5368 MOLECULAR MEDICINE REPORTS 17: 5368-5375, 2018 Knockout of Toll-like receptor 4 improves survival and cardiac function in a murine model of severe sepsis DAN ZHOU1*, YUN ZHU1*, MIN-ZHI OUYANG1, MING ZHANG1, KUI TANG1, CHENG-CHENG NIU1 and LING LI2 1Department of Ultrasound Diagnosis, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011; 2Medical Basic Teaching Experiment Center, College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, P.R. China Received August 15, 2017; Accepted December 19, 2017 DOI: 10.3892/mmr.2018.8495 Abstract. Toll-like receptor 4 (TLR4) is a transmembrane The present study reported that TLR4 aggravates severe pattern-recognition receptor expressed in immune cells and sepsis-induced cardiac impairment by promoting the release the heart. Activation of TLR4 signaling during sepsis results of proinflammatory cytokines and neutrophil infiltration in in the release of cardiac depression mediators that may impair hearts. heart function. The present study aimed to determine whether TLR4 contributes to development of severe sepsis-induced Introduction myocardial dysfunction. A cecum ligation and puncture (CLP) procedure was employed to establish severe sepsis models. Severe sepsis and septic shock account for 20% of all admis- Wild type (WT) and TLR4 knock-out (TLR4-KO) mice sions to intensive care units and remains the most common were divided into four groups: WT-sham, TLR4-KO-sham, cause of mortality resulting from nosocomial infections (1,2). WT-CLP, and TLR4-KO-CLP. Cardiac function of these Severe sepsis is characterized by acute organ dysfunc- animals was evaluated at various time points following the tion, including heart, lung and liver. Cardiac dysfunction surgical procedure. The expression levels of proinflamma- is conferred to impaired myocardial function and collapsed tory cytokines in the heart tissues were detected by reverse circulation, and has been demonstrated to be the highest risk transcription-semi quantitative polymerase chain reaction factor for severe sepsis-linked mortality (3). The mechanisms (RT‑PCR). Myocardial neutrophil and macrophage infiltration underlying severe sepsis-induced acute cardiac dysfunction were investigated by histopathological examination, as well as are considered to involve an excessive inflammatory response a myeloperoxidase activity assay in heart tissue by RT-PCR. leading to the overexpression and release of proinflammatory Myocardium Fas cell surface death receptor/Fas ligand and cytokines, in addition to neutrophil hyperactivity (4). It has caspase-3 were also analyzed by RT-PCR. Additionally, been reported that injured cardiomyocytes release excessive myeloid differentiation primary response 88 M, toll or proinflammatory cytokines, including tumor necrosis factor interleukin-1 receptor-domain-containing adapter-inducing (TNF)-α, interleukin (IL)-1 and IL-6, thus leading to marked interferon-β and nuclear factor-κB expression levels were neutrophil aggregation and filtration in the heart in severe observed in the myocardium of all four groups. WT-CLP sepsis (4,5). mice exhibited increased mortality rates, more severe cardiac Toll-like receptor (TLR) 4 is a transmembrane dysfunction and had increased levels of interleukin (IL)-1β, pattern-recognition receptor, which is a key component of IL-6 and tumor necrosis factor-α in heart tissues and increased the innate immune system and is involved in the modulation neutrophil infiltration compared with TRL4‑KO‑CLP mice. of the sepsis‑induced inflammatory response. TLR4 detects pathogen-associated molecular patterns and then binds to bacterial lipopolysaccharide (LPS). Activation of TLR4 has been reported to induce inflammatory responses involved in the impairment of cardiac contractility. Therefore, TLR4 Correspondence to: Dr Ming Zhang, Department of Ultrasound has been proposed as a potential therapeutic target to control Diagnosis, The Second Xiangya Hospital, Central South University, the inflammatory response and improve cardiac function (6). 139 Renmin Road, Changsha, Hunan 410011, P.R. China Numerous studies revealed that TLR4 promotes cardiac E-mail: [email protected] dysfunction, induced by severe sepsis, particularly in the *Contributed equally presence of high-dose endotoxin (7,8). Severe sepsis is charac- terized by numerous bacterial infections and can be mimicked Key words: toll-like receptor 4, sepsis, myocardial dysfunction, in animal models. However, accumulating evidence has inflammation response, apoptosis demonstrated that the inhibition of TLR4 during inflamma- tion may alleviate heart failure by suppressing inflammatory responses mediated by the TLR4-myeloid differentiation ZHOU et al: KNOCKOUT OF TOLL-LIKE RECEPTOR 4 IMPROVES SURVIVAL