Anti-Hyperlipidemic and Anti-Oxidative Effects of Gelsemine in High-Fat-Diet-Fed Rabbits

Cell Biochem Biophys (2015) 71:337–344 DOI 10.1007/s12013-014-0203-2

ORIGINAL PAPER

Anti-hyperlipidemic and Anti-oxidative Effects of Gelsemine in High-Fat-Diet-Fed Rabbits

Tao Wu • Guoping Chen • Xiaolong Chen • Qiqi Wang • Gang Wang

Published online: 12 September 2014 Ó Springer Science+Business Media New York 2014

Abstract The present study investigated the anti-hyper- indicated by the increased activity of superoxide dismutase lipidemic proprieties of a natural alkaloid, gelsemine, in a and catalase, and reduction in serum nitric oxide, and high-fat-fed rabbit model. Animals were randomly divided malondialdehyde concentrations in hyperlipidemic animals into five groups and fed normal diet, hypercholesterolemic that received gelsemine supplementation. Dietary supple- diet (1 % cholesterol), or hypercholesterolemic diet (1 % mentation with gelsemine may, therefore, reverse the effect cholesterol) supplemented with gelsemine (1, 5, or 25 mg/ of the lipogenic diet on lipid profile and hepatic enzymes in kg). After 60 days, serum concentrations of total choles- hyperlipidemic rabbits, and protect tissues from oxidative terol (TC), LDL-C, HDL-C, triglycerides, apolipoproteins stress, caused by high-fat diet. A and B, SGOT, SGPT, glucose, and insulin were measured in all experimental groups. Hypercholesterolemic Keywords Gelsemine Á High-fat diet Á Hyperlipidemia Á diet resulted in significantly elevated levels of TC, TG, Oxidative stress LDL-C, SGOT, and SGPT, and reduced HDL-C compared to the normocholesterolemic diet group. Gelsemine treatment significantly improved lipid profile parameters, Introduction affected by hyperlipidemia, while having no effect on the levels of apolipoproteins, glucose, and insulin. Further- Cardiovascular and cerebrovascular diseases are the most more, gelsemine treatment decreased hyperlipidemia- frequent causes of morbidity and mortality in the US and induced oxidative stress in a dose-dependent manner, as other western countries. Atherosclerosis and subsequent formation of lesions as a result of accumulation of lipids and fibrous elements in the sub endothelial space of large & T. Wu ( ) Á Q. Wang arteries are considered the primary causes of these vascular Department of Cardiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China diseases [1–4]. Pathogenesis of atherosclerosis includes e-mail: [email protected] both modifiable and non-modifiable risk factors that col- lectively contribute to the development, progression, and G. Chen rupture of atherosclerotic plaque [1–4]. Growing evidence Department of Endocrinology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, indicates that chronic and acute overproduction of reactive China oxygen species (ROS) under pathophysiologic conditions has a causal role in atherosclerosis and other cardiovascular X. Chen diseases. Oxidative stress results from an imbalance School of Pharmacy, Shanghai Jiaotong University, Shanghai 200030, China between radical-generating and radical-scavenging systems, i.e., increased free radical production, reduced G. Wang activity of antioxidant defenses or both [5]. Recent studies Tumor Institute of Integrated Chinese and Western Medicine, suggest that the role of oxidative stress in the pathogenesis Zhejiang Academy of Traditional Chinese Medicine, Tongde Hospital of Zhejiang Province, Hangzhou 310007, Zhejiang, of atherosclerosis is due not only to the generation of free China oxygen radicals, but also to nonenzymatic protein 123 338 Cell Biochem Biophys (2015) 71:337–344 glycosylation, auto-oxidation of glucose, impaired gluta- effects on cardiac dysfunction induced by hyperglycemia/ thione metabolism, alteration in the function of antioxidant hypercholesterolemia through decreasing cardiac lipid enzymes, and lipid peroxides formation [5]. The defense accumulation and promoting glucose transport [11]. It is mechanisms against free radicals include glutathione possible that other natural alcaloids may have similar (GSH), superoxide dismutase (SOD), glutathione peroxi- pharmacological effect in hypercholesterolemic animal dase (GPx), and catalase (CAT), which contribute to models. Hence, our experiments were aimed to explore the elimination of superoxide, hydrogen peroxide, and hydro- effects of gelsemine on serum lipid profile, liver enzymes, xyl radicals [5]. Recent advances in the field of free radical glucose, and insulin, and to evaluate its protective propri- research have confirmed that foods rich in antioxidants eties against oxidative stress, induced by high-fat diet in a play an important role in the prevention of cardiovascular rabbit model of hyperlipidemia. disease [2, 6–8], and there is an accumulating amount of evidence, indicating that botanical dietary supplements can prevent the development of atherosclerosis [2]. Therefore, Materials and Methods plant derived antioxidants are now receiving special attention. Gelsemine is a natural alkaloid extracted from Reagents Gelsemium, a small genus of the family Loganiaceae, which comprises three popularly known species: Gelse- All reagents were