Role of activating the nuclear factor kappa B signaling pathway in the development of septic cardiomyopathy in rats with sepsis

Abstract

BACKGROUND:

Despite advances in the treatment of sepsis over time, this condition remains both a serious threat and a cause of death among critical patients.

OBJECTIVE:

This study aimed to explore the role of the nuclear factor kappa B (NF- $\kappa$ B) signaling pathway in the development of septic cardiomyopathy in rats with sepsis.

METHOD:

A total of 32 Sprague Dawley rats were randomized into a sham operation group and three groups with sepsis, which were tested at one of the following time-points: 3, 6, or 12 h. Each group included eight rats. Sepsis models were created via cecal ligation and puncture procedures. All the study rats had the following cardiac parameters and serum levels measured at either 3, 6, or 12 h after the operation (according to their assigned group): heart rate, left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure rise ( $+$ dP/dt ${}_{\max}$ ) and fall ( $-$ dP/dt ${}_{\max}$ ), tumor necrosis factor alpha (TNF- $\alpha$ ), interleukin 1 beta (IL-1 $\beta$ ), interleukin 6 (IL-6), and cardiac troponin I (cTnI). The myocardium of the left ventricle was collected and subjected to hematoxylin and eosin staining to observe the changes in pathological morphology. The expression of toll-like receptor 4 (TLR4) and NF- $\kappa$ B in the myocardium were detected by western blot analysis.

RESULTS:

Compared with the sham operation group, the rats in the sepsis subgroups exhibited significantly lower values for all the cardiac parameters measured, including the heart rate (sham operation group $=$ 386.63 $\pm$ 18.62 beats per minute [bpm], sepsis 3-h group $=$ 368.38 $\pm$ 12.55 bpm, sepsis 6-h group $=$ 341.75 $\pm$ 17.05 bpm, sepsis 12-h group $=$ 302.13 $\pm$ 21.15 bpm), LVSP (sham operation group $=$ 125.50 $\pm$ 11.45 mmHg, sepsis 3-h group $=$ 110.88 $\pm$ 7.51 mmHg, sepsis 6-h group $=$ 100.00 $\pm$ 15.06 mmHg, sepsis 12-h group $=$ 91.38 $\pm$ 14.73 mmHg), $+$ dp/dtmax (sham operation group $=$ 7137.50 $\pm$ 276.44 mm Hg/sec, sepsis 3-h group $=$ 5745.00 $\pm$ 346.16 mm Hg/sec, sepsis 6-h group $=$ 4360.00 $\pm$ 312.04 mm Hg/sec, sepsis 12-h group $=$ 2871.25 $\pm$ 443.99 mm Hg/sec), and $-$ dp/dtmax (sham operation group $=$ 6363.75 $\pm$ 123.86 mm Hg/sec, sepsis 3-h group $=$ 6018.75 $\pm$ 173.49 mm Hg/sec, sepsis 6-h group $=$ 5350.00 $\pm$ 337.89 mm Hg/sec, sepsis 12-h group $=$ 4085.00 $\pm$ 326.76 mm Hg/sec). They also displayed significantly higher levels of serum cytokines, including TNF- $\alpha$ (sham operation group $=$ 14.72 $\pm$ 2.90 pg/mL, sepsis 3-h group $=$ 34.90 $\pm$ 4.79 pg/mL, sepsis 6-h group $=$ 24.91 $\pm$ 2.57 pg/mL, sepsis 12-h group 22.06 $\pm$ 3.11 pg/mL), IL-1 $\beta$ (sham operation group $=$ 42.25 $\pm$ 16.91, 3-h group $=$ 112.25 $\pm$ 13.77, sepsis 6-h group $=$ 207.90 $\pm$ 22.64, sepsis 12-h group $=$ 157.18 $\pm$ 23.06), IL-6 (sham operation group $=$ 39.89 $\pm$ 5.74, sepsis 3-h group $=$ 78.27 $\pm$ 9.31, sepsis 6-h group $=$ 123.75 $\pm$ 13.11, sepsis 12-h group $=$ 93.21 $\pm$ 8.96), and cTnI (sham operation group $=$ 0.07 $\pm$ 0.03 ng/mL, sepsis 3-h group $=$ 0.18 $\pm$ 0.06 ng/mL, sepsis 6-h group $=$ 0.67 $\pm$ 0.19 ng/mL, sepsis $=$ 12-h group 1.28 $\pm$ 0.10 ng/mL). The rats in the sepsis groups exhibited pathological changes in the myocardium, which deteriorated gradually over time. The animals in all the sepsis groups exhibited significantly higher levels of TLR4 and NF- $\kappa$ B protein expression compared with the sham group. The TLR4 protein expressions were 0.376 in the sham operation group, 0.534 in the sepsis 3-h group, 0.551 in the sepsis 6-h group, and 0.719 in the sepsis 12-h group. The NF- $\kappa$ B protein expressions were 0.299 in the sham operation group, 0.488 in the sepsis 3-h group, 0.516 in the sepsis 6-h group, and 0.636 in the sepsis 12-h group.

