLAC) (Shanghai, China) and housed in stainless-steel cages in a room maintain- The effects of SP and NKA on inflammatory ed at 22 ± 1°C and a 12-hour light/dark cy- responses are mediated by the neurokinin 1 cle controlled environment with free access and 2 (NK1, NK2) receptors [13]. Both recep- to food and water. Animal housing conditions tors are widely expressed in peripheral tis- and experimental procedures conformed to sues. In addition, the expression of NK recep- institutional regulations and were in accor- tors could be induced by inflammatory stimu- dance with the National Institute of Health lations. Accumulated data reported that the guidelines for animal care, and all experiments activation of NK1 and NK2 receptors could were conducted were approved by the local trigger a number of biological responses, in- Animal Care and Use Committee. cluding microvascular leakage [14], mucus se- cretion [15], and inflammatory cell response Induction of acute lung injury [16, 17]. Elevated levels of SP and NKA have Mice were anesthetized by an intraperitoneal been observed in the airways of patients with injection of thiopental (37 mg/kg), and then chronic obstructive pulmonary disease and were fixed on operation platforms. Next LPS asthma. Preclinical studies have suggested (60 μg in 60 μl of PBS; Sigma-Aldrich, St. that the tachykinin NK1 and NK2 receptors Louis, MO) was treated intratracheally. Neuro- play an important role in bronchoconstriction, tensins (Nts) treated started at 1 hour after airway hyperresponsiveness and airway in- ALI induction. flammation caused by allergic and nonaller- gic stimuli [18]. Therefore, the antagonists that All animals were randomly divided into five could block these tachykinin receptors hole groups: (1) LPS+ Vehicle group (n=20, male promise for the treatment of airways diseases, =10, female =10), mice were subjected to LPS- like COPD or ALI. induced ALI as described above and received saline via tail vein injection 1 hour after the Neurotensin (NT) is a bioactive tridecapeptide induction of ALI. (2) LPS+20 mg Nts group that is widely distributed through the brain and (n=20, male =10, female =10), mice were sub- the gastrointestinal tract [19, 20]. It has been jected to LPS-induced ALI and treated with 20 shown to regulate a wide range of biological mg/kg Nts via tail vein injection at 1 hour after functions like inflammatory process in the lung ALI induction. (3) LPS+40 mg Nts group (n=20, [21]. Moreover, NT could interact with a number male =10, female =10), mice were subjected to of immune cells, including leukocytes, perito- LPS-induced ALI and treated with 40 mg/kg neal mast cells, and dendritic cells [22, 23]. In Nts via tail vein injection at 1 hour after ALI particular, due to the direct interaction of NT induction. (4) LPS+80 mg Nts group (n=20, with macrophages, NT are important in modu- male =10, female =10), mice were subjected to lating macrophage function and inhibiting the LPS-induced ALI and received 80 mg/kg Nts production of proinflammatory cytokines, sug- treatment via tail vein injection. (5) Control gesting a protective effect in inflammatory con- group (n=20, male =10, female =10), identical ditions [24, 25]. to the LPS+ vehicle group, but mice received intra-tracheal administration of saline, instead Therefore, in present study, we aimed to inves- of LPS. tigate the role of NT during acute lung inflam- mation using the LPS-induced acute lung injury Bronchoalveolar lavage (BAL) fluid collection (ALI) model, a well accepted model for ARDS. Our results demonstrated that NT has a poten- Twenty-four hours after ALI induction, the bron- tially protective effect on LPS-induced ALI choalveolar lavage (BAL) fluid was collected as through interacting and blocking tachykinin previously described [26]. Briefly, Mice were receptors in ALI mouse. euthanised and the tracheas were immediately 7293 Int J Clin Exp Pathol 2017;10(7):7292-7302 Neurotensins protects acute lung injury cannulated with an I.V. polyethylene catheter Real-time PCR assay equipped with a twenty-four-gauge needle on a 1-ml syringe. Next, lungs were lavaged once The lung tissues were removed and the total with 1.0 ml PBS for three times. The reco- mRNA was extracted using the Trizol reagent very percentage were >95%. Then the collect- (Invitrogen, Carlsbad, CA). Next the cDNA was ed BALF were centrifuged at 1000 g for 5 synthesized using a Prime-Scrip RT-PCR kit min at 4°C, the supernatants were stored at (Takara Bio Company, Shanghai, China), and -80°C for further experiments and the pellet- the expressions of NK1, NK2, and NK3 were ed cells were resuspend with 0.5 ml PBS to examined with the polymerase chain reaction determine the total BAL cells. The amount (PCR) using an ABI7900fast qPCR detection of proteins in the BALF was measured using a system (Applied Bio System). Primer sequen- Bradford Protein assay kit (Beyotime Biotech ces as follows: NK1, Forward, 5’-TTCCCCAAC- Inc.,
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