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Ginkgetin ameliorates experimental atherosclerosis in rats
Biomedicine & Pharmacotherapy 102 (2018) 510–516
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Ginkgetin ameliorates experimental atherosclerosis in rats Naqi Lian, Jing Tong, Wenwen Li, Jingzhen Wu, Yu Li
⁎
T
School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing 210023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis Ginkgetin Matrix metalloproteinases Nitric oxide Nitric oxide synthase
Atherosclerosis is a common disease seriously detrimental to human health. Natural products are important sources of therapeutic candidates for atherosclerosis. We here evaluated the effects of ginkgetin on experimental atherosclerosis in rats and explored the underlying mechanisms. Atherosclerosis was induced by high-fat diet for 12 weeks combined with single intraperitoneal injection of vitamin D3 in rats. The atherosclerotic rats were then treated with ginkgetin at 25, 50 and 100 mg/kg/d or simvastatin at 2 mg/kg/d for 8 weeks. Blood and thoracic aortas were collected for analyses of histopathology, lipid deposition, serum biochemistry, matrix metalloproteinases (MMPs), and nitric oxide (NO)/NO synthase (NOS) system. We found that ginkgetin improved thoracic aortic intima structure, reduced intima-media thickness and intima/media ratio, and attenuated lipid deposition in aorta of atherosclerotic rats. Ginkgetin also decreased the serum levels of total cholesterol, triglyceride and low-density lipoprotein cholesterol, but restored the serum levels of high-density lipoprotein cholesterol in atherosclerotic rats. Additionally, ginkgetin reduced the mRNA and protein expression of MMP-2 and MMP-9 in thoracic aortas of rats with atherosclerosis. Further examinations showed that ginkgetin increased the NO and NOS levels in serum and thoracic aortas. Ginkgetin also unregulated the expression of endothelial NOS and downregulated the expression of inducible NOS at both mRNA and protein levels in thoracic aortas of atherosclerotic rats. Altogether, ginkgetin showed therapeutic effects on experimental atherosclerosis associated with improving lipid profile and modulating the MMPs and NO/NOS systems in rats. Ginkgetin could be a promising candidate for the treatment of atherosclerosis.
1. Introduction Atherosclerosis represents one of the most common causes of cardiovascular diseases including unstable angina, myocardial infarction and ischemic heart failure. It is characterized by smooth muscle cell (SMC) proliferation, endothelial dysfunction, inflammatory responses, extracellular matrix alteration and thrombosis [1]. Development of atherosclerosis leads to insufficient blood supply to the coronary arteries and subsequent myocardial ischemia, which are basic pathological processes of coronary heart disease [2]. Although atherosclerosis has been studied for decades, the underlying mechanisms remain poorly understood. Many risky factors such as dyslipidemia, hypertension and diabetes have been proposed, among which dyslipidemia is thought to play a key role in the pathogenesis of atherosclerosis. Patients with dyslipidemia usually have higher serum levels of low-density lipoprotein-cholesterol (LDL-C), which can be oxidized and converted into oxidized LDL, resulting in oxidative stress, inflammation and injury in the endothelium [3].
Accumulating evidence indicates that the nitric oxide (NO) and NO synthase (NOS) system in vascular SMCs are important regulators of local vascular injury associated with atherosclerosis. In healthy vessels, NO is produced from the endothelium by the endothelial isoform of NOS (eNOS), and functions as an endothelium-derived relaxing factor critical for maintaining vascular homeostasis [4]. Impairment of endothelium-dependent relaxation occurs in atherosclerotic vessels and represents the reduced eNOS-derived NO bioavailability, promoting the development of atherosclerosis [5]. Under injury and atherosclerotic circumstances, the inducible isoform of NOS (iNOS) is commonly unregulated to compensate for the loss of functional endothelium and eNOS [6]. Increased expression of iNOS has been described in atherosclerotic human plaques in macrophages, endothelial cells and SMCs, supporting the role for iNOS in promoting the pathogenesis of atherosclerosis [7]. In the advanced atherosclerotic plaques, smooth muscle and macrophage-derived iNOS may continually promote a pathogenic environment by enhancing oxidative and nitrosative stress [8]. Current knowledge suggests the NO and NOS system as an attractive target for
Abbreviations: eNOS, endothelial isoform of NOS; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL-C, high-density lipoprotein cholesterol; HE, hematoxylin-eosin; iNOS, inducible isoform of NOS; LDL-C, low-density lipoprotein-cholesterol; MMPs, matrix metalloproteinases; NO, nitric oxide; NOS, NO synthase; SMC, smooth muscle cell; TC, total cholesterol; TG, triglyceride ⁎ Corresponding author. E-mail address: [email protected] (Y. Li). https://doi.org/10.1016/j.biopha.2018.03.107 Received 3 November 2017; Received in revised form 17 March 2018; Accepted 17 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Ginkgetin improves histology and reduces lipid deposition in rats with atherosclerosis. (A) Scheme of induction of atherosclerosis in rats for 12 weeks and treatments with Ginkgetin or simvastatin for 8 weeks. After 12 weeks, atherosclerosis was confirmed by HE staining with thoracic aortas. (B) HE staining with thoracic aortas for histological assessment after 8-week treatment. (C) Determination of intima-media thickness and intima/media ratio. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group. (D) Oil red O staining with aortic arch and thoracic aorta, and quantification of the plaque size (% of total aorta). Significance: **P < 0.01 versus control group; ##P < 0.01 versus model group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2. Materials and methods
the treatment of atherosclerosis. Herbal medicines have gained tremendous attention due to their therapeutic benefits for atherosclerosis. Ginkgo biloba belongs to the botanical family of Ginkgocea, and its leaves contain high levels of flavonoids, bioflavonoids, and terpenoids [9]. Notably, ginkgetin, one of primary biflavone compounds isolated from Ginkgo biloba leaves, has been reported to exhibit anti-inflammatory, anti-fungal, neuroprotective, and anti-tumor activities [10], highlighting ginkgetin as an attractive therapeutic candidate. Given the clinical evidence of using Ginkgo biloba extract as a therapy for cardiovascular and cerebrovascular diseases [11], we here evaluated the ameliorative effects of ginkgetin on the experimental atherosclerosis in rats and explored the underlying mechanisms.