CONCLUSION:

Sepsis can lead to myocardial injury and cardiac dysfunction. This may be related to the activation of the NF- $\kappa$ B intracellular signal transduction pathway and the release of inflammatory factors as a result of lipopolysaccharides acting on TLR4 during the onset of sepsis.

Keywords

Septic cardiomyopathy TLR4 NF-κB signaling pathway tumor necrosis factor-α interleukin-1β interleukin-6

1. Background

Sepsis is a life-threatening organ dysfunction that results from a host’s disordered response to an infection [1]. Despite advances in the treatment of sepsis over time, this condition remains both a serious threat and a cause of death among critical patients. This condition involves multiple organ dysfunction and accounts for 6% of inpatients and 15% of hospital deaths [2]. Epidemiological studies have shown that the prevalence of septic cardiomyopathy (SCM) in general population varies from 10% to 70%. The two main factors that may explain this considerable difference in reported prevalence are a lack of consensus on the diagnostic criteria for SCM and the diversity of techniques used to evaluate cardiac function [3]. The prevalence of SCM increases as the severity of the sepsis increases [4, 5]. Septic cardiomyopathy is characterized by left ventricular dilatation and a reduced left ventricular ejection fraction, with a return to normal cardiac function within 7–10 days [6]. As a reversible myocardial dysfunction in patients with sepsis, SCM has gradually gained more attention. For this reason, research into the pathogenesis, prevention, and treatment of this disease is clinically important and has the potential to improve the prognosis of patients with sepsis and to lower their overall mortality rate.

The pathogenesis of SCM is extremely complicated and is still not entirely understood. The occurrence of the disease is associated with several factors, including the body’s immune response, cytokine production, mitochondrial dysfunction, oxidative stress injury, the presence of exosomes in cardiomyocytes, cell apoptosis, and gene regulation [7, 8, 9]. Inflammatory cytokines in particular may play a major role in the pathogenesis of SCM. At the time of sepsis onset, lipopolysaccharides (LPSs) are delivered to the surface of a variety of immune cells, such as monocyte macrophages, and bind to the CD14/TLRs-MD2 complex on the membrane, activating nuclear factor kappa B (NF- $\kappa$ B) and other intracellular signaling pathways. Studies have shown that the activation of the NF- $\kappa$ B signaling pathway is associated with an increase in sepsis morbidity and mortality [10]. The activation of NF- $\kappa$ B may also promote myocardial dysfunction caused by sepsis [11].

Due to the complexity and heterogeneity of systemic immune response in sepsis, it is important to develop appropriate animal models and optimize research conditions to reveal the biological mechanism of sepsis. Cecal ligation and puncture (CLP) is a standard animal model of sepsis [12], which is usually used to study the pathogenesis and treatment target of sepsis. It is a simple and reliable method to control the puncture count in the CLP mouse model, which can achieve a consistent survival rate. The contents of the cecum continue to diffuse into the abdominal cavity, causing bacteria to enter the blood, eventually leading to systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Therefore, the CLP mouse model can partially represent the progress of pathophysiological phenomena similar to human, such as the changes in the early stage of inflammation, and show cytokine dynamics similar to clinical sepsis [13, 14]. However, CLP model can only reproduce the immune, hemodynamic and metabolic stages of clinical sepsis to a certain extent. Whether it can simulate human sepsis is still unknown. Compared with the diagnostic criteria of human sepsis, the basic parameters and indicators of CLP model still lack comprehensive understanding [15].

