Healthy male Sprague-Dawley (SD) rats (~100 g of body weight) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Approval from the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xinjiang Medical University was obtained prior to all animal experiments.

A total of 50 SD rats were randomly assigned into two groups: a sham group (n=10) and a sepsis group (n=40). All rats were acclimatized for 3–5 days before the experiments. The rat sepsis model was established using CLP according to a previously described (20). CLP induced intra-abdominal infection and the release of LPS, resulting in SIRS and sepsis. Briefly, the rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (3 ml/kg; Sigma, St. Louis, MO, USA) and fixed in a supine position. A 1.5-cm longitudinal incision was made along the abdominal midline, then the caecum was exposed and separated. For rats in the sepsis group, the caecum below the ileocaecal valve was ligated using 3.0 suture. Then, a 18-gauge needle was used to puncture through the caecum 3 times, and the feces was extruded by gently squeezing the caecum. Lastly, the separated caecum was returned to the abdominal cavity and the incision was sutured layer by layer. Rats in the sham group were treated with similar procedures but without ligation or puncture. The entire operation was carried out under aseptic conditions. The rats were sacrificed 24 h post-operatively, and the heart tissues were collected for the following experiments.

CMVECs were isolated from SD rats following a previously reported method (21). In brief, two SD rats were anesthetized and then intraperitoneally injected with heparin (300 IU/g; Sigma) for 20 min. Then, the rats were sacrificed by cervical dislocation. After sterilization with 75% alcohol, the thoracic cavity was opened. Then, the heart was removed and rinsed with sterile D-Hanks' containing heparin. Next, the connective tissue, atria, right ventricle and interventricular septum were removed, and epicardial and endocardial cells from the left ventricle were devitalized by immersing in 75% ethanol for 20–30 sec. The remaining tissue was minced and rinsed with D-Hanks', and then incubated in 0.02% collagenase type II (dissolved in D-Hanks') for 30 min and 0.02% trypsin (both from Sigma) for another 10 min at 37°C. The dissociated cells were centrifuged at 1,000 rpm for 10 min, and then resuspended in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Cambridge, MA, USA) containing 15% fetal bovine serum (FBS). Lastly, the cells were cultured in laminin-coated dishes (Corning, Inc., Corning, NY, USA).

LPS was used to induce the sepsis cell model. To determine the optimum inducement time and concentration, the primary CMVECs were treated with various concentrations (0, 10, 50, 100 ng/ml, 1 and 2 µg/ml) for 0, 3, 6, 9 and 12 h, respectively. To investigate the effect of ulinastatin on the LPS-induced CMVECs, the CMVECs were treated with LPS and LPS + ulinastatin (1000, 3000 or 5000 U/l), respectively. In addition, the treated cells were also transfected with the lncRNA MALAT1 overexpression vector (named as pc-MALAT1; RiboBio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen).

To evaluate cell permeability, transendothelial electrical resistance (TER) was measured using STX2 electrode and EVOM2 meter (World Precision Instruments, Sarasota, FL, USA). CMVECs cells were seeded on fibronectin-coated Transwell membrane inserts (0.4-µm pore size; Corning, Inc.). Then, confluent CMVECs were treated with LPS, LPS + ulinastatin or LPS + ulinastatin + lncRNA MALAT1 overexpression vector, respectively. The value of TER was calculated as the resistance value multiplied by the membrane area.

The Annexin V-FITC apoptosis detection kit (Invitrogen) was used to quantify cell apoptosis. Briefly, the treated cells were collected by digestion with Trypsin and centrifugation at 1,500 rpm for 6 min. After being washed with phosphate-buffered saline (PBS), the cells were mixed with FITC-Annexin V and propidium iodide (PI) for 15 min at 25°C in the dark. Cells were then added to 400 µl 1X binding buffer and detected using a flow cytometer. Annexin V-positive and PI-negative cells were considered as apoptotic cells.

The ROS level was assessed by 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma). After being washed with PBS, the treated cells were incubated with serum-free DMEM containing 10 µM DCFH-DA for 20 min at 37°C in the dark. Next, the cells were collected and analyzed using a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The fluorescent intensities were measured at a 535 nm absorbance.

Total RNA extraction was carried out using TRIzol reagent (Invitrogen), and complementary DNA was obtained by the reverse transcription of total RNA using the MultiScribe RT kit (Applied Biosystems, Foster City, CA, USA). Then, the levels of target genes, including lncRNA MALAT1, B-cell lymphoma-2 (Bcl-2), Bax, caspase-3, EZH2, DAB2IP and Brachyury were detected using SYBR^® Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Dalian, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal reference. Primers for these genes are listed in Table I. The relative quantification of these genes was carried out using the comparative threshold (Ct) cycle method (2^−ΔΔCt).

RIPA buffer (Sangon Biotech, Shanghai, China) was used to extract protein from the heart tissues and cells. The measurement of protein concentration was conducted using BCA Protein Quantitative assay (Pierce, Appleton, WI, USA). Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then blotted onto nitrocellulose membranes (Millipore, Billerica, MA, USA). After being blocked with 5% nonfat milk in Tris-buffered saline Tween-20 (TBST), the membranes were incubated with rat anti-Bcl-2, Bax, caspase-3, EZH2, DAB2IP, Brachyury and GAPDH monoclonal antibodies (1:500–1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C, respectively. After being washed with TBST, the membranes were incubated with goat anti-rat secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Ultimately, the proteins were detected with Enhanced chemiluminescence (Millipore, Bedford, MA, USA).

Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore) was used to perform RIP assay following the manufacturer's instructions. In brief, CMVECs were treated with LPS for 6 h. Then, the cells were collected and treated with RIPA buffer. After centrifugation, the supernatant was incubated with protein-A/G-Sepharose beads, anti-EZH2 monoclonal antibody and IgG overnight at 4°C. Subsequently, co-precipitated RNA was extracted and then detected by RT-qPCR.

ChIP assay was carried out using QuikChIP kit (Imgenex, San Diego, CA, USA). In brief, CMVECs were transfected with two siRNAs targeting MALAT1 (si-MALAT1-1 and si-MALAT1-2; Dharmacon, Mickleton, NJ, USA) using Lipofectamine 2000 for 48 h. Then, protein-chromatin cross-linking of cells was induced using 1% formaldehyde for 10 min at 37°C and quenched by glycine. Next, the cells were lysed in lysis buffer and chromatin DNA from cell lysates was sonicated into fragments. Next, chromatin DNA fragments were subjected to immunoprecipitation with EZH2 or methyltransferase catalyzing histone H3 lysine 27 trimethylation (H3K27me3) antibody. After reverse cross-linking, DNA fragments were purified and then detected by RT-qPCR.

Statistical analyses were carried out using SPSS 19.0 (IBM Corp., Armonk, NY, USA). The results of all experiments are expressed as the mean ± SD and analyzed by t-test. A statistically significant difference was defined as a p-value <0.05.

The results of TER showed that compared with the untreated cells, cell permeability was significantly increased (p<0.05) in the LPS-induced cells in a dose- and time-dependent manner (Fig. 1A and B). Notably, cell permeability was elevated when the dose of LPS exceeded 50 ng/ml and the treatment time was >6 h (Fig. 1A and B). Thus, the optimum inducement time and concentration was determined as 6 h and 50 ng/ml. In addition, the presence of ulinastatin markedly inhibited (p<0.05) cell permeability in LPS-induced cells, and 5,000 U/l ulinastatin led to the more obvious improvement (Fig. 1C).

Next, we analyzed the effect of ulinastatin on CMVEC apoptosis. The results revealed that compared with the untreated cells, the percentage of apoptotic cells was markedly increased in the LPS-induced cells (p<0.01), while it was obviously decreased after treatment with ulinastatin (p<0.05) (Fig. 2A). Meanwhile, the expression levels of cell apoptosis-related proteins, including Bcl-2, caspase-3 and Bax, were detected by RT-qPCR and western blotting, respectively. The results showed that LPS-induced cells showed a lower Bcl-2 level as well as higher Bax and caspase-3 levels than the untreated cells (p<0.05) (Fig. 2B). After treatment with ulinastatin, the mRNA and protein levels of Bcl-2 were significantly increased, and the levels of caspase-3 and Bax were markedly decreased in the LPS-induced cells (p<0.05) (Fig. 2B). Also, compared with the untreated cells, LPS induced a higher ROS level (p<0.01) (Fig. 2C), while ulinastatin markedly inhibited the increase in ROS level induced by LPS (p<0.05) (Fig. 2C).

To investigate the mechanism of ulinastatin in LPS-induced CMVECs, the expression levels of lncRNA MALAT1 and EZH2 were detected. The results revealed that the levels of lncRNA MALAT1 and EZH2 were significantly upregulated in the hearts of rat from the sepsis group in comparison with these levels in the sham group (p<0.01) (Fig. 3A). Similarly, in vitro experiments showed that LPS markedly elevated the expression levels of lncRNA MALAT1 and EZH2 compared with the levels in the untreated cells (p<0.01) (Fig. 3B), while ulinastatin inhibited the upregulation of lncRNA MALAT1 and EZH2 (p<0.05) (Fig. 3B).

RTP assay was performed to evaluate the relationship between lncRNA MALAT1 and EZH2. The results revealed that lncRNA MALAT1 could bind to EZH2 (Fig. 4A), indicating the interaction of lncRNA MALAT1 and EZH2. Then, the lncRNA MALAT1 level was successfully inhibited by the transfection of si-MALAT1-1 and si-MALAT1-2 (p<0.05) (Fig. 4B). Silencing of lncRNA MALAT1 markedly inhibited the expression of EZH2 target genes, DAB2IP and Brachyury (p<0.05) (Fig. 4B) compared with that in the untreated cells. Furthermore, the effect of lncRNA MALAT1 on EZH2 was evaluated by ChIP assay. The results revealed that when lncRNA MALAT1-knockdown cells were precipitated with EZH2 or H3K27me3 (the composition of EZH2) antibody, the expression levels of DAB2IP and Brachyury were both inhibited compared with levels in the untreated cells (p<0.05) (Fig. 4C and D).

The above results showed that ulinastatin induced the downregulation of lncRNA MALAT1 in the LPS-induced cells. Thus, we investigated the effect of overexpression of lncRNA MALAT1 on CMVEC permeability and apoptosis following treatment with ulinastatin and LPS. Compared with the untreated cells, the lncRNA MALAT1 level was significantly increased (p<0.01) (Fig. 5A) in cells transfected with pc-MALAT1. Then, the results revealed that upon treatment with ulinastatin, upregulation of lncRNA MALAT1 markedly increased CMEC permeability (Fig. 5B) and apoptosis (Fig. 5C) in the LPS-induced cells (all p<0.05).