purchased from Sigma (Sigma-Aldrich, mium elegans Benth. (G. elegans), which is native to China St. Louis, MO, USA) unless otherwise stated. and Southeast Asia, Gelsemium sempervirens Ait. (G. sempervirens) and Gelsemium rankinii Small (G. rankinii), Grouping and Treatments native to North America. G. sempervirens has long been used as a traditional folk medicine to alleviate pain and Adult male rabbits of New Zealand strain weighing reduce anxiety, and has shown promising results in vivo, as 1.25–2.50 kg were purchased from Shanghai Laboratory even very low doses of G. sempervirens extract reduce Animal Center, Chinese Academy of Sciences. Rabbits anxiety in animal models [9, 10]. G. elegans is a well- were maintained in animal stainless steel mesh-bottomed known plant in Asia, and although it can be toxic, it has cages for 2 weeks at 21–24 °C and 12 h light/dark cycle. been used in Chinese medicine as an anti-inflammatory, Animals were fed a standard basal diet for 2 weeks for analgesic, and antineoplastic agent [9, 10]. The alkaloids adaptation. Following the initial 2 weeks, nourishment was gelsemine, koumine, gelsenicine, and gelsevirine constitute done by standard grain food purchased from Shanghai the primary active molecules of G. elegans. Gelsemine is Laboratory Animal Center, containing 15 % protein, the only common alkaloid found in both G. elegans and G. 40–50 % carbohydrates, 2 %vegetable fat, and 15–25 % sempervirens, while koumine, gelsenicine, and gelsevirine fiber. Animals were randomly divided into five groups of are unique for G. elegans. Recently, a number of studies eight animals each to be fed with normal diet, hypercho- have focused on the pharmacology of the G. elegans lesterolemic diet (1 % cholesterol), or hypercholesterol- alkaloids. Similar to alkaloids extracted from G. semper- emic diet (1 % cholesterol) ? gelsemine (1, 5, or 25 mg/ virens, gelsemine, koumine, gelsevirine, and gelsenicine kg). Gelsemine treatment was administered as previously that are extracted from G. elegans by high-speed counter- described [11]. Briefly, gelsemine (99 % purity; Chengdu current chromatography (HSCCC) and pH-zone-refining Biopurify Phytochemicals Ltd, Chengdu, China) was counter-current chromatography (CCC), were reported to reconstituted in 10 % Tween 80 solution for intragastric have analgesic effect [9, 10]. Previous studies reported that administration (i.g.). The study protocol was approved by a single dose of gelsemine, far below the LD50, had a the Medical Ethics Committee of Nanchang University. potent anxiolytic, but not antidepressant effect in mice that could be antagonized by strychnine, a glycine receptor Biochemical Measurements antagonist. These studies suggested that G. elegans alkaloids may be a promising tool in treating anxiety-related Fasted blood samples were collected to determine serum psychiatric disorders [9, 10]. concentrations of total cholesterol (TC), high-density Recent studies have discovered that natural alkaloids, lipoprotein cholesterol (HDL-C), low-density lipoprotein such as berberine, isoquinoline alkaloid, derived from cholesterol (LDL-C), and triglycerides (TG), liver enzymes medicinal herbs including Berberis, Hydrastis canadensis, (serum glutamate oxaloacetate transaminase (SGOT) and Coptis chinensis Franch., and Cortex phellodendri Chin- serum glutamate pyruvate transaminase (SGPT), insulin, ensis, can improve metabolic dysfunction, decrease ven- glucose, and apolipoproteins A and B. Serum insulin levels tricular premature complexes in the patients with were determined by ELISA using a commercially available congestive heart failure [11, 12], and have a protective kit (Monobind Inc., CA, USA). Other biochemical factors 123 Cell Biochem Biophys (2015) 71:337–344 339 were measured by routine enzymatic methods using com- Measurement of Serum Nitric Oxide (NO) mercial kits (Monobind Inc., CA, USA) and analyzed using a Hitachi 902 autoanalyzer (Tokyo, Japan). Serum NO levels were determined spectrophotometrically by measuring the accumulation of its stable degradation products, nitrite and nitrate. The serum nitrite level was Measurement of Serum Reduced Glutathione (GSH) determined by the Griess reagent as described by Hortelano et al. (1995). Briefly, the Griess reagent, a mixture (1:1) of Serum GSH levels were determined as described by Ell- 1 % sulfanilamide in 5 % phosphoric acid and 0.1 % man (1959). Briefly, 10 % of trichloroacetic acid (TCA) 1-naphthyl ethylenediamine gives a red violent diazo color was added to the serum and centrifuged. 1.0 ml of the in the presence of nitrite. The color intensity was measured collected supernatant was treated with 0.5 ml of Ellman’s at 540 nm. Results were expressed as lmol/l using a reagent (19.8 mg of 5,5’-dithiobisnitro benzoic acid NaNO calibration graph [8]. (DTNB) in 100 ml of 0.1 % sodium nitrate) and 3.0 ml of 2 phosphate buffer (0.2 M, pH 8.0). The absorbance was read Measurement of Serum Protein Content at 412 nm. Protein content was determined by the method of Lowry Measurement of Serum Thiobarbituric Acid Reactive et al. (1951) [16], using bovine serum albumin (BSA) as a Species (TBARS) standard.