In this study, sepsis rat models were created using the cecal ligation and puncture (CLP) method to explore the role of NF- $\kappa$ B signaling pathway activation in the onset and development of SCM.

2. Method and materials

2.1 Preparing the animals and the sepsis model for the experiment

A total of 32 healthy adult male Sprague Dawley rats (specific pathogen-free grade), each with a body weight of 350–420 g, were provided by the animal laboratory of Xuanwu Hospital, Capital Medical University, Beijing, China. The rats had been raised in a standard pathogen-free animal room, with 12 h alternate light/dark cycles before the experiment; water and standard rodent feed were provided without restriction. Before the experiment, the animals were acclimated to the environment where the study was conducted for one week. Based on recommendations derived from international expert consensus on preclinical sepsis research, the CLP sepsis model was used for this study [16, 17].

Before the surgery, the animals were anesthetized using an intraperitoneal administration of 20% urethane (5 mL/kg). The effect of urethane is reversible when used in the concentration range of 10–300 mmol/L [18]. The lower quadrants of the abdomen were shaved using a trimmer, and the area was disinfected with alcohol prep pads. Next, a longitudinal skin midline incision was made with a scalpel under aseptic conditions, avoiding any penetration of the peritoneal cavity. After the initial incision, small scissors were used to increase the length of the incision to 3–4 cm and to enter the peritoneal cavity. The cecum was located, isolated, and exteriorized using blunt anatomical forceps. The ligation was achieved with surgical sutures at the point that marked the beginning of the distal third of the cecum. Before cecal perforation, the cecal contents were pushed gently toward the distal cecum. The cecum was perforated by three through-and-through punctures with a sterile 20-mL syringe needle in a mesenteric-to-antimesenteric direction, midway between the ligation point and the tip of the cecum. After needle removal, a drop of stool was extruded from the mesenteric puncture and the reverse mesenteric puncture to ensure patency. The cecum was then returned to the abdominal cavity. Next, the incision was sterilized again with iodophor, and the abdominal wall incision was sutured layer by layer. The animals were administered 5 mL of normal saline subcutaneously after the procedure as a means of preventive fluid replacement.

2.2 Experimental grouping

All the rats were fasted overnight for 12 h before the experiment, although they had free access to water. They were randomized into four groups, each of which contained eight animals, as described in the following paragraphs.

(1)
The sham surgery group ( $n=$ 8): The animals in this group were only laparotomized for cecum examination, and CLP was not performed. Subsequently, they were monitored for cardiac function at 12 h following the closure of the abdomen. In addition, serum samples and myocardial tissue were collected for testing at the same time point.
(2)
Subgroups examining the response to sepsis at 3 ( $n=$ 8), 6 ( $n=$ 8), and 12 h ( $n=$ 8): Sepsis rat models were created via CLP and randomized into three subgroups. The animals in these groups were monitored for cardiac function at 3, 6, or 12 h after CLP. In addition, serum specimens and myocardial tissue were collected for testing at the time point assigned by their subgroup.

2.3 Measuring cardiac function in rats

The anesthetized rats were fixed in a supine position; a polyethylene catheter, connected to a BL-420F biological signal acquisition system (Chengdu TME Technology Co. Ltd., Chengdu, China), was inserted through the common carotid artery into the left ventricle. The following indices were recorded once the rats’ condition had been stabilized: heart rate (HR), left ventricular systolic pressure (LVSP), and the maximum rate of left ventricular pressure rise ( $+$ dP/dt ${}_{\max}$ ) and fall ( $-$ dP/dt ${}_{\max}$ ).

2.4 Collecting the serum specimens and measuring cardiac troponin I, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6

The rats in all the groups were subjected to blood sampling (5 mL) via the arterial line after their cardiac function measurements had been completed at the assigned time point (3, 6, or 12 h after CLP), and the blood samples were stored at $-$ 20 ${}^{\circ}$ C following centrifugation. The serum levels of cardiac troponin I (cTnI), tumor necrosis factor alpha (TNF- $\alpha$ ), interleukin 1 beta (IL-1 $\beta$ ), and interleukin 6 (IL-6) were ascertained using a sandwich enzyme-linked immunosorbent assay (kit supplied by Neobioscience, Shenzhen, China).

