Fereshteh Asgharzadeh ¹, Rahimeh Bargi ¹, Farimah Beheshti ⁴, Mahmoud Hosseini ², Mehdi Farzadnia ³, Majid Khazaei ^{1 *}

Thymoquinone (TQ) is one of the active ingredients of herbal plants such as Nigella sativa L. (NS) which has beneficial effects on the body. The beneficial effects of TQ on the cardiovascular system have reported. This study aimed to investigate the effect of TQ on cardiac fibrosis and permeability, serum and tissue concentration of inflammatory markers and oxidative stress status in chronic lipopolysaccharide exposure in male rats.

Seventy male Wistar rats were randomly divided into five groups as follows: (1) control; (2) LPS (1 mg/kg/day); (3-5) LPS + TQ with three doses of 2, 5 and 10 mg/kg (n=14 in each group). After 3 weeks, serum and cardiac levels of IL-1β, TNF-α and nitric oxide (NO) metabolites, and cardiac levels of malondialdehyde (MDA), total thiol groups, catalase (CAT) and superoxide dismutase (SOD) activities, permeability of heart tissue (evaluated by Evans blue dye method) and myocardial fibrosis were determined, histologically.

LPS administration induced myocardial and perivascular fibrosis and increased cardiac oxidative stress (MDA), inflammatory markers and heart permeability, while, reduced anti-oxidative enzymes (SOD and CAT) and the total thiol group. Administration of TQ significantly attenuated these observations.

TQ improved myocardial and perivascular fibrosis through suppression of chronic inflammation and improving oxidative stress status and can be considered for attenuation of cardiac fibrosis in conditions with chronic low-grade inflammation.

thymoquinone, inflammation, cardiac, fibrosis

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Inflammation is a sort of non-specific immune reaction in response to damage or disease. The acute phase response is a complex systemic initiated by infection, injury, stress, and inflammation. If the replication is not properly staged, in any case, the procedure can form into a subclinical inflammatory state that may trigger distinctive diseases under pathological conditions [1]. Chronic low-grade inflammation or subclinical inflammation is correlated with several chronic inflammatory diseases such as atherosclerosis, diabetes, and autoimmune diseases [2]. It described by 2-3 fold increment in systemic concentrations of cytokines, such as TNF-α, IL-6, and C-reactive protein, and associated with increased oxidative stress markers [3]. However, the exactly related consequences of circulating low dose endotoxemia and the mechanisms especially on the heart tissue are still not clear. Administration of lipopolysaccharide (LPS) (endotoxin, a component of the cell membrane of gram-negative bacteria) is a known model for the induction of sepsis [4]. It induced a systemic inflammation which has been shown to partially mimic the myocardial and systemic cardiovascular effects of sepsis. LPS depresses myocardial and cardiomyocyte function [5] and induces cardiomyocyte apoptosis [6]. At the point of LPS binding to its receptors, particularly Toll-like receptor (TLR)-4, downstream intracellular signaling pathways are started; a definitive result is the activation of innate immunity [7]. Because of the advancement of treatment complications, for example, drug resistance and adverse effects, traditional medicine are still deficient to provide a complete treatment of specific infections; all things considered, proceeding to discover new medications is expected to give alternative therapy, either to supplement nor supplant existing conventional medicine. The activity of herbal plants contain N. Sativa L. seeds is ascribed to its vital oil part which is predominantly (30 - 48%) thymoquinone [8]. Thymoquinone (TQ), with its anti-carcinogenic, anti-inflammatory and anti-oxidant properties has attracted the consideration of researchers to explore its molecular mechanisms and potential use in the treatment of inflammatory diseases [9]. It has several effects on antioxidant enzymes, pro-inflammatory cytokines and some factors involved in cellular signalings such as nuclear factor kappa B or mitogen-activated protein kinase (MAPK) [10]. It also reported that TQ has beneficial effects on the cardiovascular system, respiratory disease, diabetes, hyperlipidemia, and and cancer [9]. In the present study, we used a murine model of recurrent exposure to subclinical LPS and aimed to investigate the effect of TQ on inflammation-induced cardiac fibrosis in chronic low-grade inflammation model in rats.

