Twenty-four adult female Wistar albino rats weighing 250–350 g were housed at 21–22°C and 60% humidity with a 12-hour light–12-hour dark cycle, and were given free access to standard commercial feed and water. Three groups of eight rats each (control, LPS, and LPS+IRB) were created at random.

In group 1 (control, n=8) 0.5 ml of saline was administered intraperitoneally (ip) from the right inguinal region. One hour after this application, 0.5 ml of saline was administered ip from the right inguinal region.

In group 2 (LPS, n=8), LPS (048K4126; Sigma-Aldrich, Stockholm, Sweden) was administered ip at a dose of 5 mg/kg and in a volume of 0.5 ml from the right inguinal region.20 Solid LPS was dissolved in 0.9% NaCl. One hour after LPS administration, 0.5 ml of saline was administered ip from the right inguinal region.

In group 3 (LPS+IRB, n=8) LPS at a dose of 5 mg/kg was administered ip in a 0.5 ml volume from the right inguinal region. One hour after LPS administration, 3 mg/kg IRB (Karvea, Sanofi-Aventis) was administered ip in a volume of 0.5 ml from the right inguinal region.21

Rats were sacrificed under anesthesia six hours after LPS administration using ketamine and xylazine. For biochemical analysis, blood samples were taken from the inferior vena cava and placed into biochemistry tubes containing gel. Before biochemical and genetic investigation, half of the left ventricles were kept at −80°C. The aorta and the remaining portion of the left ventricles were preserved in 10% buffered formalin for histological and pathological studies.

After being left at room temperature overnight, sections of 5 μm thickness were cut from paraffin blocks, stained with hematoxylin-eosin and examined under a microscope. Microscopic alterations of myocardial tissue were assessed based on the degree of hyperemia, hemorrhage, cloudy degeneration, muscle striation and necrosis. The severity of all histopathological findings was graded semiquantitatively depending on the degree and extent of the alteration, as follows: 0=normal, 1=mild, 2=moderate, and 3=severe. Ten areas from each animal were assessed under a 40× objective.

Subsequently, slices from paraffin blocks were immunohistochemically stained for VEGF (JH121: sc-57496, 1/100 dilution) and cas-3 (anti-caspase-3 antibody [E-8]: sc-7272, 1/100 dilution) (Santa Cruz, Texas, USA) in accordance with the manufacturer's instructions. Following a 60-min incubation period with primary antibodies, the sections underwent immunohistochemistry analysis using the Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (ab80436) (Abcam, Cambridge, UK). Negative controls used antigen dilution solutions instead of primary antibodies. Another pathologist also performed a blind review of the analyses.

For immunohistochemical analysis, sections were separately investigated for each antibody. To classify the severity of the immunohistochemical reaction of cells with markers, semiquantitative analysis was performed using a grading score ranging from 0 to 3 as follows: 0=negative, 1=focal weak staining, 2=diffuse weak staining, and 3=diffuse strong staining. For the assessment, 10 different areas in each section were examined under 40× objective magnification. Morphometric analyses and microphotography were performed using the cellSens Life Science Imaging software system (Olympus Co., Tokyo, Japan). The results were saved and statistically analyzed.

After the collected rat blood was centrifuged, serum was collected and kept at −80°C for later analysis. Serum creatine kinase (CK), creatine kinase-myocardial band (CK-MB), and lactate dehydrogenase (LDH) levels were measured spectrophotometrically (Beckman Coulter, USA). Bar-Or et al.’s method was used to calculate IMA levels to assess serum oxidative stress. The serum samples were spectrophotometrically analyzed at a wavelength of 470 nm, and the outcomes were reported as absorbance units.

Rat heart tissues weighing about 200 mg each were homogenized using an ULTRA-TURRAX T-25 homogenizer (Janke & Kunkel, IKA® Werke, Germany) using phosphate-buffered saline at a ratio of 1:9 (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4). Homogenized heart tissues were then centrifuged for 10 min at 10000rpm. Using Erel's colorimetric method, the supernatants obtained after homogenization with an automatic analyzer (Beckman Coulter, USA) were used to calculate TAS and TOS values. The OSI value was then calculated using the formula OSI=[(TOS, μmol H2O2 equiv./l)/(TAS, mmol Trolox equiv./l)×100].18

RNA was isolated from homogenized heart tissues with the GeneAll Ribospin RNA isolation kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The quantity and purity of RNA samples were determined using a NanoDrop device (Thermo Fisher Scientific, USA). The concentration of each isolated RNA sample was standardized at 500 ng/μl and they were stored at −80°C for use in the cDNA synthesis step. The A.B.T.™ cDNA Synthesis Kit (Atlas Biotechnology, Turkey) protocol was used for cDNA synthesis, which was carried out in a thermal cycler (Thermo Fisher Scientific, USA) with a total reaction volume of 20 μl for each sample. Specific mRNA sequences were found on the US National Center for Biotechnology Information website, and potential primer sequences were then tested (Table 1). The A.B.T.™ 2X real-time quantitative polymerase chain reaction (qPCR) SYBR-Green MasterMix (Atlas Biotechnology, Turkey) was used to assess expression levels using the CFX96 real-time qPCR instrument (Bio-Rad, CA, USA). Real-time qPCR conditions were established according to the manufacturer's instructions. Each sample was run in triplicate, and the GAPDH gene's expression was used to normalize the results.