2.5 Preparation of the collected myocardial tissue for histopathological examination and western blot analysis to determine the expression of toll-like receptor 4 and nuclear factor kappa B

After the serum samples had been collected from the rats in the study, their chests were opened and their left ventricular myocardium was collected. A small part of the myocardium was stored in liquid nitrogen for subsequent western blot analysis to determine the levels of toll-like receptor 4 (TLR4) and NF- $\kappa$ B expression. The remaining tissue was fixed in 10% formalin and stained with hematoxylin and eosin for histopathological examination.

2.6 Statistical methods

The measurement data were expressed as mean $\pm$ standard deviation. The data were analyzed using the IBM SPSS Statistics software program (version 22.0). An independent sample $t$ -test and a one-way analysis of variance were conducted to compare the means of two and of multiple samples, respectively; a $P$ -value below 0.05 was considered statistically significant.

3. Results

3.1 General condition of the rats in each group

The rats in the sham operation group were essentially healthy following recovery from anesthesia, with slow movement and acceptable reactions. They were capable of eating and drinking at will, showed an absence of obvious abnormality in their second stool samples, and the color and luster of their fur were normal. The rats in the sepsis groups exhibited mental fatigue, a significant decrease in activity, a reduced reaction capacity, rigors, opaque discharge around the eyes, and noticeably darker urine following recovery from anesthesia. Observation by laparotomy, following the death of the sepsis rats, found pale and bloody peritoneal exudate, a fecal odor, intestinal wall congestion, and dark purple edema (and purple-black necrosis in severe cases) in the ligated segment of the cecum. The above findings were most obvious in the 12-h group.

3.2 Mortality of the rats in each group

All the rats in the sham operation group survived (mortality $=$ 0). In the 3, 6, and 12-h sepsis groups, 0, 2, and 4 rats, respectively, died naturally. This yielded mortality rates of 0%, 25%, and 50%. The overall mortality for the sepsis groups was 25% (the rats in each group that did not survive to the observation time point were replenished).

3.3 Hemodynamic results of the rats in each group

The HR of the rats in the three sepsis groups was much lower than those in the sham operation group (sham operation group $=$ 386.63 $\pm$ 18.62 beats per minute [bpm], sepsis 3-h group 368.38 $\pm$ 12.55 bpm, sepsis 6-h group $=$ 341.75 $\pm$ 17.05 bpm, sepsis 12-h group $=$ 302.13 $\pm$ 21.15 bpm), and it gradually decreased over time. The difference in HR between the sham operation group and the sepsis groups was statistically significant ( $P<$ 0.05) (see Table 1).

Table 1
HR (times/min) and LVSP (mmHg) of rats in each group

Item	Sham operation group	Sepsis 3 h group	Sepsis 6 h group	Sepsis 12 h group
HR (times/min)	386.63 $\pm$ 18.62	368.38 $\pm$ 12.55 ${}^{*}$	341.75 $\pm$ 17.05 ${}^{**}$	302.13 $\pm$ 21.15 ${}^{**}$
LVSP (mmHg)	125.50 $\pm$ 11.45	110.88 $\pm$ 7.51 ${}^{**}$	100.00 $\pm$ 15.06 ${}^{**}$	91.38 $\pm$ 14.73 ${}^{**}$

Note: $P<$ 0.05 ${}^{*}$ ; $P<$ 0.01 ${}^{**}$ . The data were subjected to statistical analysis using the SPSS Statistics 22.0 software program. An independent sample $t$ -test and a one-way analysis of variance were conducted to compare the means of two and multiple samples, respectively. HR: heart rate; LVSP: left ventricular systolic pressure.

The LVSP values of the rats in the three sepsis groups were much lower than those in the sham operation group (sham operation group $=$ 125.50 $\pm$ 11.45 mmHg, sepsis 3-h group $=$ 110.88 $\pm$ 7.51 mmHg, sepsis 6-h group $=$ 100.00 $\pm$ 15.06 mmHg, sepsis 12-h group $=$ 91.38 $\pm$ 14.73 mmHg), and they gradually decreased over time. The difference in LVSP values between the sham operation group and the sepsis groups was statistically significant ( $P<$ 0.05) (see Table 1).