In this experiment, 70 male Wistar rats, eight weeks old, weighing between 200-250 g, were used. They kept under standard conditions (temperature 22±2°C and 12hours of light/dark cycle) and free access to food and water. The local ethical committee of animal studies approved the study. The animals randomly divided into five groups that treated as follows: (1) control; (2) LPS; (3-5) LPS+TQ with three doses of 2, 5 and 10 mg/kg (n=14 in each group).

Lipopolysaccharide, DTNB (5,5´-dithiobis-2- nitrobenzoic acid) and TQ (2-isopropyl-5-methyl-p-benzoquinone) purchased from Sigma (Sigma Chemical Co., USA). LPS dissolved in saline and TQ dissolved in saline supplemented with ethanol 96%. Tris, TBA (2-thiobarbituric acid), HCl (Hydrochloric acid), EDTA (Ethylene di amine tetra acetic acid), TCA (Trichloro acetic acid), DMSO (5,5-dithio-bis-(2-nitrobenzoic acid), diethyl ether, formamide and Evans blue obtained from Merck (Merck Co., Germany).

For induction of chronic low-grade inflammation, LPS groups received 1 mg/kg/day, intraperitoneally (ip), for three weeks [11, 12]. The animals of the control group received saline instead of LPS. TQ-treated groups were received TQ (2, 5 or 10 mg/kg/day; ip) from the beginning of the study [13]. After three weeks, the animals anesthetized with diethyl ether and blood samples were taken from orbital sinus for biochemical analysis. The blood samples centrifuged at 3000 rpm for 20 minutes, and serums were collected and stored at -70°C for further analysis. Then, half of the animals in each group sacrificed. The hearts were isolated, washed with saline and the left ventricles used for histopathological assessment. The right ventricles were also dissected to homogenize for determination of tissue oxidative, anti-oxidative, nitrite and inflammatory markers. In the rest of animals, the permeability of the heart tissues evaluated as described below.

The specific rat ELISA kits (ebioscience Co, San Diego, CA, USA) used for serum and heart homogenate TNF-α and IL-1β measurements according to the manufactory instructions.

For the measurement of serum and heart nitrite, Griess reagent method was used (Promega Corp, USA) [14]. In brief, sulfanilamide and NED solutions put in room temperature. First, 50 μl of the sample (serum or tissue homogenate) added to the wells. Then, 50 μl of sulfanilamide solution added to all samples into the wells and incubated for 5-10 minutes at room and away from light. Next, NED solution poured to all samples. At this stage, color purple appeared. Optical densities of samples were measured using a microplate reader at wavelength 520-550 nm. The mean optical densities of each sample obtained by the reading device by using the standard curve nitrite concentrations.

MDA level is an index of lipid peroxidation. For its evaluation, the target tissue was weighed and homogenized immediately with a phosphate buffer solution. Then, 1 ml of 10% homogenates with two mL of TBA + TCA + HCL solution mixed for 45 minutes in a boiling water bath. After cooling to room temperature, they centrifuged for 10 minutes. Then, the absorbance at 535 nm read and MDA was calculated based on the following formula [15].

To measure the total thiol group, DTNB (Di-Tio nitro benzoic acid) reagent used. This represents the yellow color complex reacts with SH groups. 1 ml of Tris-EDTA buffer (pH = 8.6) added to tissue homogenate and the specimen absorbance measured at 412 nm against Tris-EDTA buffer alone (A1). Next, 20 μl of DTNB reagents were added to the solution and kept at room temperature for 15 minutes. Then, the sample absorbance read again (A2). The absorbance of DTNB reagent was read alone as a blank (B). Total thiol concentration (mM) calculated from the following formula [16].

Superoxide dismutase (SOD) was measured using a Ransod kit (Randox Laboratory, UK). Catalase action was measured by a method as previously described (Aebi, 1984). In brief, the principle of this method determined on the hydrolyzation of H2O2 in phosphate buffer, pH 7.0, and requisite volume of sample and decreasing absorbance at 240 nm. The Conversion of H2O2 into H2O and O2 in 1 minute under standard condition was thought to be the enzyme response velocity.