2.1. Chemicals and antibodies Ginkgetin (purity > 96%) was obtained from Nanjing Puyi Biological Technology Co., Ltd. (Nanjing, China). Simvastatin was obtained from Hangzhou MSD Pharmaceutical Co., Ltd. (Hangzhou, China). Vitamin D3, propylthiouracil and sodium taurocholate were provided by Shanghai Fuxing Zhaohui Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol was purchased from Shanghai Lanji Science and Technology Development Co., Ltd. (Shanghai, China). The following primary antibodies were used for Western blot assays: MMP-2 and MMP-9 (Proteintech Group, Chicago, IL, USA); eNOS, iNOS, and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibody Anti-Rabbit IgG H&L (HRP) was obtained from Abcam (Cambridge, UK). 511
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Fig. 2. Ginkgetin improves serum biochemical parameters in rats with atherosclerosis. Biochemical analyses of serum levels of TC (A), TG (B), LDL-C (C), and HDL-C (D). Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
(1 mm3) was fixed in 10% phosphate-buffered formaldehyde for 2 h. After washing with phosphate-buffered saline, the fixed tissues were dehydrated in graded ethanol and embedded in paraffin. Paraffin sections of 4–6 μm were sliced using a sliding microtome, and paraffin was removed using xylene. Tissue sections were stained with HE reagents according to standard procedures. Photographs were blindly taken at random fields under a microscope (ZEISS Axio vert. A1, Germany). The intima-media thickness and ratio of intima/media were measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). In addition, the aortic arch and abdominal aorta were stained en face with Oil red O (60% saturated Oil red O in 40% deionized water; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The atherosclerotic lesion areas were measured using the Image-Pro Plus 6.0 software. Representative views were shown.
2.2. Animal procedures and treatments All experimental procedures were approved by the Institutional and Local Committee on the Care and Use of Animals, and all animals received humane care according to the National Institutes of Health (USA) guidelines. Seventy-five male Sprague-Dawley rats (180–200 g body weight) were provided by Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). They were exposed to a 12 h light/dark cycle and had free access to drinking water. After 1 week adaptive feeding with normal diet, the rats were divided into two groups, namely, control group (n = 15) and atherosclerosis group (n = 60). The control rats were received an intraperitoneal injection with 0.9% saline solution, and were fed normal diet during the whole course of experiments. The rats in atherosclerosis group were intraperitoneally injected with vitamin D3 at 600,000 IU/kg and then were fed an atherogenic diet containing 3% cholesterol, 0.5% sodium cholate, 0.2% propyl thiouracil, 5% refined sugar, 10% lard, and 81.3% chow diet. After 12 weeks, 3 rats in the control group and 5 rats in the atherosclerosis group were randomly selected and scarified, and their thoracic aortas were collected for hematoxylin-eosin (HE) staining to confirm the formation of atherosclerosis. Subsequently, the atherosclerotic rats were randomly divided into 5 groups (11 rats in each group), namely, model group, simvastatin treatment group (2 mg/kg/d), ginkgetin treatment groups at low (25 mg/kg/d), moderate (50 mg/kg/d), and high (100 mg/kg/d) doses. Rats in treatment groups were administrated with simvastatin or ginkgetin by gavage daily. Rats in control and model groups were given equal amount of 0.9% saline solution by gavage daily. After 8-week treatment, all rats were sacrificed after being anesthetized by intraperitoneal injection with pentobarbital (50 mg/kg). Blood samples were collected from the abdominal aorta and the thoracic aortas were harvested for analyses.
2.4. Serum biochemical analyses Blood samples from the abdominal aorta were drawn (10 ml), and the serum was collected and stored at −20 °C. Serum levels of total cholesterol (TC), triglyceride (TG), LDL-C, and high-density lipoprotein cholesterol (HDL-C) were measured using an Automatic Biochemistry Analyzer (HICATHI) according to the manufacturer’s instructions. 2.5. Determination of NO and NOs levels Levels of NO and NOS in serum and thoracic aortas were determined using corresponding enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the protocol provided by the manufacture. 2.6. Real-time PCR
2.3. Histopathology
Total RNA was extracted from the thoracic aortas using TRI reagent according to the protocol provided by the manufacturer (SigmaAldrich, St. Louis, MO, USA). Total RNA (1 μg) was treated with DNase I
After removal of epicardial adipose tissues, the thoracic aortas 512
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Fig. 3. Ginkgetin reduces the expression of MMP-2 and MMP-9 in thoracic aortas in rats with atherosclerosis. (A) Real-time PCR analyses of the mRNA expression of MMP-2 and MMP-9. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group. (B) Western blot analyses of the protein expression of MMP-2 and MMP9 with quantification. Significance: **P < 0.01 versus control group; ##P < 0.01 versus model group.
CTTCACCACCTTCT-3′. mRNA levels were expressed as fold changes after normalization with GAPDH.