The $\pm$ dp/dt ${}_{\max}$ values of the rats in the three sepsis groups were much lower than those in the sham operation group ( $+$ dp/dtmax: sham operation group $=$ 7137.50 $\pm$ 276.44 mm Hg/sec, sepsis 3-h group $=$ 5745.00 $\pm$ 346.16 mm Hg/sec, sepsis 6-h group $=$ 4360.00 $\pm$ 312.04 mm Hg/sec, sepsis 12-h group $=$ 2871.25 $\pm$ 443.99 mm Hg/sec; -dp/dtmax: sham operation group $=$ 6363.75 $\pm$ 123.86 mm Hg/sec, sepsis 3-h group $=$ 6018.75 $\pm$ 173.49 mm Hg/sec, sepsis 6-h group $=$ 5350.00 $\pm$ 337.89 mm Hg/sec, sepsis 12-h group $=$ 4085.00 $\pm$ 326.76 mm Hg/sec), and they gradually decreased over time. The differences in the $\pm$ dp/dt ${}_{\max}$ values between the sham operation group and the sepsis groups were statistically significant ( $P<$ 0.05) (see Table 2).

Table 2

$\pm$ dp/dt ${}_{\max}$ of rats in each group (mmHg/ms)

Cardiac function	Sham operation group	Sepsis 3 h group	Sepsis 6 h group	Sepsis 12 h group
$+$ dp/dt ${}_{\text{max value}}$	7137.50 $\pm$ 276.44	5745.00 $\pm$ 346.16 ${}^{**}$	4360.00 $\pm$ 312.04 ${}^{**}$	2871.25 $\pm$ 443.99 ${}^{**}$
$-$ dp/dt ${}_{\text{max value}}$	6363.75 $\pm$ 123.86	6018.75 $\pm$ 173.49 ${}^{**}$	5350.00 $\pm$ 337.89 ${}^{**}$	4085.00 $\pm$ 326.76 ${}^{**}$

Note: $P<$ 0.05 ${}^{*}$ ; $P<$ 0.01 ${}^{**}$ . The data were subjected to statistical analysis using the SPSS Statistics 22.0 software program. An independent sample $t$ -test and a one-way analysis of variance were conducted to compare the means of two and multiple samples, respectively. $+$ dP/dtmax: maximum rate of pressure rise; $-$ dP/dtmax: maximum rate of pressure fall.

3.4 Test results of the samples of rat serum in each group

The mean serum TNF- $\alpha$ levels of the rats in the three sepsis groups were significantly higher than the mean levels of the sham operation group ( $P<$ 0.05), with the 3-h group demonstrating the highest level. The mean serum IL-1 $\beta$ levels of the rats in the three sepsis groups were significantly higher than the mean levels in the sham operation group ( $P<$ 0.05), with the 6-h group demonstrating the highest level. The mean serum cTnI levels of the rats in the three sepsis groups were significantly higher than the mean levels in the sham operation group ( $P<$ 0.05), and it gradually increased over time (see Table 3).

Table 3
Test results of rat serum samples in each group

Item	Sham operation group		Sepsis 3 h group		Sepsis 6 h group		Sepsis 12 h group
TNF- $\alpha$ value (pg/mL)	14.72	$\pm$ 2.90	34.90	$\pm$ 4.79 ${}^{**}$	24.91	$\pm$ 2.57 ${}^{**}$	22.06	$\pm$ 3.11 ${}^{**}$
IL-1 $\beta$ value (pg/mL)	42.25	$\pm$ 16.91	112.25	$\pm$ 13.77 ${}^{**}$	207.90	$\pm$ 22.64 ${}^{**}$	157.18	$\pm$ 23.06 ${}^{**}$
IL-6 value (pg/mL)	39.89	$\pm$ 5.74	78.27	$\pm$ 9.31 ${}^{**}$	123.75	$\pm$ 13.11 ${}^{**}$	93.21	$\pm$ 8.96 ${}^{**}$
cTNI value (ng/mL)	0.07	$\pm$ 0.03	0.18	$\pm$ 0.06 ${}^{*}$	0.67	$\pm$ 0.19 ${}^{**}$	1.28	$\pm$ 0.10 ${}^{**}$

Figure 1.

