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Lycopene has a protective effect on septic shock‑induced cardiac injury in rats.

OBJECTIVE
The aim of the present study is to investigate the cardioprotective effect of lycopene, known for its antioxidant and anti-inflammatory effect, in a rat sepsis model induced by lypopolysaccharide (LPS).


METHODS
The oxidative stress parameters, antioxidant parameters and cytokine levels with or without lycopene treatment in LPS‑induced septic rats as well as in controls were measured in serum and tissue. Histologic examinations of the cardiac tissues were also performed. The Kruskal-Wallis and the Bonferroni-adjusted Mann-Whitney U Test was used for analysis. A p value < 0.05 was considered significant.


RESULTS
The data of this study showed that lycopene pretreatment reduced the oxidative stres parametersand , proinflammatory cytokines as well as increased the antoxidant enzyme activities in both serum and cardiac tissues in LPS‑induced septic rats.. Moreover, hyperaemia and haemorrhage in the epicardium, myocardium and endocardium were lower in the lycopene pretreated group as compared to the LPS alone group.


CONCLUSION
These results suggest that lycopene could be beneficial for the prevention of cardiac injury caused by sepsis through reducing the cytokine levels and oxidative stress parameters (Tab. 4, Fig. 1, Ref. 35).


Introduction
Sepsis continues to be a major cause of death among hospitalised patients. In the presence of severe sepsis and septic shock, a supportive treatment in addition to causal therapy is mandatory (1). Depending on the extent of the infl ammatory response, the clinical presentation of an affected patient may involve multiple organ failure, including that of the heart, lungs and liver, acute kidney injury, coagulopathy and, eventually, death (2). In the course of sepsis, the cardiac dysfunction may manifest as systolic heart failure or diastolic dysfunction (3). Sepsis-induced myocardial injury is caused by various mechanisms, cytokines, prostanoids and nitric oxide (NO). All have been implicated as major potential factors. Lipopolysaccharides (LPS) are extremely strong stimulators of infl ammatory reactions, act at very low concentrations, and are involved in the pathogenesis of sepsis and septic shock. Interleukin (IL)-1β, and tumour necrosis factor (TNF)-α are proinfl ammatory cytokines that act as potent drivers of septic shock induced by LPS (4).
Lycopene, primarily found in tomatoes and several other fruits and vegetables, has been tested for its benefi cial effects owing to its antioxidant and anti-infl ammatory effect (5) Recent epidemiological studies show that an increased serum lycopene level is associated with a reduction in cardiovascular diseases. Animal studies have corroborated this fi nding by demonstrating the beneficial effect of lycopene in experimental models such as myocardial ischemia reperfusion injury and post-myocardial infarction (6,7). However, there is currently no solid evidence for the protective effect of lycopene in sepsis-induced heart injury.
We thought that lycopene may have a cardioprotective function in sepsis due to its antioxidant and anti-infl ammatory properties. In order to elucidate the mechanism of its action, we measured the levels of oxidative stress and infl ammatory markers, both in serum and heart tissue samples, and examined histopathological changes.

Material and methods
This study was undertaken according to the recommendations in the Guide for the Care  Study animals were divided into three groups per 7 rats as follows: Group I, Control; Group II, LPS (s.c. 10 mg/kg); Group III, lycopene (i.p. 50 mg/kg) for 5 days and LPS (s.c. 10 mg/kg) thereafter. Twelve hours after LPS administration, the rats were anaesthetized. The heart was removed following blood sampling from the vena cava. Sera were separated after centrifugation of blood samples. Heart tissues and sera were kept at -80 °C until the time of biochemical analysis. Serum and tissue levels of IL-1β, TNF-α, NO, LOOH and SH were measured using a commercially enzyme-linked immunosorbent assay (ELISA) kit (Ray Biotech Inc., Diaclone, Cayman).
Serum TOS and TAS were determined using a novel automated measurement method (8). Samples obtained from the heart tissue were fi xed in a 10% formaldehyde solution. Following a routine tissue process, 4-μ-thick sections were obtained from paraffi n-embedded blocks and stained with haematoxylin-eosin (H&E) stain. Then, tissue sections were examined under a light microscope, and photographs were taken.