to eliminate genomic DNA contamination, followed by synthesis of the first strand using the reverse transcription system (Promega, Madison, Wisconsin, USA). Reverse transcription was carried out as follows: 42 °C for 30 min, 95 °C for 5 min and 4 °C for 5 min (one cycle). Real-time PCR was performed in 25 μl of reaction solution containing 12.5 μl 2 × iQSYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, USA), 300 nM primers and complementary DNA. The cycles for PCR were as follows: 95 °C for 7 min, 40 cycles of 95 °C for 20 s, 54 °C for 30 s and 72 °C for 30 s. Melting curves were determined by heat-denaturing PCR products over a 35 °C temperature gradient at 0.2 °C/s from 60 to 95 °C. Fold changes in the mRNA levels of target genes were related to the invariant control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were calculated as described [12]. The following primers (GenScript, Nanjing, China) were used in real-time PCR: MMP-2: (F) 5′-GCTGATACTGACACTGGTACTG-3′, (R) 5′-CAATCTTTT CTGGGAGCTC-3′; MMP-9: (F) 5′-AAGGATGGTCTACTGGCAC-3′, (R) 5′-AGAGATTCTCACTGGGGC-3′; eNOS: (F) 5′-GGAAGCTGTCCCTGAT CCGAGC-3′, (R) 5′-AACTTGGAGCCGTTGTGCGCTC-3′, iNOS: (F) 5′-TGGAGCCCCTGAAGAG-3′, (R) 5′-AAGTGCGTTGTGCGGTAGC-3′, and GAPDH: (F) 5′-GGCCCCTCTGGAAAGCTGTG-3′, (R) 5′-CCGCCTG
2.7. Western blot analysis Total proteins were prepared from pieces of thoracic aortas using ice-cold radioimmunoprecipitation assay lysis buffer containing 150 mM NaCl, 50 mM Tris, 0.1% sodium dodecyl sulphate, 1% Nonidet P-40, and 0.5% deoxycholate supplemented with protease inhibitors. Protein concentrations were determined using the BCA protein assay kit according to the protocol provided by the manufacturer (Pierce Chemical, Rockford, Illinois, USA). Forty micrograms of total protein was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, Massachusetts, USA), and blocked with 5% skim milk in Tris-buffered saline containing 0.1% tween. Target proteins were detected by their primary antibodies, respectively, and horseradish peroxidase-conjugated secondary antibodies. GAPDH was probed as an internal control. Protein bands were visualized using chemiluminescence reagent (Amersham, Chalfont St Giles, Bucks, UK). 513
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Fig. 4. Ginkgetin increases the levels of NO and NOs in serum and thoracic aortas in rats with atherosclerosis. Measurement of NO levels in serum (A) and thoracic aortas (B), and measurement of NOS levels in serum (C) and thoracic aortas (D). Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
but ginkgetin reduced the plaque size dose-dependently in atherosclerotic rats (Fig. 1D). The positive drug simvastatin also significantly improved the histology of thoracic aortas (Fig. 1B–D). Furthermore, biochemical analyses showed that the serum levels of TC, TG and LDL-C were significantly elevated in atherosclerotic rats, but the serum levels of HDL-C were significantly decreased (Fig. 2A–D). However, ginkgetin dose-dependently decreased the serum levels of TC, TG and LDL-C and restored the serum levels of HDL-C (Fig. 2A–D). Simvastatin also corrected these lipid-related parameters (Fig. 2A–D). Taken together, these data indicated that ginkgetin attenuated atherosclerosis in rats.
The densities of bands were normalized to GAPDH. The levels of target protein bands were densitometrically determined by using Quantity Ones 4.4.1 (Bio-Rad, Hercules, CA, USA). The variation in the density was expressed as fold changes compared with the control in the blot. 2.8. Statistical analyses Data were presented as mean ± SD, and results were analyzed using SPSS16.0 software. The significance of difference was determined by one-way ANOVA with the post-hoc Dunnett’s test. Values of P < 0.05 were considered to be statistically significant.
3.2. Ginkgetin reduces the expression of MMP-2 and MMP-9 in thoracic aortas in rats with atherosclerosis
3. Results
We next investigated the effects of ginkgetin on the expression of matrix metalloproteinases (MMPs) in rat thoracic aortas. These molecules serve as important regulators for extracellular matrix alteration in the pathogenesis of atherosclerosis. Real-time PCR analyses demonstrated that the mRNA expression of MMP-2 and MMP-9 was significantly increased in thoracic aortas of atherosclerotic rats, but ginkgetin downregulated their mRNA levels dose-dependently (Fig. 3A). Moreover, Western blot analyses revealed that MMP-2 and MMP-9 were upregulated at the protein level in thoracic aortas of atherosclerotic rats, but ginkgetin reduced their protein expression dose-dependently (Fig. 3B). Treatment with simvastatin also downregulated the expression of MMP-2 and MMP-9 at both gene and protein levels (Fig. 3A and B). Collectively, ginkgetin modulated the MMPs system contributing to the attenuation of atherosclerosis in rats.
3.1. Ginkgetin improves histology, reduces lipid deposition, and corrects serum biochemical parameters in rats with atherosclerosis We established an experimental atherosclerosis model in rats fed an atherogenic diet combined with single intraperitoneally injection with vitamin D3. After 12 weeks, the thoracic aortas from selected rats of each group were isolated for histological examination. Obvious lipid infiltration beneath ascular intima was observed in the model rats (Fig. 1A), confirming the formation of atherosclerosis. Subsequently, the atherosclerotic rats were treated with ginkgetin or the positive drug simvastatin for 8 weeks. Histopathological examinations exhibited significantly thickened arterial wall and widened intimal space concomitant with deposition of cholesterol and lipids and formation of calcification in the thoracic aortas of atherosclerotic rats. However, treatment with ginkgetin reduced lipid deposition and improved thoracic aortic intima structure dose-dependently, and the most significant ameliorative effects were produced by ginkgetin at the high dose (Fig. 1B). Determination of intima-media thickness and intima/media ratio gave consistent results (Fig. 1C). Additionally, oil red O staining with aortic arch and abdominal aorta showed that the atherosclerotic lesion areas in model rats were significantly larger than that of control,
3.3. Ginkgetin regulates NO/NOs system in rats with atherosclerosis We subsequently examined the effects of ginkgetin on the NO/NOS system in atherosclerotic rats. We found that the NO contents were significantly decreased in serum and thoracic aortas in atherosclerotic rats, but ginkgetin increased the NO levels dose-dependently (Fig. 4A 514
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Fig. 5. Ginkgetin upregulates eNOs expression and downregulates iNOs expression in thoracic aortas of rats with atherosclerosis. (A) Real-time PCR analyses of the mRNA expression of eNOS and iNOS in thoracic aortas. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group. (B) Western blot analyses of the protein expression of eNOS and iNOS in thoracic aortas with quantification. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
