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Zn(II)–curcumin protects against hemorheological alterations, oxidative stress and liver injury in a rat model of acute alcoholism Chuan Yu, Xue-Ting Mei, Yan-Ping Zheng, Dong-Hui Xu ∗ Laboratory of Traditional Chinese Medicine and Marine Drugs, Department of Biochemistry, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China

a r t i c l e

i n f o

a b s t r a c t

Article history:

Curcumin can chelate metal ions, forming metallocomplexes. We compared the effects of

Received 14 November 2013

Zn(II)–curcumin with curcumin against hemorheological alterations, oxidative stress and

Received in revised form

liver injury in a rat model of acute alcoholism. Oral administration of Zn(II)–curcumin

9 February 2014

dose-dependently prevented the ethanol-induced elevation of serum malondialdehyde

Accepted 12 February 2014

(MDA) content and reductions in glutathione level and superoxide dismutase (SOD) activity.

Available online 20 February 2014

Zn(II)–curcumin also inhibited ethanol-induced liver injury. Additionally, Zn(II)–curcumin dose-dependently inhibited hemorheological abnormalities, including the ethanol-induced

Keywords:

elevation of whole blood viscosity, plasma viscosity, blood viscosity at corrected hematocrit

Zn(II)–curcumin

(45%), erythrocyte aggregation index, erythrocyte rigidity index and hematocrit. Compared

Alcohol

to curcumin at the same dose, Zn(II)–curcumin more effectively elevated SOD activity, ame-

Oxidative stress

liorated liver injury and improved hemorheological variables. These results suggest that

Hemorheology

Zn(II)–curcumin protected the rats from ethanol-induced liver injury and hemorheological

Liver injury

abnormalities via the synergistic effect of curcumin and zinc. © 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Alcohol abuse has become a global social, economic and health issue. Alcohol abuse results in oxidative stress, leading to the modification of all biological structures and serious malfunction of the cells and tissues (Kaphalia and Calhoun, 2013). Ethanol ingestion elevates the level of malondialdehyde (MDA) and perturbs the plasma enzymatic antioxidant system, including enzymes such as superoxide dismutase (SOD). Furthermore, alcohol abuse contributes to the depletion of glutathione (GSH) by reducing its synthesis and metabolism

∗

Corresponding author. Tel.: +86 20 84113651; fax: +86 20 84113651. E-mail address: [email protected] (D.-H. Xu).

http://dx.doi.org/10.1016/j.etap.2014.02.011 1382-6689/© 2014 Elsevier B.V. All rights reserved.

(Liang et al., 2013). GSH is a major cellular antioxidant and redox regulator, and plays an important role in preventing the oxidation of cellular constituents (Brocardo et al., 2011). Depletion of hepatic GSH makes hepatocytes more vulnerable to the oxidative stress induced by alcohol, and contributes to alcoholic liver disease. The pathogenesis of alcoholic liver disease is closely related to alcohol-induced oxidative stress (Radosavljevic et al., 2009). Moreover, chronic alcohol consumption can lead to an abnormal erythrocyte morphology and increased erythrocyte fragility as a result of the oxidation and cross-linking of erythrocyte ghost proteins (Tyulina et al., 2006). Increased

