Heart failure (HF) is a clinical syndrome caused by structural and/or functional cardiac modifications resulting in impairment of the ventricles to fill with or eject blood [1
]. HF comprises three main subtypes: HF with normal left ventricular ejection fraction (LVEF) (LVEF ≥ 50%), also called HF with preserved ejection fraction (HFpEF), HF with reduced LVEF (LVEF < 40%), also called HFrEF and HF with mid-range LVEF (LVEF = 40%–49%), also called HFmrEF [1
]. In general, HF is a major cause of morbidity and mortality in the developed world, with a high prevalence in the general population (1%–2%) [1
]. HF prognosis is concerning, with around 2%–17% of patients dying during the first hospital admission and more than 50% within the first five years [2
]. Moreover, HF is a complex disease with a multifactorial etiology, arterial hypertension being among the most prevalent causes for all HF subtypes [1
]. One animal model with clinical relevance in the study of HF is the abdominal aortic banding (AAB) in rats. AAB closely mimics the development of HF in humans as a cause of hypertension and can be used to study new therapeutic approaches in specific states of the disease [3
]. In this model, pressure overload gradually increases in intensity as the rat matures and leads to structural and functional abnormalities characteristic for HF [4
]. Also, inflammation, immune activation and oxidative stress have been identified as important causative processes in HF development and progression. Independently of the etiology, the pathophysiologic mechanisms that influence immune activation can be identical [5
]. Extensive activation of renin–angiotensin–aldosterone system in HF is a major pathophysiologic mechanism in HF and recently, aldosterone has been found to increase the expression of proinflammatory cytokines in macrophages, whereas natriuretic peptides have been identified to display anti-inflammatory effects in vivo [5
]. Excess mitochondrial production of reactive oxygen species, in comparison to antioxidant defense, has been observed as an important step in the pathophysiology of cardiac remodeling and HF [6
]. Thus, treatment with antioxidant complexes could be an interesting possible option to decrease HF progression and offer cardioprotection.
L. (L. barbarum
, known as goji berry or wolfberry) is a member of the Solanaceae family, well known in traditional herbal medicine, especially in China as a renowned Yin strengthening agent [7
]. Scientific investigations have focused on antioxidative and immunomodulatory effects in the context of atherosclerosis, neurodegenerative diseases and diabetes [7
]. The therapeutic effects of L. barbarum
have been attributed to its active polysaccharide complex (LBP), comprising six monosaccharides (galactose, rhamnose, glucose, mannose, arabinose and xylose). Cardioprotective effects of LBPs have already been observed in models of ischemia-reperfusion myocardial damage [8
], doxorubicin-induced cardiotoxicity [9
] and transgenic models, such as mice with microRNA-1 overexpression [12
Thus, the aim of our research was to evaluate the effects of LBPs in a pressure overload-induced HF model by echocardiography and analysis of inflammation and oxidative stress markers in rats.
L. (L. barbarum
) has been used in traditional Chinese medicine for a long time. Polysaccharides extracted from L. barbarum
comprise 6 sugars (galactose, rhamnose, glucose, mannose, arabinose and xylose; molar ratios 2.43, 4.22, 1.38, 0.95, 1 and 0.38, respectively), with furan and pyran rings and alpha and beta anomeric configurations [13
]. LBPs are important active constituents that have demonstrated pharmacological functions including antioxidative, immunomodulatory, antitumor, antiaging, neuroprotective, hypoglycemic and hypolipidemic effects [7
]. However, there are limited reports regarding effects of LBPs in the context of cardiovascular diseases. Of all cardiovascular diseases, HF has a high prevalence, with over 26 million people diagnosed worldwide. Despite advances in disease management, a diagnosis of HF carries significant risk of morbidity and mortality [14
]. Thus, the need for new therapeutic approaches in HF is still high and requires both the use of animal models that reproduce the human disease and also the testing of remedies with potential beneficial effects.