Results of the histopathology sections of rat myocardium in each group. A: Sham operation group, B: Sepsis 3-h group, C: Sepsis 6-h group, D: Sepsis 12-h group.

3.5 Histopathological findings from the rat myocardium samples from each group

Compared with the sham operation group, the sepsis groups exhibited granular degeneration of the myocardial fibers, edema, and faded staining. These pathological changes were more pronounced at each subsequent time point, and some of the specimens from the 12-h group demonstrated myocyte necrosis and the disintegration and fracture of muscle fibers (see Fig. 1).

3.6 Western blot analysis results of toll-like receptor 4 and nuclear factor kappa B expression in the myocardium for each group

Figure 2.

Expression of proteins of TLR4 and NF- $\kappa$ B in the myocardium of rats from different groups. Representative western blotting images showing protein expression of TLR4 and NF- $\kappa$ B (A1) in the myocardium of rats from each group. Results from the western blot analysis showing TLR4 (B1) and NF- $\kappa$ B (B2) expression. Protein expression is shown as relative densitometric absorption units (left axis). Data are expressed as the mean $\pm$ standard deviation (each group, $n=$ 6). ${}^{*}P<$ 0.05 vs. sham group; ${}^{\#}P<$ 0.05 vs. 3-h group and 6-h group. TLR4, toll-like receptor 4; NF- $\kappa$ B, nuclear factor-KB. A1: Sham operation group; sepsis 3-h group; sepsis 6-h group; sepsis 12-h group.

As shown in Fig. 2, the western blot analysis reflected a weak expression of both TLR4 and NF- $\kappa$ B in the sham operation group with weak bands.

The animals in all the sepsis groups exhibited significantly higher levels of TLR4 and NF- $\kappa$ B protein expression compared with the sham group. The TLR4 protein expressions were 0.376 in the sham operation group, 0.534 in the sepsis 3-h group, 0.551 in the sepsis 6-h group, and 0.719 in the sepsis 12-h group. The NF- $\kappa$ B protein expressions were 0.299 in the sham operation group, 0.488 in the sepsis 3-h group, 0.516 in the sepsis 6-h group, and 0.636 in the sepsis 12-h group.

In the sepsis groups, the relative expression of TLR4 and NF- $\kappa$ B gradually increased over time, and the 12-h group displayed the highest level. The difference in expression was statistically significant when compared with the sham operation group ( $P<$ 0.05). Additionally, the relative expression of TLR4 in the 12-h sepsis group was significantly higher than in the 3 and 6-h groups ( $P<$ 0.05).

4. Discussion

Complications associated with sepsis include multiple organ dysfunction that can involve the heart, lungs, and kidneys [14]. Studies have shown SCM to be independently associated with early mortality and morbidity in cases of cardiovascular disease following discharge [15]. Septic cardiomyopathy is characterized by myocardial systolic/diastolic dysfunction, cardiac enlargement, reduced ejection fraction, poor systolic response to volume loading, and a reduced peak systolic pressure/end-systolic volume ratio [16]. Approximately 50% of patients with SCM suffer from diastolic cardiac dysfunction, which is closely linked with high mortality rates [17].

In the current study, the hemodynamic parameters of the rats in each group were measured using left ventricular catheterization. The LVSP and $+$ dp/dt ${}_{\max}$ values are common indicators that can sensitively reflect myocardial systolic function, and $-$ dp/dt ${}_{\max}$ is a typical indicator of myocardial diastolic function [18]. The results of this study suggest that ventricular systolic and diastolic functions were reduced in the early stages of sepsis. As the course of the disease progressed with time, the cardiac dysfunction in the rats was gradually exacerbated, resulting in a much higher mortality rate.

The cTnI is a specific marker of myocardial injury and is secreted into the blood immediately after the myocardial cell membrane is damaged [15, 19]. Existing studies reveal that the release of cTnI during sepsis is highly associated with left ventricular dysfunction and a poor prognosis [20, 21, 22]. Sepsis-induced cardiac dysfunction and a reduced cardiac output lead to reduced tissue perfusion, thereby increasing the risk of death. Abraham et al. [15] suggest that 67% of septic patients in intensive care units experienced an elevated cTnI level within days of hospitalization, indicating myocardial injury. This was closely related to early mortality and postdischarge cardiovascular events. In the current study, all sepsis groups recorded an elevated cTnI, beginning in the 3-h group and peaking in the 12-h group, suggesting that myocardial cell damage occurred in the early stage of sepsis. As the sepsis progressed with time, the damage gradually worsened, and the cardiac diastolic and systolic functions decreased. Thus, an elevated cTnI level during sepsis serves as an important indicator that patients are at high risk of experiencing cardiovascular complications.