The formation of lipid peroxides was measured in the Statistical Analysis serum. The formation of malondialdehyde (MDA), an end product of fatty acid peroxidation, was measured by One-way analysis of variance (ANOVA) was performed spectrophotometry at 532 nm, using a thiobarbituric acid and Tukey post hoc test was used for multiple comparisons. reactive substance (TBARS) as described in by Genet et al. Statistical analyses were performed using the SPSS 13.0. (2002). Results are expressed as nmol of MDA formed/mg The results are expressed as mean ± SEM. The results protein [13]. originated from analysis of serum. Linear correlation tests were also performed. Differences of P \ 0.05 were considered significant. Measurement of Serum Superoxide Dismutase (SOD) Activity Results The activity of SOD was determined by the level of inhibition of pyrogallol autoxidation at pH 8 as previously Effect of Gelsemine on Lipid Metabolism, Liver described [14]. The specific activity of SOD was expressed Function and Glucose Metabolism as units per mg protein per minute. Rabbits maintained on hypercholesterolemic diet contain- Measurement of Serum Catalase (CAT) Activity ing 1 % cholesterol for 8 weeks exhibited significantly elevated levels of TC, TG, LDL-C (Fig. 1a–c), SPGT, and

Catalase activity was assayed by H2O2 consumption, fol- SGOT (Fig. 2a, b) (over 1.5-, 3-, 2.3-, 2.5-, and 2.7-fold, lowing Aebi’s (1984) protocol, modified by Pieper et al. respectively, P \ 0.05), and over 3.5-fold decrease in the (1995). Briefly, ethanol was added (1:100 v/v) to the serum levels of HDL-C (Fig. 1d) (P \ 0.05), compared to the and incubated for 30 min in an ice bath. 1 % Triton X-100 normocholesterolemic diet group. In contrast, there was no (1:10 v/v) (Sigma) was then added, and the mixture was difference in the serum concentrations of glucose, insulin, incubated in an ice bath for 15 min. Five hundred micro- apo A, and apo B between hypercholesterolemic and nor- liter of the mixture was placed into a glass cuvette, and the mocholesterolemic diet groups (Fig. 1e, f) (P [ 0.05). reaction was started by adding 250 llof30mMH2O2 in Supplementing the diet with gelsemine (1, 5, or 25 mg/kg) phosphate buffer (50 mM, pH 7.0). After 15 s, the absor- did not cause any significant alteration in serum parame- bance at 240 nm was read every 15 s for 45 s. CAT ters, apolipoproteins, hepatic transaminases, glucose, and activity was expressed as mmol H2O2/min/mg protein. An insulin compared to the hypercholesterolemic diet group enzyme unit was defined as the amount of enzyme that (P [ 0.05). On the other hand, gelsemine supplementation catalyzes the release of one lmol of H2O2 per min. Specific resulted in a marked dose-dependent decrease in circulat- activity was calculated in terms of units per mg of protein. ing concentrations of TG, TC, LDL-C, SGOT, and SGPT The assay was performed at 25 °C[6, 15]. (P \ 0.05), and an increase in HDL-C levels (P \ 0.05), 123 340 Cell Biochem Biophys (2015) 71:337–344

Fig. 1 Effect of gelsemine on serum concentrations of lipid profile groups and compared to untreated control animals. Values are the parameters and apolipoproteins in experimental groups. Rabbits were mean ± SEM. Statistical significance for the difference between the fed lipogenic diet to induce hyperlipidemia diet (model group). data of the control group (normal diet) versus model group Alternatively, diet was supplemented with increased doses of (hypercholesterolemic diet): **P \ 0.01, ***P \ 0.001. Statistical gelsemine (GM) as described in ‘‘Materials and Methods’’ section. significance for the difference between the data of untreated model Serum concentrations of total cholesterol (TC), triglycerides, LDL-C, group versus treated groups: ##P \ 0.01, ###P \ 0.001 HDL-C, and apolipoproteins a, b were measured in all experimental compared to the animals that were sustained on hyper- concentrations in the gelsemine-fed group (P \ 0.05). cholesterolemic diet alone (Figs. 1, 2). Comparison of None of the evaluated biochemical parameters did signifi- gelsemine-supplemented versus normocholesterolemic diet cantly differ between gelsemine-supplemented groups groups revealed an elevation of TG and reduction in apo A (Figs. 1, 2)(P [ 0.05).

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Fig. 2 Effect of gelsemine on serum concentrations of liver enzymes, difference between the data of the control group (normal diet) versus glucose, and insulin in experimental groups. Animals are treated as model group (hypercholesterolemic diet): **P \ 0.01, ***P \ 0.001. described in Fig. 1. GM gelsemine, SGOT serum glutamate oxaloac- Statistical signiﬁcance for the difference between the data of etate transaminase, SGPT serum glutamate pyruvate transaminase. untreated model group versus treated groups: ##P \ 0.01, Values are the mean ± SEM. Statistical signiﬁcance for the ###P \ 0.001

Effects of Gelsemine on the Serum Levels of MDA, effect is dose dependent. We next tested the activity levels GSH, SOD, and CAT of SOD and CAT in experimental groups by ELISA. Hypercholesterolemic diet led to significantly decreased We next evaluated oxidative stress parameters in the serum activity of SOD and CAT compared to control (ovcer 1.5- of hyperlipidemic rabbit model by measuring serum levels and 3-fold, respectively, P \ 0.001) (Fig. 3c, d). Gelsemine of GSH and MDA, an aldehydic product of lipid peroxi- supplementation resulted in a significant dose-dependent dation that reacts quickly with biomolecules, such as pro- rise in serum SOD activity, compared to hypercholesterol- teins, lipids, and nucleic acids and affects liver and b- emic diet group, with SOD levels steadily increasing with pancreatic cell function, hence disturbing glucose regula- the increase in gelsemine dosage (P \ 0.05, P \ 0.001 and tion [7]. Hypercholesterolemic diet resulted in over-pro- P \ 0.001 for 1, 5, and 25 mg/kg gelsemine, respectively). duction of lipid peroxidation product (MDA), which is of Twenty five milligram per kilogram gelsemine effectively six fold level compared to control group. Moreover, restored serum SOD activity to control levels. Similarly, hypercholesterolemic diet also produced low-level of GSH, CAT activity in hyperlipidemic animals, supplemented with which is only one quarter of control group’s 5 and 25 mg/kg gelsemine was significantly higher than in (P \ 0.001)(Fig. 3a, b). Gelsemine in higher dosage (5 and the hyperlipidemic group (P \ 0.05 and P \ 0.01, respec- 25 mg/kg) significantly attenuated serum MDA levels and tively) (Fig. 3). Both CAT and SOD activity levels in ani- increased GSH compared with the untreated control groups mals treated with the highest gelsemine dose (25 mg/kg) (P \ 0.01 and P \ 0.001, respectively). Moreover, sup- were significantly higher than the group receiving the lower plementing the hypercholesterolemic diet with 25 mg/kg (5 mg/kg) gelsemine dose (Fig. 3c, d; P \ 0.05). These gelsemine effectively restored serum MDA and GSH levels results strongly suggest that the effect of gelsemine on SOD to values similar to control (Fig. 3), suggesting that the and CAT is dose dependent.

