Male Sprague-Dawley rats (weight, 200–250 g; age, 6–8 weeks; n=50), were purchased from Shanghai Experimental Animal Breeding Co. (Shanghai, China). Following adaptation to the environment for 1 week, the rats were randomly divided into five groups: sham (n=10), vehicle (VEH; n=10), LOS (n=10), LOS+IL-6 (n=10) and IL-6 (n=10). In the sham group, the rats received no treatment. The rats were housed in at 22±2°C and 40–60% humidity and light-controlled room 12 h light/dark with free access to water. The experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (17).

Animals were anesthetized with intravenous infusion of sodium pentobarbital [50 mg/kg⁻¹; intraperitoneal (i.p.)]. The rats underwent implantation of PVN cannulae, following which coronary artery ligation was performed to induce HF. At 24 h post-surgery, they were administered with artificial cerebrospinal fluid lasting for 4 weeks in the VEH group. At the same time, the rats were administered with AT1-R antagonist, LOS (200 µg/day) in the LOS group (n=10; Merck Millipore; Darmstadt, Germany). The rats were administered with LOS (200 µg/day) and IL-6 (1 µg/day) in the LOS+IL-6 group (n=10; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The rats were administered with IL-6 (1 µg/day) in the IL-6 group (n=10). Osmotic mini-pumps were implanted subcutaneously and connected with the cannulae for the continuous infusion of LOS, IL-6 and LOS+IL-6 or artificial cerebrospinal fluid directly into the PVN (0.1 ml/h pumping, once a day for 4 weeks). A stainless steel double cannula (Plastics One, Inc., Roanoke, VA, USA) was implanted into the PVN. Left ventricular (LV) function was assessed using echocardiography 24 h following recovery from surgery. At 4 weeks, the rats were anesthetized for echocardiograph examination and were then sacrificed by inducing an air embolism via intravenous infusion of 1 ml air, tissue and plasma for further analyses was subsequently obtained.

Alzet miniosmotic pumps (model 2004; Durect Corporation, Cupertino, CA, USA) were used to enable the continuous infusion of drugs. The rats were placed into a stereotaxic apparatus following anesthesia (sodium pentobarbital; 50 mg/kg⁻¹; i.p.) The method for PVN cannulation has been described previously (7). In brief, a stainless steel double cannula with a center-to-center distance of 0.5 mm was implanted into the PVN (2.0 mm posterior to the bregma and 8.5 mm ventral from the skull surface). HF was induced by ligation of the left anterior descending coronary artery, as previously described. In the sham group, surgery was performed in the same manner without ligating the coronary artery. Following surgery, the animals were administered with benzathine penicillin (30,000 units; IM).

Echocardiography was performed using an Acuson Sequoia clinical imager (Siemens AG, Munich, Germany) fitted with an 8-MHz sector-array probe, which generates 2-dimensional images at a rate of 100/sec. The method for assessment LV function has been described previously (7). The animals were sedated with sodium pentobarbital (25 mg kg⁻¹; i.p.) to facilitate positioning for echocardiographic examination. The animal was positioned in the left lateral recumbent position to optimize the windows for echocardiography. The anterior chest was shaved and pre-warmed acoustic coupling gel was applied. Short-axis images were acquired parallel to the mitral valve plane to obtain the largest cross-sectional image of the LV. Long-axis views were obtained perpendicular to the mitral valve plane. Images were stored for subsequent offline analysis. LV end-diastolic volume (LVEDV), LV end-systolic volume, LV ejection fraction (LVEF), LV stroke volume and LV mass were computed. The region of the LV, which exhibited akinesis was planimetered electronically and expressed as a percentage of the total LV silhouette to estimate the size of the ischemic zone. Only animals with large infarctions (ischemic zone ≥40%) were used in the examination.

The animals were sacrificed by decapitation, and their brains were rapidly removed and frozen on dry ice. Serial sections (300 mm) of the brain were obtained using a cryostat maintained at −10°C. The sections were transferred to coverslips, which were placed on a cold stage set at −10°C. The PVN was microdissected out using Palkovits' microdissection technique (18).

