Thymoquinone protects against hyperlipemia-induced cardiac damage in low-density lipoprotein receptor-deficient (LDL-R−/−) mice CURRENT POSTED

Background Hyperlipemia is a risk factor for cardiac damage and cardiovascular disease. Several studies have shown that thymoquinone (TQ) can protect against cardiac damage. The aim of this study was to investigate the possible protective effects of TQ against hyperlipemia-induced cardiac damage in low-density lipoprotein receptor deficient (LDL-R−/−) mice. Methods: Eight-week-old male LDL-R−/−mice were randomly divided into the following three groups: the control group fed a normal diet (ND group), the high fat diet (HFD) group, and the HFD mixed with TQ (HFD+TQ) group. All groups were fed the different diets for 8 weeks. Blood samples were obtained from the inferior vena cava, collected in serum tubes, and stored at -80 °C until use. Cardiac tissues were fixed in 10% formalin and then embedded in paraffin for histological evaluation. The remainder of the cardiac tissues was snap-frozen in liquid nitrogen for mRNA preparation or immunoblotting. Results The levels of metabolism-related factors, such as total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-c), and high-sensitivity C-reactive protein (hs-CRP), were decreased in the HFD+TQ group compared with that in the HFD group. Periodic acid-Schiff staining demonstrated that lipid deposition was lower in the HFD+TQ group than that in the HFD group. The expression of pyroptosis indicators (NOD-like receptor 3 [NLRP3], interleukin [IL]-1β, IL-18 and caspase-1), pro-inflammation factors (IL-6 and tumour necrosis factor alpha [TNF-α]), and macrophage markers (cluster of differentiation [CD]68) was significantly downregulated in the HFD+TQ group compared with that in the HFD group. Conclusions Our results indicate that TQ may serve as a potential therapeutic agent for hyperlipemia-induced cardiac damage. group, but were significantly increased in the HFD + TQ group. Our data establish that TQ contributes to the mitigation of hyperlipidaemia-induced cardiac damage, as shown by reduced lipid deposition and pyroptosis and downregulated pro-inflammatory cytokine expression. These findings provide new insights into the role of TQ in hyperlipidaemia-induced cardiac damage and introduce the possibility of a novel therapeutic intervention for treating CVDs.


Introduction
Hyperlipemia is a critical damage-inducing element in cardiovascular disease (CVD) [1]; individuals with hyperlipidaemia have a higher risk of CVD compared with those with normal cholesterol levels [2]. Furthermore, increasing evidence has shown that dyslipidaemia-related cardiac damage is associated with lipid accumulation, oxidative stress, and inflammation [3,4].
Several researchers have investigated various drugs for treating hyperlipidaemia such as statins; however, as these are related to the development of resistance in cells and as these are associated with adverse effects, new methods for treating hyperlipidaemia are needed. Thymoquinone (TQ) is the major constituent of Nigella sativa [5], commonly known as black seed or black cumin, and is globally used in folk (herbal) medicine for treating and preventing a number of diseases and conditions [6]. Previous studies have reported that TQ suppresses chronic cardiac inflammation [7], and regulates the expression of factors, such as vascular endothelial growth factor and nuclear factorerythroid-2-related factor 2 (Nrf2), thereby improving the antioxidant potential of the cardiac muscle.
In addition, TQ alleviates diabetes-associated oxidative stress in cardiac tissues [8]. Additionally, several studies have shown that the protective effect of TQ against cardiac damage such as in case of ischemic damage [9] and acute abdominal aortic ischemia-reperfusion injury [10]is mediated via the pyroptosis pathway [11]. Recently, pyroptosis, an inflammatory form of programmed cell death [12], has been gaining increasing attention, especially during hyperlipemia [13,14]; however, the pathophysiological mechanisms underlying the relationship between hyperlipemia and cardiac damage are not yet fully understood. Therefore, in this study, we investigated the role of TQ in hyperlipidaemia-induced cardiac damage in a low-density lipoprotein receptor-deficient (LDL-R⁻/⁻) mouse model.

Animal model
LDL-R ⁻/⁻ mice were purchased from Beijing Vital River Lab Animal Technology CO., LTD. (Beijing, China). All mice were bred in a room with a 12/12-h light-dark cycle at a controlled temperature (24 -26 °C). Male LDL-R⁻/⁻ mice (8-week-old) were randomly divided into the following three groups: mice fed a normal diet (ND group, n = 8), mice fed a high-fat diet (HFD group, n = 8), and mice fed a highcholesterol diet + 50 mg/kg/day of TQ (HFD+TQ group, n = 8). The experimental diet was purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). Mice in all groups were fed with the appropriate diet for 8 weeks. Blood samples were acquired from the inferior vena cava, collected in serum tubes, and stored at -80 °C until use. Cardiac tissues were fixed in 10% formalin and embedded in paraffin for histological evaluation. The remaining cardiac tissues were snap-frozen in liquid nitrogen for mRNA isolation and immunoblotting analyses. The animal experiment was approved by the Animal Ethics Committee of Beijing Hospital.