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Fig. 3 Effects of gelsemine on MDA (a), GSH (b), SOD (c), and group (hypercholesterolemic diet): **P \ 0.01, ***P \ 0.001. Sta- CAT (d). Rabbits are treated as explained in Fig. 1. GM gelsemine. tistical signiﬁcance for the difference between the data of untreated Values are the mean ± SEM. Statistical signiﬁcance for the differ- model group versus treated groups: ##P \ 0.01, ###P \ 0.001 ence between the data of the control group (normal diet) versus model

Effects of Gelsemine on Serum NO

We evaluated the effect of gelsemine on the nitric oxide production in hyperlipidemic rabbit model. Animals sustained on hypercholesterolemic diet alone exhibited over 1.8-fold increase of NO levels, compared to the control group (P \ 0.001) (Fig. 4). Gelsemine (1, 5, and 25 mg/ kg/day)-treated model groups presented signiﬁcantly decreasing serum NO levels, compared to hyperlipidemic group (P \ 0.001). The effect of gelsemine on serum NO was dose dependent, with animals on higher dose gelsemine diet exhibiting serum NO levels signiﬁcantly lower than those receiving the low gelsemine dose (P \ 0.001).

Fig. 4 Effects of gelsemine on serum NO. Serum concentrations of nitric oxide (NO) were measured using Griess reagent. GM gelsemine. Values are the mean ± SEM. Statistical signiﬁcance for Discussion the difference between the data of the control group (normal diet) versus model group (hypercholesterolemic diet): **P \ 0.01, ***P \ 0.001. Statistical signiﬁcance for the difference between the Oxidative stress plays an important role in the pathogen- data of untreated model group versus treated groups: ##P \ 0.01, esis and the complications of atherosclerosis. Numerous ###P \ 0.001 studies have shown that hyperlipidemia results in the