In the statistical analysis, the histological and immunohistochemical results, serum CK, CK-MB, LDH and IMA, tissue TAS, TOS, and OSI levels, as well as genetic parameters, were compared between the groups. The Shapiro-Wilk W test was used to determine normality of distributions. For the statistical analysis, one-way ANOVA with Bonferroni's test was used with GraphPad Prism v. 5 (GraphPad Software, Inc., La Jolla, CA, USA). The level of significance was set at p<0.05.

The histopathological examination revealed normal heart architecture in the control group. The LPS group presented myocardial cell degeneration, minor hemorrhage, edema, and significant hyperemia, while the LPS+IRB group saw significant recovery after receiving IRB therapy.

Histological analysis of the aorta revealed normal tissue appearance, characterized by a uniformly stained and regularly arranged smooth endothelial layer, in the control group, as well as regular and wavy elastic fibers. The aortas of the LPS group showed loss of and injury to endothelial cells, as well as damage to elastic fibers. IRB markedly improved the histopathological findings in the aortas (Figure 1).

Figure 1.

Representative microscopic images of the heart and aorta in the three groups showing loss of and injury to endothelial cells, as well as damage to elastic fibers (arrows) in the LPS group. CON: control; LPS: lipopolysaccharide; LPS+IBR: lipopolysaccharide and irbesartan.

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Immunohistochemically, a marked increase in cas-3 and VEGF expression was observed in the LPS group, most highly expressed in endothelial and myocardial cells. IRB treatment caused marked amelioration in the LPS+IRB group (Figure 2A and B). The graph in Figure 2C depicts immunohistochemical scoring and statistical analysis of cas-3 and VEGF between groups.

Figure 2.

Immunohistochemical findings for caspase-3 (A) and vascular endothelial growth factor (B) showing moderate myocardial hemorrhage (arrows) and endothelial cell loss (arrows) with elastic fiber damage in the aorta in the LPS group and scoring between groups (C). The differences between the means of the groups with different letter combinations were statistically significant (**p<0.001 and *p<0.05). Scale bars=50 μm; hematoxylin-eosin stain; streptavidin-biotin-peroxidase method. CON: control; LPS: lipopolysaccharide; LPS+IBR: lipopolysaccharide and irbesartan; VEGF: vascular endothelial growth factor.

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Myocardial architecture was normal in the control group. In the LPS group, there was moderate myocardial hemorrhage and endothelial cell loss with elastic fiber damage in the aorta, while histopathological findings improved in the LPS-IRB group.

There was negative expression of VEGF in the control group but a marked increase in myocardial and endothelial cells in the LPS group, and decreased expression in the LPS+IRB group. The differences between the means of the groups with different letter combinations were statistically significant.

CK, CK-MB and LDH, serum levels of which rise as a result of cardiac injury, were significantly increased in the LPS group, while they decreased significantly in the IRB-treated group. TAS, TOS, and OSI levels were used to measure oxidative damage in tissues, with TAS levels being significantly lower in the LPS group compared to the control group, whereas TOS and OSI levels were significantly higher. TAS, TOS, and OSI levels in the LPS+IRB group were also close to those in the control group. In addition, we determined that while IMA, which is an indicator of serum oxidative stress, increased in the LPS group, it decreased significantly in the group treated with IRB (Table 2).

We found that expression of apoptotic genes (BAX and p53) increased and expression of antiapoptotic genes (SIRT1 and Bcl-2) decreased in the LPS group compared to the control group. After treatment with IRB, BAX and p53 expression decreased and SIRT1 and Bcl-2 expression increased significantly, to close to the control level (Figure 3).

Figure 3.

Relative mRNA expression results and statistical comparison between groups. BAX: Bcl-2-associated X protein; BCL2: B-cell lymphoma 2; IRB: irbesartan; LPS: lipopolysaccharide; SIRT1: sirtuin 1. Values are presented as means±standard deviation. *p<0.05, **p<0.001.

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The results of this study showed that LPS damages the heart and aorta, and IRB treatment ameliorates histopathological findings and also has a positive effect on heart damage by reducing oxidative stress and apoptotic effects.