successfully established the experimental atherosclerosis model in rats showing typical pathological alterations during the 12-week period by feeding high-fat diet combined with single intraperitoneal injection of vitamin D3, followed by 8-week drug intervention. Although there is a possibility that the atherosclerosis could be recovered spontaneously to a certain extent, our data at the end of experiments demonstrated that the serum levels of TC, TG and LDL-C were all increased significantly and the serum levels of HDL-C were decreased significantly in the model rats. In addition, the aortic tunica intima of rats exhibited serious pathological changes, including increased intima-media thickness and apparent atheromatous plaque. Therefore, we presumed that the atherosclerosis in the model rats was not spontaneously reduced during the 8-week treatment in current settings. The obtained findings could reflect the anti-atherosclerotic effects of drugs examined in this study. We then used this model to evaluate the potential therapeutic effects of ginkgetin, a major active constituent of Ginkgo biloba that is useful for preventing and treating cardiovascular diseases [15]. Epidemiological evidence has revealed that elevation of serum cholesterol levels is an important factor in atherogenesis, and that there is a positive correlation between the severity of atherosclerosis and serum levels of cholesterol and LDL-C [16]. In addition, it was reported that the clinical complications of atherosclerosis could be suppressed, and the lifetime could be prolonged by reducing the serum levels of lipids such
and B). Further measurements of NOS levels showed that the atherosclerotic rats had significantly decreased NOS levels in serum and thoracic aortas, but rats received ginkgetin and simvastatin treatments exhibited increased NOS levels (Fig. 4C and D). We subsequently determined the expression of eNOS and iNOS, and observed that the mRNA and protein expression of eNOS was significantly decreased in thoracic aortas of atherosclerotic rats, but the mRNA and protein levels of iNOS were significantly upregulated (Fig. 5A and B). However, treatments with ginkgetin or simvastatin restored eNOS expression and reduced iNOS expression at both mRNA and protein levels in thoracic aortas of atherosclerotic rats (Fig. 5A and B). Altogether, regulation of NO/NOs system was involved in ginkegtin attenuation of atherosclerosis in rats. 4. Discussion Atherosclerosis is a chronic disease with lipids and inflammation playing key roles in all stages of the disease from initiation through progression ultimately to thrombotic complications [13]. Increasing epidemiological evidence reveals that the incidence of atherosclerosis is closely related to metabolic disorder of blood lipids. Lipids are deposited to induce inflammatory cell infiltration and phagocytosis, and plaque calcification after endothelium injury [14]. In current study, we 515
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concerning this article.
as TC, TG and LDL-C [17]. However, atherosclerosis can be affected by increasing the HDL-C levels [18], and the serum HDL-C levels were inversely correlated with the risk of atherosclerosis [19]. In the present study, we demonstrated that treatment with ginkgetin significantly improved hyperlipidemia, suggesting that ginkgetin could be beneficial for the whole lipid profiles, and attenuate the initiation and progression of atherosclerosis. Atherosclerosis involves the accumulation of vascular SMCs, white blood cells and modified lipids in the intima layer of medium- and large-sized arterial blood vessels [20]. The SMCs accumulating at the tunica intima of arteries can migrate from the tunica media of the blood vessels, and their proliferation in the initma help form atheroma plaque. The MMPs system has been implicated in induction of proliferation and migration of SMCs both in vivo and in vitro [21]. The key role of MMPs in this process is to degrade the elastic lamina barrier of extracellular matrix through its proteolytic activity, resulting in pathological conditions such as rheumatoid arthritis and vascular disease [22]. MMP-2 is a member of the MMP family that has been extensively studied, which plays a prime role in increasing SMC migration and vascular remodeling [23]. MMP-9 is produced by various matrix cells and macrophages, which mainly participates in degradation of active substance in extracellular matrix in different tissues [24]. Our current data revealed that the expression of MMP-2 and MMP-9 in thoracic aortas of atherosclerotic rats was reduced by ginkgetin. These effects might attenuate the remodeling of vascular extracellular matrix and reduce the migration of SMCs, thus contributing to the improvement of atherosclerosis in rats. NO is a well-characterized endothelium-derived vasodilator synthesized by eNOS, or iNOS following stimulation by cytokines. A large number of studies have demonstrated that NO suppresses the development of atherosclerosis by reducing monocyte-endothelial cell interaction, preventing platelet adherence and aggregation, and inhibiting SMC proliferation. Our current data showed that the NO levels in serum and thoracic aortas were noticeably increased in atherosclerotic rats treated with ginkgetin. These effects contributed to ginkgetin attenuation of atherosclerosis in rats. We subsequently examined the generators of NO. Dysfunctional eNOS has been deeply involved in the impaired endothelium-dependent relaxation in atherosclerotic vessels [5]. In the present study, the eNOS expression in serum and thoracic aortas was decreased in atherosclerotic rats, confirming the notion that unavailability of NO promotes the pathogenesis of atherosclerosis. Ginkgetin could increase the expression of eNOS in atherosclerotic rats. Furthermore, iNOS can be induced considerably in vascular SMCs by cytokines released from infiltrating inflammatory cells after injury [8]. Upregulation of iNOS was also found in the majority of samples as early as the fatty streak stage and in all of the advanced stages of plaques [7], indicating that iNOS expression and activity were correlated with the progression of atherosclerosis. We herein observed that ginkgetin diminished the elevation of iNOS expression in both serum and thoracic aortas of atherosclerotic rats. Our data revealed that modulation of NO/NOS system could be an important mechanism underlying ginkgetin amelioration of atherosclerosis in rats. It would be attractive to use the SMC culture system to investigate the signaling pathways involved in ginkgetin regulation of MMPs and NO systems for treatment of atherosclerosis. In conclusion, we provided the evidence that ginkgetin had therapeutic effects on experimental atherosclerosis in rats. These effects were associated with improvement of lipid profile and regulation of MMPs and NO/NOS systems. These discoveries suggested that ginkgetin could be further exploited as a therapeutic option for atherosclerosis.