The method for immunohistochemistry has been previously described (19). Paraffin sections of the artery were deparaffinized and endogenous peroxidase activity was inactivated with 3% H₂O₂ for 10 min. The Fra-like (Fra-LI) primary antibody (1:200; cat. no. sc-271657; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or normal blocking serum was added and incubated overnight. Biotin-conjugated goat anti-mouse immunoglobulin G (IgG) (1:1,000; cat. no. 115-035-003; Jackson, ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was used as the secondary antibody and incubated for 30 min at 4°C. An avidin-biotin enzyme reagent was sequentially added and incubated for 20 min. A peroxidase substrate was added and incubated until desired stain intensity developed. Finally, the sections were covered with a glass cover slip and observed under a light microscope. The intensity of positive staining in tissue was analyzed by integrated optical density (IOD) using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). The Fra-LI expression was expressed as (IOD/area)x100 in accordance with a previous study (20).

The method for mRNA analysis has been described previously (19). Total RNA was extracted from the artery specimens using TRIzol^® reagent according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The total RNA (1 µg) was used as a template to produce cDNA using an RT kit (BioDev-Tech Co., Ltd., Beijing, China). The qPCR was performed by monitoring the increase in fluorescence of the SYBR Green dye using GreenMaster mix (Genaxxon BioScience, Ulm, Germany) according to the manufacturer's protocol. The primer sets used to amplify nNOS were 5′-gccatccagcataatgacccag-3′ (sense) and 5′-gagggtgatccaaagatgtcctc −3′ (antisense) (21). The primer sets used to amplify CRH were 5′-cagaacaacagtgcgggctca-3′ (sense) and 5′-aaggcagacagggcgacagag-3′ (antisense) (22). The primer sets used to amplify GAPDH were 5′-ccactttgtgaagctcatttcct-3′ (sense) and 5′-tcgtcctcctctggtgctct-3′ (antisense). PCR amplification was performed with tag polymerase for 32 cycles at 95°C for 45 sec, 62°C for 30 sec and 72°C for 1 min (for CRH, nNOS and GAPDH). The 2^−ΔΔCq method (23) was used to determine relative changes in the gene expression of CRH, nNOS and GAPDH. Values are expressed in relative quantities to the mRNA expression in the control group.

The method for western blot analysis has been described previously (19). The specimens were washed with ice-cold PB and lysed for 20 min on ice with lysis buffer (cat.no. SC-003; Invent, Lund, Sweden). Following lysis, the lysates were centrifuged for 4 min at 394 × g and the supernatants were collected in a fresh tube on ice. The protein concentrations in each sample were determined using a BCA assay. Total proteins (100 µg) were mixed with loading buffer with the anionic denaturing detergent, sodium dodecyl sulfate (SDS), boiled for 5 min and then resolved by 10% SDS polyacrylamide gel electrophoresis. The proteins were transferred onto PVDF membranes. Following blocking of the membranes in TBST containing non-fat milk for 1 h at 4°C under agitation, the membranes were washed three times in TBST and incubated for 2 h at 4°C with anti-rat nNOS antibody (1:200 dilution; cat. no. NB120-3511; Novus Biologicals, Ltd., Cambridge, UK), anti-rat CRH antibody (1:200 dilution; cat. no. NBP1-42614; Novus Biologicals, Ltd.) or GAPDH monoclonal antibody (1:200 dilution; cat. no. NB100-56875; Novus Biologicals, Ltd.). Following washing three times in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:1,000; cat. no. AB501-01A; Novoprotein, Shanghai, China) at temperature for 1 h at 4°C and then washed three times with TBST. The immunobands were detected using a streptavidin amplification reagent (cat. no. WBKL SOO 50; EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol.

The method for the analysis of IL-6 and NE in plasma has been previously described (19). From all animals, fresh blood samples (3 ml) were obtained via the femoral vein and centrifuged at 2,465 × g for 10 min at 4°C. The supernatant was placed in a clean centrifuge tube and frozen at −20°C. Plasma concentrations of IL-6 and NE were assayed using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. The minimum detectable concentration of the kits was <1.0 pg/ml; the variation in different boards was <15%. They were not cross-reactive with other soluble structural analogues.