Statistical analysis
The Kruskal-Wallis test was used to compare the groups, and the Bonferroni-adjusted Mann-Whitney U Test was used to compare the two groups for biochemical parameters. Values of less than 0.05 were regarded as statistically signifi cant. The results are given as mean ± standard deviation (SD) for biochemical measurements.

Results
Serum cytokine and NO measurements obtained from the control group, LPS group and Lycopene + LPS group are summarised in Table 1. Serum TNF-α and IL-1β values were signifi cantly higher in the LPS group as compared to the control group, whereas reduced cytokine levels were found in the Lycopene + LPS group, while being signifi cantly lower compared to the LPS group (p < 0.05). Cardiac tissue measurements were consistent with serum measurements showing a signifi cant increase in cytokine levels in the LPS group versus the control group and a signifi cant reduction in cytokine levels in the Lycopene + LPS group, as compared to the LPS group (p < 0.05) (Tab. 2). Changes in NO levels closely matched those in cytokine levels. While LPS administration resulted in increased NO levels, the lycopene administration was associated with a signifi cant fall in NO levels (p < 0.05) in both serum and cardiac tissue samples.
The measurements of serum and cardiac tissue samples showed that LOOH (indicator of oxidative stress) levels were signifi cantly high and SH levels (indicator of antioxidant capacity) were low in

Tab. 4. The levels of oxidative stress parameters in cardiac tissue for all groups.
the LPS group (p < 0.05). However, the administration of lycopene had a directly opposite effect on these two parameters. Based on measurements from serum and tissue samples, TAS was lower in the LPS group compared to lycopene group but the difference was not signifi cant whereas it was signifi cantly higher in the lycopene group versus the control. TOS was signifi cantly higher in the LPS group versus the controls and lower in the lycopene group versus the LPS group (p < 0.05) (Tabs 3 and 4).

Histopathological results
On histopathological examination, marked hyperaemia and haemorrhage in the epicardium, myocardium and endocardium were observed in both LPS and Lycopene + LPS groups, but not in the controls. However, the severity was higher in LPS group compared with Lycopene+LPS group. In brief, we can state that the severity of hyperaemia and haemorrhage in the LPS group is moderate, while being mild in the Lycopene+LPS group (Fig. 1).