Biochemical measurements
Sera were separated from the collected blood samples by centrifugation at 3000 rpm for 15 min. The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and high-sensitivity Creactive protein (hs-CRP) in the serum were detected using the Total Cholesterol, low-density lipoprotein cholesterol, and high-sensitivity C-reactive protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), as per the manufacturer's instructions.

Haematoxylin and eosin staining
The cardiac tissues were fixed with 10% buffered formalin for 30 min and then dehydrated in 75% ethanol overnight, followed by paraffin embedding. Serial sections (4 μm) were stained with haematoxylin and eosin for pathological analysis.

Periodic acid-Schiff (PAS) staining
Cardiac tissues from each group were stored in 10% formalin, dehydrated in an ascending alcohol series (75, 85, 90 and 100% alcohol, 5 min each) and then embedded in paraffin wax. Paraffin sections (4-μm-thick), sliced from these paraffin-embedded tissue blocks, were then de-paraffinized via immersion in xylene (three times, 5 min each) and rehydrated using a descending alcohol series (100, 90, 85 and 75% alcohol, 5 min each). Samples were stained with PAS stain to investigate the changes in cardiac morphology. Red staining indicated lipid deposition.
The relative signal intensity was quantified using NIH ImageJ software.

RNA isolation and real-time PCR (qPCR)
Total RNA was isolated from cardiac tissues and complementary DNA (cDNA) was synthesised using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen, Beijing, China) according to the manufacturer's protocol. Gene expression was quantitatively analysed by qPCR using the TransStart Top Green qPCR SuperMix kit (Transgen). β-Actin was amplified and quantitated in each reaction in order to normalise the relative amounts of the target genes. Primer sequences are listed in Table 1.  Abbreviations: TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; NLRP3, the nucleotide-binding and oligomerization domain-like receptor 3; IL-18, interleukin-18; IL-1β, interleukin-1β

Statistical analysis
All data are presented as the mean ± standard error of mean (SEM). Statistical analysis was performed using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA). Inter-group variation was measured using one-way analysis of variance (ANOVA) and subsequent Tukey's test. The minimal level for statistical significance was set at P < 0.05.

Metabolic Characterisation
The metabolic characteristics of LDL-R⁻/⁻ mice after 8 weeks of different treatments are summarised in Table 2. The heart/body weight ratio did not change in the three groups. TC, LDL-c and hs-CRP levels were markedly increased in the HFD group, but significantly decreased in the HFD + TQ group.

TQ reduced HFD-induced cardiac damage
To evaluate inflammatory cell infiltration into the cardiac tissue, haematoxylin and eosin staining was performed (Fig. 1). HFD+TQ group mice showed markedly reduced inflammatory cell infiltration in their cardiac tissue compared with that in the HFD group mice, indicating that TQ reduced HFDinduced cardiac damage.
To evaluate lipid accumulation in cardiac tissue, we evaluated PAS staining and the expression of CD36 and CD68 (Fig. 2). Increased lipid retention was detected in the cardiac tissues of HFD group mice. Interestingly, HFD +TQ group mice showed markedly reduced lipid deposition in the cardiac tissue compared with that in HFD group mice.

TQ reduced HFD-induced expression of pro-inflammatory cytokines in mouse cardiac tissues
To examine the involvement of pro-inflammatory cytokines in the cardiac tissues of the three groups of mice, mRNA expression of IL-6 and tumour necrosis factor alpha (TNF-α) was measured using qPCR ( Fig. 3). Although IL-6 and TNF-α mRNA were upregulated in the HFD group, this upregulation was attenuated in the HFD+TQ group.

TQ reduced HFD-induced pyroptosis in cardiac tissues
To evaluate pyroptosis in cardiac tissues, we examined the mRNA and protein expression of pyroptosis indicators NLRP3, caspase-1, IL-1β, and IL-18 (Fig. 4). NLRP3, caspase-1, IL-1β and IL-18 mRNA was significantly downregulated in the HFD+TQ group compared with that in the HFD group ( Fig. 4a). Western blotting (Fig. 4b) demonstrated that the levels of NLRP3, caspase-1, IL-1β, and IL-18 were markedly reduced in the cardiac tissues of the HFD+TQ group compared with that in the HFD group ( Fig. 4b-c). These results indicate that TQ reduced HFD-induced upregulation of NLRP3, caspase-1, IL-1β, and IL-18 expression.