In the present study, HF was induced by banding of the descending abdominal aorta. This procedure induced a gradual increase in left ventricular after-load pressure over time, as previously described [4
]. This particular model is similar to the pathological evolution from hypertension to HF in humans; thus, this model is useful for the investigation of the effects of different therapeutic approaches in this particular context [15
]. So far, studies showed that at about 18 weeks after abdominal aortic banding, rats exhibit high pressure-overload, which in time induced structural and functional abnormalities indicative of HF [3
]. The present study confirms that at approximately 24 weeks post-surgery, AWT, PWT and LV mass increase, suggesting LV hypertrophy. Also, at this timepoint, LVESD and LVEDD increase and EF decreases, suggesting LV dilatation and systolic dysfunction. All these modifications are indicative of HFmrEF. After echocardiographic confirmation of HF, treatment with LBPs was initiated and terminated 12 weeks thereafter, at 36 weeks of the study. The aim of our study was to evaluate the effects of LBPs on inflammation and oxidative stress markers after the onset of the cardiac pathology and not the preventive effects. At the end of the treatment period (week 36), in the no treatment group (control AAB group) EF, an important marker of systolic function, decreased with ~10% compared to week 24, whereas in the treatment groups (AAB_100 and AAB_200), EF remained constant, suggesting possible prevention of cardiac structural and functional alteration. Similarly, in a mouse model of structural remodeling and cardiac contractile dysfunction induced by over-expression of microRNA-1, treatment with LBPs for two months resulted in cardiac contractile function improvement, more precisely cardiac output (CO), end-systolic pressure (ESP), end-systolic volume (ESV) and maximum derivative of change in systolic volume (dP/dtmax
) improvement [12
Also, our results show marked increases in circulating cytokines, including TNF-α and IL-6 and oxidative stress markers, such as malondialdehyde in AAB rats compared to controls. These results confirm preexisting clinical and preclinical studies that demonstrated the prevalence of oxidative stress, inflammation and immune system activation during HF [2
More specifically, similar to our findings, LV dilation and systolic dysfunction were found in other studies to be associated with a marked increase in circulating cytokines, including TNF-α and IL-6 [4
]. The present study shows a significant decrease of TNF-α and IL-6 after 12 weeks of treatment with 200 mg/kg bw/day LBPs. On the other hand, the lack of modifications in white blood cell count in AAB rats compared to control rats could signify that the increase in plasma cytokines does not originate from a systemic inflammatory state induced by white blood cells, but from more complex cardiac cellular signaling involving cytokines. To the best of our knowledge, our results represent the first reporting of LBPs effects on proinflammatory cytokines levels in a cardiovascular disease context. Such effects have been demonstrated before for LBPs, but mostly related to cancer immunotherapy [7
Moreover, our results show that HFmrEF is associated with significant oxidative stress expressed by significantly increased plasma MDA levels, the end-product of lipid peroxidation, in AAB rats compared to controls. Studies show that because cardiomyocytes have lower levels of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione-peroxidase (GSH-Px), the heart is more prone to oxidative stress [21
]. Twelve weeks of treatment with LBPs demonstrated a significant beneficial reducing effect over MDA levels for the 200 mg/kg bw/day dose of LBPs. The powerful antioxidant effect of L. barbarum
has been used for a long time in traditional Chinese medicine in the context of age-related diseases and has been demonstrated to derive primarily from LBPs [7
]. The antioxidant effects of LBPs have been detected in various in vitro and in vivo assays. In vitro antioxidant activity has been observed in the β-carotene/linoleic acid assay, the scavenging activity towards the superoxide anion and reducing capacity and the inhibition of AAPH [2,2′-azobis(2-amidinopropane)dihydrochloride]-induced hemolysis assay [7
]. In vivo antioxidant effects were observed in the streptozotocine-induced diabetes in rats, heat-induced damages in rat testes, high cholesterol diet-fed rabbits and mice, ischemia-reperfusion damage in rat hearts and doxorubicine-induced cardiotoxicity [7
]. In the doxorubicine model, MDA levels were high, whereas SOD and GSH-Px levels were lower in the doxorubicine-treated rats. Cardiotoxicity originates from mitochondrion damage, with cytochrome C release into the cytoplasm and caspase-3 induction of apoptosis. The beneficial effects of LBPs are thought to partially come from the induction of the expression of antiapoptotic protein Bcl-2, with this effect contributing to their cardioprotective potential by attenuating doxycycline-induced cardiac myofibrillar disarrangement in rats [9
It is possible that together, the antioxidant and immunomodulatory effects have a positive effect on systolic function and lead to cardioprotection. This cardioprotective potential of LBPs and the specific pharmacologic mechanisms involved should be further investigated.
4. Materials and Methods
4.1. Animals and Experimental Protocol
Four-week-old male Wistar rats (n = 38; average body weight 150 g) were provided by the Experimental Medicine and Practical Skills Center of the “Iuliu Hațieganu” University of Medicine and Pharmacy in Cluj-Napoca, Romania. Rats were kept in standard laboratory conditions, with 12-h light/dark cycles, a constant environmental temperature and received standard pellets and water ad libitum.
All experiments and procedures were performed in conformance with the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures [22
] and with the Universities Federation for Animal Welfare guidelines. The experimental protocol was approved by the Ethical Committee of the “Iuliu Hațieganu” University of Medicine and Pharmacy in Cluj-Napoca, Romania.