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Disclosure of interest The authors declare that they have no conflicts of interest
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Ginkgetin ameliorates experimental atherosclerosis in rats Naqi Lian, Jing Tong, Wenwen Li, Jingzhen Wu, Yu Li
⁎
T
School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing 210023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Atherosclerosis Ginkgetin Matrix metalloproteinases Nitric oxide Nitric oxide synthase
Atherosclerosis is a common disease seriously detrimental to human health. Natural products are important sources of therapeutic candidates for atherosclerosis. We here evaluated the effects of ginkgetin on experimental atherosclerosis in rats and explored the underlying mechanisms. Atherosclerosis was induced by high-fat diet for 12 weeks combined with single intraperitoneal injection of vitamin D3 in rats. The atherosclerotic rats were then treated with ginkgetin at 25, 50 and 100 mg/kg/d or simvastatin at 2 mg/kg/d for 8 weeks. Blood and thoracic aortas were collected for analyses of histopathology, lipid deposition, serum biochemistry, matrix metalloproteinases (MMPs), and nitric oxide (NO)/NO synthase (NOS) system. We found that ginkgetin improved thoracic aortic intima structure, reduced intima-media thickness and intima/media ratio, and attenuated lipid deposition in aorta of atherosclerotic rats. Ginkgetin also decreased the serum levels of total cholesterol, triglyceride and low-density lipoprotein cholesterol, but restored the serum levels of high-density lipoprotein cholesterol in atherosclerotic rats. Additionally, ginkgetin reduced the mRNA and protein expression of MMP-2 and MMP-9 in thoracic aortas of rats with atherosclerosis. Further examinations showed that ginkgetin increased the NO and NOS levels in serum and thoracic aortas. Ginkgetin also unregulated the expression of endothelial NOS and downregulated the expression of inducible NOS at both mRNA and protein levels in thoracic aortas of atherosclerotic rats. Altogether, ginkgetin showed therapeutic effects on experimental atherosclerosis associated with improving lipid profile and modulating the MMPs and NO/NOS systems in rats. Ginkgetin could be a promising candidate for the treatment of atherosclerosis.
1. Introduction Atherosclerosis represents one of the most common causes of cardiovascular diseases including unstable angina, myocardial infarction and ischemic heart failure. It is characterized by smooth muscle cell (SMC) proliferation, endothelial dysfunction, inflammatory responses, extracellular matrix alteration and thrombosis [1]. Development of atherosclerosis leads to insufficient blood supply to the coronary arteries and subsequent myocardial ischemia, which are basic pathological processes of coronary heart disease [2]. Although atherosclerosis has been studied for decades, the underlying mechanisms remain poorly understood. Many risky factors such as dyslipidemia, hypertension and diabetes have been proposed, among which dyslipidemia is thought to play a key role in the pathogenesis of atherosclerosis. Patients with dyslipidemia usually have higher serum levels of low-density lipoprotein-cholesterol (LDL-C), which can be oxidized and converted into oxidized LDL, resulting in oxidative stress, inflammation and injury in the endothelium [3].
Accumulating evidence indicates that the nitric oxide (NO) and NO synthase (NOS) system in vascular SMCs are important regulators of local vascular injury associated with atherosclerosis. In healthy vessels, NO is produced from the endothelium by the endothelial isoform of NOS (eNOS), and functions as an endothelium-derived relaxing factor critical for maintaining vascular homeostasis [4]. Impairment of endothelium-dependent relaxation occurs in atherosclerotic vessels and represents the reduced eNOS-derived NO bioavailability, promoting the development of atherosclerosis [5]. Under injury and atherosclerotic circumstances, the inducible isoform of NOS (iNOS) is commonly unregulated to compensate for the loss of functional endothelium and eNOS [6]. Increased expression of iNOS has been described in atherosclerotic human plaques in macrophages, endothelial cells and SMCs, supporting the role for iNOS in promoting the pathogenesis of atherosclerosis [7]. In the advanced atherosclerotic plaques, smooth muscle and macrophage-derived iNOS may continually promote a pathogenic environment by enhancing oxidative and nitrosative stress [8]. Current knowledge suggests the NO and NOS system as an attractive target for
Abbreviations: eNOS, endothelial isoform of NOS; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL-C, high-density lipoprotein cholesterol; HE, hematoxylin-eosin; iNOS, inducible isoform of NOS; LDL-C, low-density lipoprotein-cholesterol; MMPs, matrix metalloproteinases; NO, nitric oxide; NOS, NO synthase; SMC, smooth muscle cell; TC, total cholesterol; TG, triglyceride ⁎ Corresponding author. E-mail address: [email protected] (Y. Li). https://doi.org/10.1016/j.biopha.2018.03.107 Received 3 November 2017; Received in revised form 17 March 2018; Accepted 17 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. Ginkgetin improves histology and reduces lipid deposition in rats with atherosclerosis. (A) Scheme of induction of atherosclerosis in rats for 12 weeks and treatments with Ginkgetin or simvastatin for 8 weeks. After 12 weeks, atherosclerosis was confirmed by HE staining with thoracic aortas. (B) HE staining with thoracic aortas for histological assessment after 8-week treatment. (C) Determination of intima-media thickness and intima/media ratio. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group. (D) Oil red O staining with aortic arch and thoracic aorta, and quantification of the plaque size (% of total aorta). Significance: **P < 0.01 versus control group; ##P < 0.01 versus model group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2. Materials and methods
the treatment of atherosclerosis. Herbal medicines have gained tremendous attention due to their therapeutic benefits for atherosclerosis. Ginkgo biloba belongs to the botanical family of Ginkgocea, and its leaves contain high levels of flavonoids, bioflavonoids, and terpenoids [9]. Notably, ginkgetin, one of primary biflavone compounds isolated from Ginkgo biloba leaves, has been reported to exhibit anti-inflammatory, anti-fungal, neuroprotective, and anti-tumor activities [10], highlighting ginkgetin as an attractive therapeutic candidate. Given the clinical evidence of using Ginkgo biloba extract as a therapy for cardiovascular and cerebrovascular diseases [11], we here evaluated the ameliorative effects of ginkgetin on the experimental atherosclerosis in rats and explored the underlying mechanisms.