123 Cell Biochem Biophys (2015) 71:337–344 343 overproduction of oxygen free radicals, thus producing plasma CAT and SOD enzymatic activity in gelsemine- imbalance between plasma oxidant and antioxidant con- treated hyperlipidemic rabbits. SOD is responsible for tent [5]. Furthermore, hyperlipidemia leads to adipocyte removal of superoxide radicals and CAT decomposes dysfunction and decrease in inhibition of the release of hydrogen peroxide to water and oxygen. These enzymes, free acid into the plasma [5, 7]. The consequent increase therefore, may contribute to the modulation of the redox in circulating free fatty acids (FFA) contributes to an state of plasma [7]. increase in hepatic TG and subsequent increase in the Our recent study indicates that gelsemine is effective in production of atherogenic small dense LDL. This pathway preventing hyperlipidemia that results from lipogenic diet. may constitute the predominant mechanism of lipogenic Gelsemine inhibits the increase in the serum lipid profile diet-induced complications of atherosclerosis [5, 7]. In our by controlling oxidative and nitrosative systems. We may experimental rabbit model of hyperlipidemia, lipogenic speculate that the possible mechanism of this activity of diet led to a significant decrease in plasma GSH content gelsemine may involve its ability to reduce levels of and CAT and SOD activities, accompanied by a signifi- MDA and oxygen free radicals, and at the same time to cant increase in MDA, NO and overall lipid profile, activate SOD. Hyperlipidemia, caused by lipogenic diet indicating an increased plasma oxidative stress which may and, to a lesser extent, by insulin resistance, is charac- be also present in other tissues in hyperlipidemic rabbits. terized by increased generation of ROS and oxidative These results are in agreement with the previous reports, stress [7]. It often is accompanied by lipid abnormalities showing that lipogenic diet in rabbit model is accompa- such as elevated LDL-C and cholesterol, as was also nied by an increased susceptibility to lipid peroxidation demonstrated in this in vivo study. These abnormalities [17–19]. The enhanced interaction of superoxide with NO may be further exacerbated by the increased oxidizing in the oxidant environment of hyperlipidemia increases environment that enhances the formation of oxidized the formation of peroxynitrite, which in turn, oxidizes LDLs (oxLDLs), glycated LDL and oxysterols (formed tetrahydrobiopterin (BH4, a cofactor of endothelial nitric from the oxidation of cholesterol). It has been suggested oxide synthase, eNOS), leading to eNOS uncoupling [7]. that the binding of oxidized lipid products to specific In addition to eNOS uncoupling, hyperlipidemic condi- receptor proteins and activation of ROS-generating tions stimulate the increase in nitric oxide synthase inflammatory proteins are important mechanisms of ROS- (iNOS) expression, which ultimately results in a dimin- induced atherosclerosis [7]. In our study, we evaluated the ished NO bioavailability [7]. pro-oxidant/antioxidant balance in hyperlipidemic rabbit Our recent data suggest that dietary supplementation model by measuring MDA and NO levels and enzymatic with gelsemine significantly reduces adverse metabolic antioxidants. Increased MDA, NO and decreased SOD, effects of the lipogenic diet in rabbit model. Gelsemine GST and CAT enzymatic activity indicated that hyper- supplementation improved overall lipid profile and the lipidemic state was associated with pro-oxidation. By oxidative stress parameters (MDA and NO) of the hyper- restoring SOD, GST, and CAT activities, gelsemine may lipidemic animals, and restored the levels of GSH, CAT, therefore contribute to the decrease in free radical gen- and SOD, affected by the hypercholesterolemic diet. These eration and increased antioxidant defenses in hyperlipi- effects suggest that gelsemine treatment effectively reduces demic animal. The hypothetical mechanism for this effect oxidative stress in the lipogenic rabbits. of gelsemine may involve modulating antioxidant gene The normalization of serum oxidative stress parameters expression and/or up-regulating mitochondrial antioxidant observed in hyperlipidemic rabbits after gelsemine sup- genes. plementation suggests a possible protective effect of gel- In summary, present study shows that dietary gelsemine semine that could be mediated through suppression of supplementation is able to reverse the adverse effect of the oxygen free radicals induced by hyperlipidemia. Gelsemine lipogenic diet on lipid profile and hepatic enzymes in markedly stimulated production of GSH, a crucial substrate hyperlipidemic rabbit model. This positive effect of gel- for GPx and glutathione-S-transferase, which reacts with semine suggests its possible therapeutic value as a safe and free radicals and is a part of the cellular defense mecha- effective dietary supplement for patients with dyslipidemia nisms against intermediate oxygen products [7]. Studies or nonalcoholic fatty liver disease. Future research is show that the GSH/GSSG ratio plays a critical role in needed to study the exact mechanism underlying the anti- glucose homeostasis in hyperlipidemia, since thiol groups hyperlipidemic effects of gelsemine, and to explore whe- are important in intracellular and membrane redox state ther these effects are exerted by a single constituent or are [7]. It is possible, that gelsemine-induced increase in due to a synergism between different phytochemicals. plasma GSH content might enhance the GSH/GSSG ratio and decrease lipid peroxidation, improving serum glucose Acknowledgments This Project is supported by: 1. Zhejiang Pro- regulation. Additionally, we reported a marked increase in vincial Natural Science Foundation of China (Grant No. Y2080374); 123 344 Cell Biochem Biophys (2015) 71:337–344

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