Histological examination can verify the presence of LPS-mediated myocardial injury. In the present study, the myocardial specimens of some rats in the 3-h sepsis group were observed to have granular degeneration of the myocardial fibers, edema, faded staining, inflammatory-cell infiltration, and other pathological changes on histopathological examination, which became more pronounced over time. Some myocardial specimens from rats in the 12-h group showed severe degeneration of the muscle fibers, edema, an increase in muscle fibril gaps, and the necrosis and disintegration of individual muscle cells, with a large amount of inflammatory-cell infiltration at the focal necrosis site. This is consistent with existing study findings [23].

Cascades of inflammatory factors play a key role in the onset and development of sepsis. As a bacterial product, LPS can stimulate monocytes, neutrophils, endothelial cells, and cardiomyocytes to initiate the inflammatory process and to produce proinflammatory cytokines. The stimulation of proinflammatory cytokine production by LPS is also one of the major underlying mechanisms of SCM [23, 24]. For example, it can lead to TNF- $\alpha$ being secreted in the early stage of sepsis, and an elevated concentration of this cytokine creates an environment in which SCM is more likely to occur. As a trigger factor for inflammation, TNF- $\alpha$ precipitates an inflammatory cascade reaction by inducing the production of various inflammatory factors, such as IL-1 $\beta$ and IL-6. This cascade leads to a sharp drop in the concentration of calcium ions within the sarcoplasmic reticulum during myocardial contraction, giving rise to myocardial systolic and diastolic dysfunction [19, 25]. According to observations, in the early stage, the rats with LPS-induced sepsis suffered from a remarkably higher concentration of inflammatory cytokines, including TNF- $\alpha$ , IL-1 $\beta$ , and IL-6, accompanied by systolic and diastolic dysfunction.

Although inflammatory cytokines play an important role in sepsis-related myocardial damage, studies indicate that the prognosis of the disease cannot be improved by interventions involving one specific factor alone [15]. However, the myocardial damage caused by sepsis may be effectively relieved by intervening in the initial stages of inflammatory cascades. This can be achieved by targeting the initial event that generated inflammatory cytokines and caused signal transduction pathways to make changes in the cells. Toll-like receptors are commonly linked to the increased production of proinflammatory cytokines, and TLR4 is a type-I transmembrane protein and an LPS receptor that is responsible for signal transmission; it plays an important role in the initiation of the inflammatory response to sepsis [26]. Nuclear factor kappa B, a transcription factor, is crucial to various conditions, such as the autoimmune and inflammatory responses and atherosclerosis. In a resting state, NF- $\kappa$ B and its inhibitor, NF- $\kappa$ light polypeptide gene enhancer in B-cells inhibitor alpha (I $\kappa$ B $\alpha$ ), form trimers in the cytoplasm and are inactive. When TLR4 is activated, it promotes the degradation of I $\kappa$ B $\alpha$ and the phosphorylation of the NF- $\kappa$ B p65 subunit to activate NF- $\kappa$ B. The activated NF- $\kappa$ B is rapidly transferred from the cytoplasm to the nucleus. This initiates the transcription and, subsequently, the production of various inflammatory cytokines, including TNF- $\alpha$ , IL-6, IL-10, endothelin-1, complement C5a, and lysosomes. Ultimately, inflammatory cytokines interact to generate a cascade of reactions [26]. Studies have shown that the phosphorylation levels of NF- $\kappa$ B and I $\kappa$ B $\alpha$ are related to the severity of the inflammatory response [27, 28]. As a result of the response, inflammatory cells infiltrate the myocardial tissue, resulting in myocardial depression and cardiac dysfunction [29]. Therefore, the activation of the TLR4/NF- $\kappa$ B signaling pathway is closely associated with elevated levels of proinflammatory cytokines both in the plasma and in tissues, and it is essential in the process of exacerbating sepsis and sepsis-induced myocardial depression [30, 31]. In this study, time-related progression of the sepsis led to an increase in the expression of TLR4 and NF- $\kappa$ B, and this was significantly higher in the sepsis groups compared with the sham operation group, which was consistent with TNF- $\alpha$ , IL-1 $\beta$ , IL-6, and other inflammatory cytokines.