2.1. Chemicals and antibodies Ginkgetin (purity > 96%) was obtained from Nanjing Puyi Biological Technology Co., Ltd. (Nanjing, China). Simvastatin was obtained from Hangzhou MSD Pharmaceutical Co., Ltd. (Hangzhou, China). Vitamin D3, propylthiouracil and sodium taurocholate were provided by Shanghai Fuxing Zhaohui Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol was purchased from Shanghai Lanji Science and Technology Development Co., Ltd. (Shanghai, China). The following primary antibodies were used for Western blot assays: MMP-2 and MMP-9 (Proteintech Group, Chicago, IL, USA); eNOS, iNOS, and GAPDH (Cell Signaling Technology, Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibody Anti-Rabbit IgG H&L (HRP) was obtained from Abcam (Cambridge, UK). 511
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Fig. 2. Ginkgetin improves serum biochemical parameters in rats with atherosclerosis. Biochemical analyses of serum levels of TC (A), TG (B), LDL-C (C), and HDL-C (D). Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
(1 mm3) was fixed in 10% phosphate-buffered formaldehyde for 2 h. After washing with phosphate-buffered saline, the fixed tissues were dehydrated in graded ethanol and embedded in paraffin. Paraffin sections of 4–6 μm were sliced using a sliding microtome, and paraffin was removed using xylene. Tissue sections were stained with HE reagents according to standard procedures. Photographs were blindly taken at random fields under a microscope (ZEISS Axio vert. A1, Germany). The intima-media thickness and ratio of intima/media were measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). In addition, the aortic arch and abdominal aorta were stained en face with Oil red O (60% saturated Oil red O in 40% deionized water; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The atherosclerotic lesion areas were measured using the Image-Pro Plus 6.0 software. Representative views were shown.
2.2. Animal procedures and treatments All experimental procedures were approved by the Institutional and Local Committee on the Care and Use of Animals, and all animals received humane care according to the National Institutes of Health (USA) guidelines. Seventy-five male Sprague-Dawley rats (180–200 g body weight) were provided by Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). They were exposed to a 12 h light/dark cycle and had free access to drinking water. After 1 week adaptive feeding with normal diet, the rats were divided into two groups, namely, control group (n = 15) and atherosclerosis group (n = 60). The control rats were received an intraperitoneal injection with 0.9% saline solution, and were fed normal diet during the whole course of experiments. The rats in atherosclerosis group were intraperitoneally injected with vitamin D3 at 600,000 IU/kg and then were fed an atherogenic diet containing 3% cholesterol, 0.5% sodium cholate, 0.2% propyl thiouracil, 5% refined sugar, 10% lard, and 81.3% chow diet. After 12 weeks, 3 rats in the control group and 5 rats in the atherosclerosis group were randomly selected and scarified, and their thoracic aortas were collected for hematoxylin-eosin (HE) staining to confirm the formation of atherosclerosis. Subsequently, the atherosclerotic rats were randomly divided into 5 groups (11 rats in each group), namely, model group, simvastatin treatment group (2 mg/kg/d), ginkgetin treatment groups at low (25 mg/kg/d), moderate (50 mg/kg/d), and high (100 mg/kg/d) doses. Rats in treatment groups were administrated with simvastatin or ginkgetin by gavage daily. Rats in control and model groups were given equal amount of 0.9% saline solution by gavage daily. After 8-week treatment, all rats were sacrificed after being anesthetized by intraperitoneal injection with pentobarbital (50 mg/kg). Blood samples were collected from the abdominal aorta and the thoracic aortas were harvested for analyses.
2.4. Serum biochemical analyses Blood samples from the abdominal aorta were drawn (10 ml), and the serum was collected and stored at −20 °C. Serum levels of total cholesterol (TC), triglyceride (TG), LDL-C, and high-density lipoprotein cholesterol (HDL-C) were measured using an Automatic Biochemistry Analyzer (HICATHI) according to the manufacturer’s instructions. 2.5. Determination of NO and NOs levels Levels of NO and NOS in serum and thoracic aortas were determined using corresponding enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the protocol provided by the manufacture. 2.6. Real-time PCR
2.3. Histopathology
Total RNA was extracted from the thoracic aortas using TRI reagent according to the protocol provided by the manufacturer (SigmaAldrich, St. Louis, MO, USA). Total RNA (1 μg) was treated with DNase I
After removal of epicardial adipose tissues, the thoracic aortas 512
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Fig. 3. Ginkgetin reduces the expression of MMP-2 and MMP-9 in thoracic aortas in rats with atherosclerosis. (A) Real-time PCR analyses of the mRNA expression of MMP-2 and MMP-9. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group. (B) Western blot analyses of the protein expression of MMP-2 and MMP9 with quantification. Significance: **P < 0.01 versus control group; ##P < 0.01 versus model group.
CTTCACCACCTTCT-3′. mRNA levels were expressed as fold changes after normalization with GAPDH.