Discussion
Sepsis is a common condition with a high mortality rate. Antisepsis treatments, though, are not at the desired levels (9, 10). As widely observed in patients with severe sepsis, sepsis frequently affects the cardiovascular system and leads to cardiac dysfunction (11). It has been reported that LPS administration induces systemic infl ammation similar to many of the initial clinical features of sepsis such as extensive proinfl ammatory cytokine productions, which leads to multiple organ failure and high mortality rate (12). Therefore, this model is used in many experimental sepsis-induced studies (13,14).
Since sepsis can result in severe organ injury by provoking infl ammatory cascades and oxidative stress, we aimed to investigate the possible benefi cial effect of lycopene in cardiac damage in sepsis by evaluating the oxidative stress parameters, antioxidant and oxidant status and also proinfl ammatory cytokine levels in LPS-induced sepsis model, (15). Additionally, the histopathologic examinations were also conducted.
In our study, the increase in ROS generation was refl ected in a signifi cant increase in TOS and LOOH levels and signifi cant decrease in SH levels. There was also a decrease in TAS but it was not signifi cant. Oxidative damage associated with the production of excessive amounts of reactive oxygen species (ROS) by activated immune cells is believed to play an integral role in the pathogenesis of sepsis (16). The prominent role of lipid peroxidation in septic shock and secondary organ dysfunction has been reported while the increased occurrence of multiple organ failure occurring in sepsis coincides with increased oxidative stress (17,18).
We demonstrated that lycopene treatment resulted in reduced LOOH levels and TOS, i.e. oxidative stress parameters, whereas SH levels and TAS, i.e. antioxidant indicators, were elevated. Based on this fi nding, one might deduce that lycopene has a protective effect against LPS-induced oxidative damage via oxidative and antioxidant capacity. Lycopene, an antioxidant carotenoid and the most effective singlet oxygen quencher, has been shown to be a potent antioxidant in both human and animal studies (19)(20)(21). In a hypoxia-ischemia study, lycopene improved mitochondrial dysfunction and reduced oxidative stress. It has been suggested that some of the benefi cial effects of lycopene in sepsis may take place via its effect on cardiomyocyte mitochondria. It has also been shown in septic patients that lycopene reduces the elevated malondialdehyde (MDA), an end-product formed during oxidative stress, (19). The oxidative stress induced by obstructive jaundice is reduced by lycopene treatment via decreasing MDA and increasing GSH levels in the liver and renal tissues (20). Lycopene (1 mg/kg) treatment has also a protective effect on renal toxicity induced by carbon tetrachloride administration. It reduces MDA levels and increases antioxidant GSH levels (21).
In our study conducted on LPS-administrated rats, we have seen an increase in both TNF-α and IL-1β levels, i.e. in infl ammatory response markers in serum and cardiac tissues as well as that lycopene remarkably attenuated the upregulation of these infl ammatory cytokines.
It was reported that TNF-α, an infl ammatory cytokine, is considered to be an endogenous mediator in LPS-induced shock (22) and that LPS leads to increased TNF-α levels, both in serum and heart tissue (23).

A B C
ditions including sepsis. It has been reported that lycopene showed anti-infl ammatory effect on macrophages by reducing proinfl ammatory cytokine and chemokine expression in LPS-induced sepsis model (25). It was also shown that, lycopene treatment attenuated cardiac dysfunction in diabetic rats via decreasing TNF-α expression (26,27). Nitric oxide (NO) and other reactive species cause hypotension, excessive vasodilatation and impair oxygen usage in sepsis (28). In our study, we observed an increase in NO levels in LPStreated rats. NO, a potent vasodilator that causes hyperpolarisation of smooth muscle plasma membranes, has a potential role in LPSinduced vascular hyperactivity (29). Peroxynitrite is important in NO-dependent pathogenic mechanisms, as well as in infl ammatory conditions such as septic shock, myocardial infarction and chronic infl ammatory diseases (30). Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by an enhancement of iNOS activity and release of nitric oxide (NO). It was demonstrated that endotoxin exposure of human myocardium impairs cardiac contractility independently from systemic endotoxin effects. This impairment of myocardial performance appears to be mediated by an enhanced release of NO (31). The data of this study also reveal a signifi cant reduction in NO levels by lycopene both in serum and cardiac tissues in LPSinduced sepsis model. It was reported that oxidative stress and NO deplete lycopene in tissues and human cells and that cardiovascular diseases are accompanied with lycopene depletion (32). A number of studies also show that lycopene suppresses infl ammation in several tissues by inhibiting the formation of NO (33). Immunohistochemical examination reveals a signifi cant increase in inducible NO synthase in contrast-induced nephropathy and signifi cant improvements in infl ammation, autophagy and apoptosis by administration of lycopene in rats (34).
Epidemiological and clinical studies suggest that dietary antioxidants may reduce the risk of cardiovascular disorders (35). The biochemical and histopathological results of this study underscore the cardioprotective effect of lycopene in an LPS-induced sepsis model in the present study.

Conclusion
Based on the results of this study, it may be suggested that lycopene supplementation-given alone or in combination with standard therapies, may reduce the oxidative stress and infl ammation. As such it may represent a novel adjunct treatment in sepsis and a potential therapeutic candidate for the treatment of cardiac damage in sepsis.