Rats were divided into two main groups: rats that were subjected to sham operation (control rats, n
= 10) and rats that were subjected to abdominal aortic banding (AAB rats, n
= 28). Abdominal aortic banding was performed as previously described [4
]. Briefly, after anesthesia (50 mg/kg ketamine and 10 mg/kg xylazine, i.m.), a suture line (3–0 silk) was placed on the abdominal aorta, above the renal arteries. In order to obtain a constant reduction of blood flow, a 23G (0.6 mm diameter) blunt needle was placed alongside the aorta and the suture line was tied around the aorta and the needle. Afterwards, the needle was removed and the abdominal cavity was closed. For the control group the abdomen was opened and closed, without placement of aortic banding.
Subsequently, AAB rats were subdivided into three groups: AAB control group (n = 12) and two other groups treated with different doses of L. barbarum polysaccharides (LBPs): AAB_100 (n = 7) and AAB_200 (n = 9).
A commercially available LBPs extract was used, standardized to 60% polysaccharides. LBPs were mixed in drinking water and administered orally to the AAB_100 group (100 mg/kg body weight/day) and the AAB_200 group (200 mg/kg body weight/day). The control AAB group did not receive any treatment. The total treatment period was 12 weeks starting with week 24 of the study. Body weight (BW) was monitored every two weeks during the experiment. Afterwards, rats were sacrificed at week 36 of the study. Organs (heart, lungs, kidneys and liver) were harvested and weighed. Blood was collected on EDTA from the retro-orbital sinus. Plasma was separated by centrifugation (4000 rpm, RT, 6 min) and samples were stored at −80 °C until use.
4.2. Echocardiography Measurements
Rats were anesthetized (30 mg/kg ketamine and 0.5 mg/kg xylazine, i.m.) and transthoracic echocardiography was performed in the supine or left lateral position, every 12 weeks. A commercially available echocardiograph, equipped with a 7.5 MHz electric transducer (Ultrasonix, Boston, MA, USA), was used to acquire two-dimensional echocardiography images at the mid-papillary muscle level. Structures were manually measured by the same observer, who was an expert in animal cardiology, using the leading-edge method of the American Society of Echocardiography [23
]. The main measured parameters included: left ventricular anterior wall thickness (AWT), left ventricular posterior wall thickness (PWT), LV end-diastolic and end-systolic diameters (LVEDd and LVEDs), LV mass and ejection fraction (EF).
4.3. Complete Blood Count
Complete blood count (CBC) was performed on fresh whole blood, before centrifugation, using Abacus Junior Vet analyzer (Diatron GmbH, Austria). The analyzer provided results for: white blood cells count (WBC), lymphocytes count (LYM), other leucocytes (except lymphocytes and granulocytes) count (MID), granulocytes count (GRA), lymphocytes percentage (LY), other leucocytes percentage (MI), granulocytes percentage (GR), red blood cells count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDWc), platelets (PLT), platelet proportion in plasma (PCT), mean platelet volume (MPV), platelet distribution width (PDWc).
4.4. Proinflammatory Plasma Cytokines
The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in plasma were analyzed using enzyme linked immunosorbent assays (ELISA kits) (ThermoFisher Scientific, Waltham, MA USA) according to the manufacturer’s instructions.
4.5. Plasma Lipid Peroxidation
Malondialdehide (MDA) analysis was performed using a Waters Acquity UPLC system coupled with Waters Acquity photo diode array detector (Waters, Milford, MA, USA), as previously described [24
]. Briefly, in order to quantify total MDA (free and protein bound) samples were submitted to a hydrolysis step at 60 °C in a water bath, in the presence of NaOH. After removing proteins with perchloric acid, MDA was derivatized with 2,4-dinitrophenylhydrazine. The derivatization product was extracted in n-hexane, followed by the evaporation of the organic layer. The residue was dissolved in mobile phase and subjected to UPLC–PDA analysis. Chromatographic separation was achieved on a BEH C18 column (50 mm × 2.1 mm i.d., 1.7 mm) from Waters (Waters, Milford, MA, USA), with a mixture of 1% formic acid/acetonitrile as the mobile phase (0.3 mL/min), and gradient elution. The total chromatographic runtime was 7.5 min. The absorbance of the eluent was monitored at 301 nm. Data acquisition and processing were performed using Empower 2 software (Waters, Milford, MA, USA).
4.6. Data Analysis
All data are expressed as mean ±SD (standard deviation). Statistical analysis was performed with MedCalc version 19.0.6 and GraphPad Prism 5. One-way ANOVA and t-students tests were applied. Differences were considered statistically significant at p < 0.05. Post-hoc analysis was applied for statistical comparisons among groups.