to eliminate genomic DNA contamination, followed by synthesis of the first strand using the reverse transcription system (Promega, Madison, Wisconsin, USA). Reverse transcription was carried out as follows: 42 °C for 30 min, 95 °C for 5 min and 4 °C for 5 min (one cycle). Real-time PCR was performed in 25 μl of reaction solution containing 12.5 μl 2 × iQSYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, USA), 300 nM primers and complementary DNA. The cycles for PCR were as follows: 95 °C for 7 min, 40 cycles of 95 °C for 20 s, 54 °C for 30 s and 72 °C for 30 s. Melting curves were determined by heat-denaturing PCR products over a 35 °C temperature gradient at 0.2 °C/s from 60 to 95 °C. Fold changes in the mRNA levels of target genes were related to the invariant control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were calculated as described [12]. The following primers (GenScript, Nanjing, China) were used in real-time PCR: MMP-2: (F) 5′-GCTGATACTGACACTGGTACTG-3′, (R) 5′-CAATCTTTT CTGGGAGCTC-3′; MMP-9: (F) 5′-AAGGATGGTCTACTGGCAC-3′, (R) 5′-AGAGATTCTCACTGGGGC-3′; eNOS: (F) 5′-GGAAGCTGTCCCTGAT CCGAGC-3′, (R) 5′-AACTTGGAGCCGTTGTGCGCTC-3′, iNOS: (F) 5′-TGGAGCCCCTGAAGAG-3′, (R) 5′-AAGTGCGTTGTGCGGTAGC-3′, and GAPDH: (F) 5′-GGCCCCTCTGGAAAGCTGTG-3′, (R) 5′-CCGCCTG
2.7. Western blot analysis Total proteins were prepared from pieces of thoracic aortas using ice-cold radioimmunoprecipitation assay lysis buffer containing 150 mM NaCl, 50 mM Tris, 0.1% sodium dodecyl sulphate, 1% Nonidet P-40, and 0.5% deoxycholate supplemented with protease inhibitors. Protein concentrations were determined using the BCA protein assay kit according to the protocol provided by the manufacturer (Pierce Chemical, Rockford, Illinois, USA). Forty micrograms of total protein was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, Massachusetts, USA), and blocked with 5% skim milk in Tris-buffered saline containing 0.1% tween. Target proteins were detected by their primary antibodies, respectively, and horseradish peroxidase-conjugated secondary antibodies. GAPDH was probed as an internal control. Protein bands were visualized using chemiluminescence reagent (Amersham, Chalfont St Giles, Bucks, UK). 513
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Fig. 4. Ginkgetin increases the levels of NO and NOs in serum and thoracic aortas in rats with atherosclerosis. Measurement of NO levels in serum (A) and thoracic aortas (B), and measurement of NOS levels in serum (C) and thoracic aortas (D). Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
but ginkgetin reduced the plaque size dose-dependently in atherosclerotic rats (Fig. 1D). The positive drug simvastatin also significantly improved the histology of thoracic aortas (Fig. 1B–D). Furthermore, biochemical analyses showed that the serum levels of TC, TG and LDL-C were significantly elevated in atherosclerotic rats, but the serum levels of HDL-C were significantly decreased (Fig. 2A–D). However, ginkgetin dose-dependently decreased the serum levels of TC, TG and LDL-C and restored the serum levels of HDL-C (Fig. 2A–D). Simvastatin also corrected these lipid-related parameters (Fig. 2A–D). Taken together, these data indicated that ginkgetin attenuated atherosclerosis in rats.
The densities of bands were normalized to GAPDH. The levels of target protein bands were densitometrically determined by using Quantity Ones 4.4.1 (Bio-Rad, Hercules, CA, USA). The variation in the density was expressed as fold changes compared with the control in the blot. 2.8. Statistical analyses Data were presented as mean ± SD, and results were analyzed using SPSS16.0 software. The significance of difference was determined by one-way ANOVA with the post-hoc Dunnett’s test. Values of P < 0.05 were considered to be statistically significant.
3.2. Ginkgetin reduces the expression of MMP-2 and MMP-9 in thoracic aortas in rats with atherosclerosis
3. Results
We next investigated the effects of ginkgetin on the expression of matrix metalloproteinases (MMPs) in rat thoracic aortas. These molecules serve as important regulators for extracellular matrix alteration in the pathogenesis of atherosclerosis. Real-time PCR analyses demonstrated that the mRNA expression of MMP-2 and MMP-9 was significantly increased in thoracic aortas of atherosclerotic rats, but ginkgetin downregulated their mRNA levels dose-dependently (Fig. 3A). Moreover, Western blot analyses revealed that MMP-2 and MMP-9 were upregulated at the protein level in thoracic aortas of atherosclerotic rats, but ginkgetin reduced their protein expression dose-dependently (Fig. 3B). Treatment with simvastatin also downregulated the expression of MMP-2 and MMP-9 at both gene and protein levels (Fig. 3A and B). Collectively, ginkgetin modulated the MMPs system contributing to the attenuation of atherosclerosis in rats.
3.1. Ginkgetin improves histology, reduces lipid deposition, and corrects serum biochemical parameters in rats with atherosclerosis We established an experimental atherosclerosis model in rats fed an atherogenic diet combined with single intraperitoneally injection with vitamin D3. After 12 weeks, the thoracic aortas from selected rats of each group were isolated for histological examination. Obvious lipid infiltration beneath ascular intima was observed in the model rats (Fig. 1A), confirming the formation of atherosclerosis. Subsequently, the atherosclerotic rats were treated with ginkgetin or the positive drug simvastatin for 8 weeks. Histopathological examinations exhibited significantly thickened arterial wall and widened intimal space concomitant with deposition of cholesterol and lipids and formation of calcification in the thoracic aortas of atherosclerotic rats. However, treatment with ginkgetin reduced lipid deposition and improved thoracic aortic intima structure dose-dependently, and the most significant ameliorative effects were produced by ginkgetin at the high dose (Fig. 1B). Determination of intima-media thickness and intima/media ratio gave consistent results (Fig. 1C). Additionally, oil red O staining with aortic arch and abdominal aorta showed that the atherosclerotic lesion areas in model rats were significantly larger than that of control,
3.3. Ginkgetin regulates NO/NOs system in rats with atherosclerosis We subsequently examined the effects of ginkgetin on the NO/NOS system in atherosclerotic rats. We found that the NO contents were significantly decreased in serum and thoracic aortas in atherosclerotic rats, but ginkgetin increased the NO levels dose-dependently (Fig. 4A 514
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Fig. 5. Ginkgetin upregulates eNOs expression and downregulates iNOs expression in thoracic aortas of rats with atherosclerosis. (A) Real-time PCR analyses of the mRNA expression of eNOS and iNOS in thoracic aortas. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group. (B) Western blot analyses of the protein expression of eNOS and iNOS in thoracic aortas with quantification. Significance: **P < 0.01 versus control group; #P < 0.05 versus model group, ##P < 0.01 versus model group.