Heart permeability was measured by quantifying the Evans blue (EB) dye bound to albumin. The animals were anesthetized ,and EB dye (20 mg/ml) injected through the tail vein before sacrifice. After 20 minutes, the hearts were dissected, weighted, and placed in 4 mL formamide solution for 48 hours at room temperature. The optical density of the solution was measured by spectrophotometry at 620 nm. Data expressed as μg EB/g of wet weight tissue [17].

The left ventricles placed in 10% buffered formalin for 24 hours preceding preparing and paraffin taking after a standard system. At that point, serially segmented into 5 μm thick samples were set up from the tissue pieces, stained with hematoxylin and eosin (H&E) and Masson's trichrome and analyzed under light microscopy. The area of collagen content (blue stained) surrounding the coronary artery was also measured and normalized to the control group.

Data expressed as the mean ± standard error of the mean (SEM). All data were analyzed using the SPSS 20 software. The comparisons between groups were evaluated using one-way ANOVA, followed by LSD posthoc test. The level of significance considered if p < 0.05.

The results showed that LPS group had lower body weight gain compared to control, although it was not statistically significant (15.4 ± 3.6 vs. 20.2 ± 6.41 g, respectively). Weight gain in the animals who received TQ with doses of 2, 5 and 10 mg/kg was: 4.2 ± 1.08, 3.5 ± 0.42 and 1.6 ± 0.45; respectively.

Measurement of serum levels of IL-1β (Fig. 1)A) and TNF-α (Fig. 1)B) showed that there were no significant differences between LPS and control groups and treatment by TQ couldn’t alter them (p > 0.050). Evaluation of cardiac levels of IL-1β and TNF-α showed that animals in the LPS group had higher levels than control. Administration of TQ by all three doses (2, 5 and 10 mg/kg) lowered serum levels of IL-1β compare to the LPS group (p < 0.001). The same results were observed in cardiac TNF-α after treatment by TQ. TQ dose-dependently reduced tissue levels of IL-6 and TNF-α (Fig. 1)C&D).

As shown in figure 2A, there was no significant difference in serum nitrite concentration between LPS and control groups (p > 0.05). Administration of TQ by three doses could not significantly change serum nitrite level (p > 0.05). However, cardiac level of nitrite in the LPS group showed higher level compare to control group (p < 0.05). Treatment by TQ reduced the level of cardiac nitrite, dose-dependently (Fig. 2)B), without changing in serum nitrite level.

Figure 3A&B illustrate that LPS group had higher MDA concentration (Fig. 3)A) and lower total thiol contents (Fig. 3)B) in cardiac tissues compare to control animals (p < 0.001). Treatment by TQ with all doses decreased cardiac MDA and increased total thiol concentration (p < 0.001). In addition, LPS administration decreased cardiac SOD (Fig. 3)C) and catalase (Fig. 3)D) activity compare to control animals (p < 0.001). Administration of TQ increased catalase and SOD activity, dose dependently.

Results showed that heart permeability of LPS animals was higher than control animals (p < 0.001). All doses of TQ decreased cardiac permeability (p < 0.001) which reached to control level by high dose of TQ (p < 0.001) (Fig. 4)

The H&E stained histopathological examination illustrated that there were no pathological changes in ventricular muscle structure in the control group (Fig. 5)A). In contrast, in LPS group, increased infiltration of inflammatory cells and disarrangement of myofibers were observed (Fig. 5)B). Administration of TQ illustrated improvement of pathological changes, dose dependently (Fig. 5)C-E). Staining with Masson’s trichrome illustrated that in LPS administration significantly increased in collagen deposition in myocardium compared to control rats (Fig. 6)A&B). TQ induced as dose dependent decrease in cardiac fibrosis (Fig. 6)C-E). Also, the increment in percent of collagen deposition in LPS group was observed compared to the control group (Fig. 6)F; p < 0.001). Administration of TQ (all doses) reduced the percent of collagen deposition in the left ventricular wall (p < 0.001). Also, chronic LPS exposure resulted in a significant perivascular collagen deposition around coronary artery which prevented by TQ treatment (Fig. 7)