TQ reduced HFD-induced increase in P-ERK levels in the cardiac tissues of mice
To investigate the effect of TQ on the regulation of the ERK signalling pathway, we analysed P-ERK levels in the respective treatment groups by western blotting (Fig. 5). P-ERK level was higher in the HFD group than that in the ND group, and the HFD+TQ group exhibited significantly low P-ERK levels than the HFD group.

Discussion
The present study demonstrates that TQ has a protective effect against hyperlipemia-induced progressive lipid deposition, pro-inflammatory cytokine expression, and pyroptosis.
Metabolic characteristic analysis indicated that the levels of TC and LDL-c were increased in the HFD group compared to that in the ND group mice. These results are in agreement with reports by Kolbus et al. [15]. Interestingly, the TC and LDL-c levels in the HFD + TQ group were significantly lower than those in the HFD group. Several clinical studies have indicated that hs-CRP can serve as a biomarker for the risk prediction of cardiovascular events [16,17]. Our results show that the HFD + TQ group had markedly reduced serum hs-CRP levels compared with that in the HFD group, indicating that TQ influences cholesterol metabolism and hs-CRP levels.
Hyperlipidaemia promotes macrophage accumulation and lipid deposition in cardiac tissues [18]. Cellular lipid homeostasis involves the regulation of influx, synthesis, catabolism, and efflux of lipids.
An imbalance in these processes can result in the conversion of macrophages into foam cells [19].
The CD68 marker identifies a population of macrophages; CD68 positive cells are often observed infiltrating cardiac tissues [18]. The results of our lipid deposition assays showed that CD36 expression and PAS staining were significantly increased in the LDL-R⁻/⁻ HFD group mice compared with that in the ApoE⁻/⁻ ND mice; however, this damage was significantly inhibited in the HFD + TQ group.
Pro-inflammatory cytokines have been reported to be highly expressed in hyperlipidaemia, and are known to contribute to cardiac damage [20,21]. Our study showed that the expression of IL-6, and TNF-α was reduced in the HFD + TQ group compared with that in the HFD group, indicating that TQ downregulated HFD-induced expression of IL-6 and TNF-α.
Pyroptosis is a novel programmed cell death mechanism. Recent studies have reported that pyroptosis contributes to the development of hyperlipidaemia. Pyroptosis induction is closely associated with the activation of the NLRP3 inflammasome, which has been linked to key cardiovascular risk factors including hyperlipidaemia [22,23]. A significant decrease in atherosclerotic lesion size has also observed at the aortic sinus of HFD-fed LDL-R⁻/⁻ mice reconstituted with NLRP3 knockout bone marrow cells [23]. In addition, previous studies have shown that NLRP3 recruits caspase-1, leading to the activation of caspase-1, maturation and secretion of IL-1β and IL-18, and initiation of pyroptosis [24][25][26][27]. Our results showed that the cardiac tissues in the HFD + TQ group expressed markedly reduced levels of NLRP3, caspase-1, IL-1β and IL-18 compared with that in the HFD group, indicating that TQ downregulated HFD-induced pyroptosis.
ERK is a cytoplasmic kinase whose activity is regulated by phosphatases [28]. Previous studies have suggested that TQ increases the phosphorylation of mitogen-activated protein kinases and ERK [29]. P-ERK modulates cellular metabolism by a series of reactive oxygen stress activities. TQ increases P-ERK levels to regulate cellular activity [30]. Our study showed that P-ERK levels decreased in the HFD group, but were significantly increased in the HFD + TQ group.

Conclusions
Our data establish that TQ contributes to the mitigation of hyperlipidaemia-induced cardiac damage, as shown by reduced lipid deposition and pyroptosis and downregulated pro-inflammatory cytokine expression. These findings provide new insights into the role of TQ in hyperlipidaemia-induced cardiac damage and introduce the possibility of a novel therapeutic intervention for treating CVDs.

Availability of data and materials
All datas generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.  Pro-inflammatory gene expression in the cardiac tissue. Relative mRNA expression of TNF-α and IL-6 in cardiac tissue of three group with different treatments. Data are given as the means ± SEM; n = 5-6 in each group. * P < 0.05 ; **P < 0.01 Figure 3 Pro-inflammatory gene expression in the cardiac tissue. Relative mRNA expression of TNF-α and IL-6 in cardiac tissue of three group with different treatments. Data are given as the means ± SEM; n = 5-6 in each group. * P < 0.05 ; **P < 0.01