successfully established the experimental atherosclerosis model in rats showing typical pathological alterations during the 12-week period by feeding high-fat diet combined with single intraperitoneal injection of vitamin D3, followed by 8-week drug intervention. Although there is a possibility that the atherosclerosis could be recovered spontaneously to a certain extent, our data at the end of experiments demonstrated that the serum levels of TC, TG and LDL-C were all increased significantly and the serum levels of HDL-C were decreased significantly in the model rats. In addition, the aortic tunica intima of rats exhibited serious pathological changes, including increased intima-media thickness and apparent atheromatous plaque. Therefore, we presumed that the atherosclerosis in the model rats was not spontaneously reduced during the 8-week treatment in current settings. The obtained findings could reflect the anti-atherosclerotic effects of drugs examined in this study. We then used this model to evaluate the potential therapeutic effects of ginkgetin, a major active constituent of Ginkgo biloba that is useful for preventing and treating cardiovascular diseases [15]. Epidemiological evidence has revealed that elevation of serum cholesterol levels is an important factor in atherogenesis, and that there is a positive correlation between the severity of atherosclerosis and serum levels of cholesterol and LDL-C [16]. In addition, it was reported that the clinical complications of atherosclerosis could be suppressed, and the lifetime could be prolonged by reducing the serum levels of lipids such
and B). Further measurements of NOS levels showed that the atherosclerotic rats had significantly decreased NOS levels in serum and thoracic aortas, but rats received ginkgetin and simvastatin treatments exhibited increased NOS levels (Fig. 4C and D). We subsequently determined the expression of eNOS and iNOS, and observed that the mRNA and protein expression of eNOS was significantly decreased in thoracic aortas of atherosclerotic rats, but the mRNA and protein levels of iNOS were significantly upregulated (Fig. 5A and B). However, treatments with ginkgetin or simvastatin restored eNOS expression and reduced iNOS expression at both mRNA and protein levels in thoracic aortas of atherosclerotic rats (Fig. 5A and B). Altogether, regulation of NO/NOs system was involved in ginkegtin attenuation of atherosclerosis in rats. 4. Discussion Atherosclerosis is a chronic disease with lipids and inflammation playing key roles in all stages of the disease from initiation through progression ultimately to thrombotic complications [13]. Increasing epidemiological evidence reveals that the incidence of atherosclerosis is closely related to metabolic disorder of blood lipids. Lipids are deposited to induce inflammatory cell infiltration and phagocytosis, and plaque calcification after endothelium injury [14]. In current study, we 515
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concerning this article.
as TC, TG and LDL-C [17]. However, atherosclerosis can be affected by increasing the HDL-C levels [18], and the serum HDL-C levels were inversely correlated with the risk of atherosclerosis [19]. In the present study, we demonstrated that treatment with ginkgetin significantly improved hyperlipidemia, suggesting that ginkgetin could be beneficial for the whole lipid profiles, and attenuate the initiation and progression of atherosclerosis. Atherosclerosis involves the accumulation of vascular SMCs, white blood cells and modified lipids in the intima layer of medium- and large-sized arterial blood vessels [20]. The SMCs accumulating at the tunica intima of arteries can migrate from the tunica media of the blood vessels, and their proliferation in the initma help form atheroma plaque. The MMPs system has been implicated in induction of proliferation and migration of SMCs both in vivo and in vitro [21]. The key role of MMPs in this process is to degrade the elastic lamina barrier of extracellular matrix through its proteolytic activity, resulting in pathological conditions such as rheumatoid arthritis and vascular disease [22]. MMP-2 is a member of the MMP family that has been extensively studied, which plays a prime role in increasing SMC migration and vascular remodeling [23]. MMP-9 is produced by various matrix cells and macrophages, which mainly participates in degradation of active substance in extracellular matrix in different tissues [24]. Our current data revealed that the expression of MMP-2 and MMP-9 in thoracic aortas of atherosclerotic rats was reduced by ginkgetin. These effects might attenuate the remodeling of vascular extracellular matrix and reduce the migration of SMCs, thus contributing to the improvement of atherosclerosis in rats. NO is a well-characterized endothelium-derived vasodilator synthesized by eNOS, or iNOS following stimulation by cytokines. A large number of studies have demonstrated that NO suppresses the development of atherosclerosis by reducing monocyte-endothelial cell interaction, preventing platelet adherence and aggregation, and inhibiting SMC proliferation. Our current data showed that the NO levels in serum and thoracic aortas were noticeably increased in atherosclerotic rats treated with ginkgetin. These effects contributed to ginkgetin attenuation of atherosclerosis in rats. We subsequently examined the generators of NO. Dysfunctional eNOS has been deeply involved in the impaired endothelium-dependent relaxation in atherosclerotic vessels [5]. In the present study, the eNOS expression in serum and thoracic aortas was decreased in atherosclerotic rats, confirming the notion that unavailability of NO promotes the pathogenesis of atherosclerosis. Ginkgetin could increase the expression of eNOS in atherosclerotic rats. Furthermore, iNOS can be induced considerably in vascular SMCs by cytokines released from infiltrating inflammatory cells after injury [8]. Upregulation of iNOS was also found in the majority of samples as early as the fatty streak stage and in all of the advanced stages of plaques [7], indicating that iNOS expression and activity were correlated with the progression of atherosclerosis. We herein observed that ginkgetin diminished the elevation of iNOS expression in both serum and thoracic aortas of atherosclerotic rats. Our data revealed that modulation of NO/NOS system could be an important mechanism underlying ginkgetin amelioration of atherosclerosis in rats. It would be attractive to use the SMC culture system to investigate the signaling pathways involved in ginkgetin regulation of MMPs and NO systems for treatment of atherosclerosis. In conclusion, we provided the evidence that ginkgetin had therapeutic effects on experimental atherosclerosis in rats. These effects were associated with improvement of lipid profile and regulation of MMPs and NO/NOS systems. These discoveries suggested that ginkgetin could be further exploited as a therapeutic option for atherosclerosis.
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Disclosure of interest The authors declare that they have no conflicts of interest
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