The data were analyzed using the SPSS 11.5 program (SPSS, Inc., Chicago, IL, USA) for Windows. Quantitative data are presented as the mean ± standard deviation. For comparison between multiple groups, data was analyzed using one-way analysis of variance, and with Student-Newman-Keuls post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

Echocardiography was used to evaluate alterations in LV function (Table I). At 24 h, the LVEDV, LVEDV/mass ratio and % infarction zone (IZ) were higher in the VEH, LOS, IL-6, and LOS+IL-6 groups, compared with those in the sham group. No differences were observed in these parameters among the HF rats assigned to the LOS, IL-6, LOS+IL-6 or the VEH treatment groups at 24 h (P>0.05). At 4 weeks, LVEDV and LVEDV/mass ratio were higher, compared with the 24-h baseline values in the LOS, LOS+IL-6 and vehicle-treated HF rats (P<0.01). At 4 weeks, LVEF was higher in the HF rats in the LOS and LOS+IL-6 treatment groups, compared with the HF rats in the VEH or IL-6 treatment groups (P<0.01). The LVEDV and LVEDV/mass in the HF rats, which received LOS or LOS+IL-6 were lower, compared with those in the rats in the VEH and IL-6 treatment groups (P<0.01), whereas no significant difference were observed between the LOS and LOS+IL-6 groups (P>0.05).

The immunohistochemistry showed that the staining intensity of Fra-LI was minimal in the sham group (Fig. 1). Fra-LI activity is an indicator of chronic neuronal excitation. Lower levels of Fra-LI immunostaining were detected in the LOS and LOS+IL-6 groups, compared with those in the VEH and IL-6 groups (P<0.01). The HF rats treated with IL-6 had increased Fra-LI-positive PVN neurons, compared with those in the VEH, LOS and LOS+IL-6 groups (P<0.01). No significant differences were observed between the LOS and LOS+IL-6 groups (P>0.05; Fig. 2).

In order to investigate the mechanism responsible for alterations in the HF mice, mRNA levels of nNOS were analyzed using RT-qPCR analysis (Fig. 3). Compared with the VEH group, the mRNA expression levels of nNOS were significantly increased in the sham group (P<0.01), LOS group (P<0.05) and LOS+IL-6 group (P<0.05). The HF rats treated with IL-6 had fewer nNOS-positive neurons, compared with the rats treated with LOS+IL-6 (P<0.01). No significant difference the expression of nNOS in PVN neurons was observed between the LOS group and LOS+IL-6 group (P>0.05).

Compared with the sham group, the mRNA expression of CRH was significantly increased in the LOS group, LOS+IL-6 group and VEH group (P<0.01). By contrast, the mRNA expression levels of CRH were decreased in the LOS and LOS+IL-6 groups, compared with that in the IL-6 group (P<0.01). The rats treated with IL-6 had a higher number of CRH-positive neurons, compared with the rats treated with LOS+IL-6 or VEH (P<0.01). No significant difference in the gene expression of CRH in PVN neurons was observed between the LOS group and LOS+IL-6 group (P>0.05; Fig. 3).

Western blot analysis was used to examine whether the protein levels of nNOS correlated with alterations in its mRNA levels (Fig. 4). Compared with the VEH group and IL-6 group, significant increases in the protein expression of nNOS were observed in the sham group, LOS group and LOS+IL-6 group in the blots (P<0.01). The rats treated with IL-6 exhibited lower expression of nNOS, compared with the rats treated with LOS+IL-6 (P<0.05). No differences in the protein expression of nNOS were found between the LOS group and LOS+IL-6 group (P>0.05).

Western blot analysis was used to examine whether the protein levels of CRH were correlated with alterations in its mRNA levels (Fig. 4). Compared with the sham group, significant increases in the expression levels of CRH were observed in the VEH group, LOS group, LOS+IL-6 group and IL-6 group in the blots (P<0.01). The rats treated with IL-6 exhibited higher expression of CRH, compared with the rats treated with LOS+IL-6 (P<0.01). No difference in the protein expression of CRH was observed between the LOS group and LOS+IL-6 group (P>0.05).

Compared with the sham group, the levels of IL-6 and NE were significantly increased following treatment (P<0.01; Fig. 5). Compared with the VEH group, treatment with LOS and LOS+IL-6 significantly decreased the expression of IL-6 and NE (P<0.01), and treatment with IL-6 increased the levels of IL-6 and NE (P<0.05). No significant differences were observed in the levels of IL-6 or NE between the LOS group and LOS+IL-6 group (P<0.05; Fig. 5).