Our results showed that Also exposure to chronic inflammation induced by LPS, cardiac fibrosis occurs as a primary effect. And TQ reduced it. TQ has beneficial effects on cardiovascular health, and its anti-inflammatory and antioxidant activity examined in both in vitro and in vivo models [10]. In this study, the effect of TQ on cardiac fibrosis and permeability and serum and tissue concentrations of inflammatory markers and oxidative stress in the subclinical LPS-induced model of inflammation studied. In this study, chronic injection of LPS did not change body weight compared to the control group. Wilbur et al. reported that no change in activity, appetite , and body weight observed in rats that exposed to LPS injection [18]. In our study, administration of TQ (all doses) in LPS group significantly reduced body weight. It showed that treatment with TQ has positive effects in reduction of body weight and metabolic syndrome factors [19]. Our study showed that the heart levels of IL-1β and TNF-α were significantly higher in the LPS group which was in line with previous studies [20]. Administration of TQ reduced tissue levels of the inflammatory cytokines, dose-dependently. The ability of TQ in reducing serum levels of inflammatory cytokines such has observed in other inflammatory models [21]. Since inflammation process is regulated lipoxygenase and cyclooxygenase, TQ plays as anti-inflammatory through inhibition of cyclooxygenase and 5-lipoxygenase membrane [22]. The production of inflammatory mediators such as IL-1β, TNF-α, NO and PGE2 is set mainly by NFκB (as regulators key of genes involved in inflammatory responses) [23]. In the present study, tissue levels of nitrite in the LPS group were higher than control and treatment by TQ decreased tissue level of NO significantly. It suggested that TQ decreased NO generation by reducing the expression of iNOS [24]. Mahmoudi et al. showed that TQ, dose-dependently, reduced NO production in the supernatant of stimulated macrophages by LPS, as well as inhibited an increase in mRNA expression of inducible nitric oxide [25]. Subclinical inflammation also is associated with increased oxidative stress [26]. NFκB transcription factor regulates the expression of inflammatory mediators that involved in oxidative stress [27], thus inhibiting the production of NFκB can prevent the events resulting in injury. The production of reactive oxygen species induced by LPS causes a wide variety of cellular responses by activating transcription factor NFκB through MAPKs and PI3K / Akt routes [28]. It indicated that TQ inhibits these transcription factors and thus reduce inflammation and oxidative stress [8]. In the present study, chronic injection of LPS accompanied with a significant increase in the concentration of MDA, lower content of thiol groups and reduced activity of SOD and catalase in the heart tissue. Interestingly, TQ reduced oxidative stress markers and increased anti-oxidative parameters in LPS groups which support the previous studies [8]. The morphological changes in endothelial cells by bacterial infections cause increased its permeability and endothelial dysfunction [29]. We observed a significant increase in permeability of heart tissue in LPS animal and injection of TQ reduced heart permeability, dose-dependently Studies have also specified that NFκB has an significant role in endothelial response to inflammatory stress [30]. Thus, it is possible that TQ improves endothelial barrier function which damaged by LPS via strong antioxidant activity possibly through its ability to inhibit NFκB. Excessive collagen by fibrocytes changed the ventricular walls structures and interferes with the normal function of the heart, as a result of increased fibrosis and heart failure [31]. The level of subclinical LPS causes cardiac fibrosis that may associated with the development of cardiovascular disease [18], and studies have shown that inflammation is a risk factor for cardiac fibrosis. It also specified that the activation of ROS might lead to cardiac fibrosis [32]. Results of the present study showed cardiac fibrosis (indicated by more collagen content) in chronic inflammation induced by LPS [2]. Interestingly, TQ improved left the ventricular wall and perivascular fibrosis as dose-dependent manner. It suggested that NFκB activation leads to the expression of inflammatory markers and fibrotic factors [33] and it is possible that TQ could inhibit this pathway resulted in the anti-fibrotic and antioxidant effect of TQ.

In conclusion, exposure to the chronic low-grade level of LPS is accompanied by fibrosis in the myocardium and perivascular tissue, heart permeability, and oxidative stress status. TQ attenuated these observations which suggest being considered in treatment in conditions with chronic low-grade inflammation.

The authors are thankful for vice chancellor of Mashhad University of Medical Sciences for their financial support (grant number: 931641).

The authors declare that there are no conflicts of interest.

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