In this study, HR, LVSP, $+$ dp/dtmax and $-$ dp/dtmax were significantly decreased in the sepsis group compared with the surgical control group, which may be related to myocardial injury during septic cardiomyopathy, consistent with previous results by Jia et al. [32]. However, it was suggested that HR increased significantly with the development of sepsis in Shang et al. [10]. The changes of heart rate in septic cardiomyopathy remain to be further explored.

The observation phase point of this study is 3 h–12 h. Prolonged observation time can better reflect the pathophysiological process of septic cardiomyopathy. If the change of cardiac function is monitored by transthoracic echocardiography, it can more comprehensively reflect the condition of cardiac function in sepsis. In the future, it is necessary to further observe the effects of TLR4 inhibitors or clinical treatments that may have related effects, and further clarify NF- $\kappa$ Change relationship between B and TLR4.

5. Conclusion

The current research status of septic cardiomyopathy is described in the introduction. The mechanism of septic cardiomyopathy is not yet fully understood. Previous studies have suggested that the occurrence of septic cardiomyopathy is associated with a variety of factors. In this study, we further investigated the relationship between TLR4-activated NF- $\kappa$ B signaling pathway and inflammatory factors in the development of septic cardiomyopathy. In the future, it is still necessary to observe the effects of TLR4 inhibitors or clinical drugs that may have related effects at the same time to further clarify the relationship between NF- $\kappa$ B and TLR4 changes. At the same time, this study mainly used CLP technology and focused on septic cardiomyopathy to further evaluate the effect of sepsis on cardiac function and myocardial injury in rats by establishing a rat model of sepsis.

In conclusion, the findings of this study indicate that first, evidence of cardiac dysfunction and myocardial injury is evident in rat models with CLP-established sepsis at 3 h following surgery, and second, the TLR4 and NF- $\kappa$ B-mediated cell signaling pathways may be relevant to the pathogenesis of SCM.

Ethics approval and consent to participate

The study was conducted with approval from the Ethics Committee of Xuanwu Hospital Capital Medical University (LYS [2019] No. 078). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Beijing Municipal Administration of Hospitals Incubating Program (No. PX2019028).

Author contributions

Conception and design of the research: XRX, JQ; Acquisition of data: LPL, YWG; Analysis and interpretation of the data: LPL, JW; Statistical analysis: LPL, XRX; Obtaining financing: XRX; Writing of the manuscript: XRX, YLL; Critical revision of the manuscript for intellectual content: YLL, JW. All authors read and approved the final draft.

Footnotes

Acknowledgments

The authors would like to thank the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

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Role of activating the nuclear factor kappa B signaling pathway in the development of septic cardiomyopathy in rats with sepsis

Abstract

BACKGROUND:

OBJECTIVE:

METHOD:

RESULTS:

CONCLUSION:

Keywords

1. Background

2. Method and materials

2.1 Preparing the animals and the sepsis model for the experiment

2.2 Experimental grouping

2.4 Collecting the serum specimens and measuring cardiac troponin I, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6

2.5 Preparation of the collected myocardial tissue for histopathological examination and western blot analysis to determine the expression of toll-like receptor 4 and nuclear factor kappa B

2.6 Statistical methods

3. Results

3.1 General condition of the rats in each group

3.2 Mortality of the rats in each group

3.3 Hemodynamic results of the rats in each group

Table 1 HR (times/min) and LVSP (mmHg) of rats in each group

Table 3 Test results of rat serum samples in each group

3.6 Western blot analysis results of toll-like receptor 4 and nuclear factor kappa B expression in the myocardium for each group

5. Conclusion

Ethics approval and consent to participate

Availability of data and materials

Competing interests

Funding

Author contributions

Footnotes

Acknowledgments

References

Table 1
HR (times/min) and LVSP (mmHg) of rats in each group

Table 3
Test results of rat serum samples in each group