AND CARDIAC FUNCTION 5369 primary response 88 (MyD88) signaling pathway and toll or mid-papillary level in the parasternal short-axis view. Strain interleukin-1 receptor-domain-containing adapter-inducing was obtained in the middle of the posterior wall on short-axis interferon-β (TRIF), another adaptor signal, which is also views during ≥3 consecutive heartbeats. Strain was analyzed associated with this inflammatory response. Therefore, the online using Software Velocity Vector Imaging (VVI, 3.5, mechanisms of TLR4 in heart dysfunction during severe Siemens Healthcare). sepsis require further investigation. Additional studies investigated the apoptotic pathway Langendorff system. Left ventricular (LV) function of the which is activated in cardiomyocytes by inflammatory media- hearts isolated from septic or sham mice were measured tors in septic cardiomyopathy (9,10). Activation of apoptosis 12 h following the surgical procedure using a Langendorff regulatory factors, including caspase 3, have been reported to perfusion apparatus as previously described (7,12). Briefly, account for cardiomyopathy following septic challenge (10). mice were heparinized (1,000 IU/kg) and anesthetized Evidence of these studies revealed that the apoptotic pathway pentobarbital sodium, 40 mg/kg, i.p.). The hearts were is associated with a partially reversible decrease in cardiac excised and immersed immediately in cold (4˚C) perfusion myocyte fractional shortening and cytokine decrease (11). fluid (Sigma‑Aldrich; Merck KGaA). The aortas were cannu- However, few reports have indicated that TLR4 is associated lated and retrograde-perfusion was performed at a constant with septic heart apoptosis. Therefore, the present study aimed flow rate (3 ml/min) with modified Krebs‑Henseleit buffer to investigate the effects of TLR4 deletion on myocardial (Sigma-Aldrich; Merck KGaA), while the heart was paced at apoptosis following cecum ligation and puncture (CLP). 7 Hz (420 beats/min). Following 20 min of coronary perfusion, In the present study, a modified procedure of CLP was LV end-systolic pressure (LVESP), LV end-diastolic pres- employed to establish severe sepsis models on wild type (WT) sure (LVEDP) and the heart rate were recorded for ≤30 min. and TLR4 deficient (TLR4‑KO) mice to investigate the role of LV developed pressure (LVDP) was calculated as follows: TLR4 signaling pathways in cardiac dysfunction during severe LVDP=LVESP-LVEDP; +dP/dtmax was defined as peak rate of sepsis. left ventricular pressure rise. Materials and methods Measurement of serum cardiac troponin I (cTnI). Blood samples were collected via the inferior vena cava of the mice Animal models. WT and TLR4-KO male mice (n=80), 12 h following CLP under anesthesia with pentobarbital weighing 20-25g and aged 6-8 weeks, were purchased sodium (40 mg/kg, i.p.). Mice were then sacrificed via cervical from the Model Animal Research Center of Nanjing dislocation. Subsequently, the blood samples were centrifuged University (Stock: J003752; Nanjing, China). TLR4-KO mice at 589 x g for 10 min at 4˚C to obtain the supernatant, which was (C57BL/10ScNJNju) were progenies of C57BL/10ScN from immediately stored at ‑20˚C until further analysis. Troponin I the Jackson Laboratory (Ben Harbor, ME, USA), harboring a (cTnI) levels in serum were measured by ELISA (Quantikine II12rb2 allele deletion. Animals were separately housed at 26˚C Mouse kit, KT29998, MSK Biotechnology Co., Ltd., Wuhan, by sex and maintained in a specific pathogen free and humid China) according to the manufacturer's protocols. (50%) environment exposed to a 12 h light/dark cycle; animals had ad libitum access to food and water. All experimental Quantification of expression levels of inflammatory cytokines procedures were approved by the medical ethical committee (IL‑1, IL‑6, TNF‑α) and MyD88, TRIF, nuclear factor‑κB of the Second Xiangya Hospital of Central South University. (NF‑κB) in heart tissues. Following euthanasia, heart tissues Bowel perforation (CLP) was used to establish severe sepsis. of mice were harvested. Total RNA was purified from heart Briefly, all mice were anesthetized with 1.5% pentobarbital tissue using TRIzol® reagent (Gibco; Thermo Fisher Scientific, sodium [40 mg/kg, intraperitoneal (i.p).; Sigma-Aldrich; Inc., Waltham, MA, USA) according to the manufacture's Merck KGaA, Darmstadt, Germany]. A 1.0 cm long incision protocols. Reverse transcription (RT) and PCR were performed was performed on the abdomen and the cecum was exposed, to amplify mouse IL-1, IL-6, TNF-α, MyD88, TRIF, NF-κB ligated by silk 4-0 below the ileocecal valve and punctured and β-actin mRNA. Using 2 µl reverse transcriptase (Promega twice with a 20-gauge needle. The sham group underwent Corporation,