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blood viscosity, erythrocyte rigidity and impaired erythrocyte flexibility have also been reported to be induced by the administration of alcohol (Guillet et al., 1991). In patients with ischemic cerebrovascular disease, ingestion of ethanol at a dose of 0.5 or 1 g/kg body weight increased whole blood viscosity and blood viscosity corrected for hematocrit, and impaired red blood cell deformability (Nagai et al., 2001). These effects of alcohol consumption can largely be ascribed to the oxidative stress induced by alcohol. Since erythrocytes have a limited biosynthetic capacity and poor repair mechanism, constant exposure to alcohol-induced oxidative stress may lead to the accumulation of physical or molecular modifications in erythrocytes. Such modifications can result in abnormal erythrocyte function and blood rheology, which directly affects the ability of the blood to deliver oxygen to the tissues and remove toxic metabolites. Notably, approximately 30–50% of individuals with alcohol dependency have a low zinc status, as alcohol consumption decreases intestinal absorption of zinc and increases urinary zinc excretion (Osredkar and Sustar, 2011). Zinc deficiency may also give rise to oxidative stress. Increased oxidative stress and oxidative stress-induced damage have been observed in humans with a sub-optimal zinc intake (Zhao et al., 2011). A significant increase in the MDA levels and decrease in the GSH content and SOD activity are observed in the liver of rats fed on a zinc-deficient diet, and zinc supplementation resulted in a decrease in the MDA levels and increases in GSH content and SOD activity (Tupe et al., 2010). Additionally, zinc deficiency is linked to alcohol-induced intestinal barrier dysfunction, as well as alveolar epithelial cell and macrophage dysfunction (Zhong et al., 2010; Joshi et al., 2009). Curcumin is the major yellow pigment extracted from the rhizome of Curcuma longa, commonly known as turmeric. Curcumin has demonstrated great potential for the prevention and treatment of a wide variety of human diseases due to its pharmacological safety and efficacy (Aggarwal and Harikumar, 2009). However, the low bioavailability of curcumin has become an obstacle to its application in clinical treatment. Phase I clinical trials suggested that curcumin is safe, even at high doses (12 g/day); however, the bioavailability of curcumin is relatively low (Anand et al., 2007). One strategy to improve its bioavailability and biological efficacy is to chelate curcumin with metal ions. Chelates of curcumin with manganese exhibit a greater capacity to protect brain lipids against peroxidation than native curcumin (Sumanont et al., 2006). Zn(II)–curcumin, a mononuclear (1:1) zinc complex of curcumin has been synthesized and processed into solid dispersions (SDs) in our laboratory. SDs of Zn(II)–curcumin have been proven to exert potent antioxidant and gastroprotective effects in rat models of gastric ulceration induced by pylorus-ligature and ethanol, respectively (Mei et al., 2009a, 2012). As Zn(II)–curcumin is a potent antioxidant and could potentially help to reverse the zinc deficiency observed in individuals with alcohol dependency, we aimed to explore whether Zn(II)–curcumin could effectively alleviate the detrimental alterations induced by ethanol in rats, such as an imbalance between oxidation and antioxidants, liver injury and abnormal hemorheology.

Fig. 1 – Molecular structure of the Zn(II)–curcumin complex.

2.

Materials and methods

2.1.

Materials

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione]; 99% pure, was manufactured by Guangdong Zhongda Greenfield Bio-tech. Co. (Guangzhou, China). Polyvinylpyrrolidone K30 (PVP) was purchased from BASF Chemical Ltd. (New Jersey, USA). All other chemicals were of reagent grade. Solid dispersions (SDs) of Zn(II)–curcumin and curcumin were prepared according to the procedures described in our previous report (Mei et al., 2009b). The molecular formula of the Zn(II)–curcumin complex is shown in Fig. 1.

2.2.

Animals

Female Sprague-Dawley rats (6–7 weeks-old; body weight, 250–300 g) were housed under a 12 h/12 h light/dark cycle at controlled temperature and humidity with free access to food and water. All procedures were carried out in accordance with guidelines approved by the Animal Ethics Committee of Sun Yat-sen University (Guangzhou, China).

2.3.

Experimental design

The rats were randomly divided into six groups (n = 8/group) and treated as follows: Group 1, control group, 800 mg/kg PVP + saline; Group 2: model group, 800 mg/kg PVP + ethanol; Group 3: 225 mg/kg Zn(II)–curcumin SDs orally (p.o.; equivalent to 25 mg/kg Zn(II)–curcumin) + ethanol; Group 4: 450 mg/kg Zn(II)–curcumin SDs p.o. (equivalent to 50 mg/kg Zn(II)–curcumin) + ethanol; Group 5: 900 mg/kg Zn(II)–curcumin SDs p.o. (equivalent to 100 mg/kg Zn(II)–curcumin) + ethanol; and Group 6: 700 mg/kg curcumin SDs p.o. (equivalent to 100 mg/kg curcumin) + ethanol. Rats were pretreated with the vehicle PVP or the drugs at the indicated doses by gavage every 12 h for a period of 7 days. From the eighth day, in addition to receiving PVP or the drugs, the rats in Groups 2–6 were administered ethanol (2.5 g/kg, 25%, w/v in saline) by intraperitoneal injection, while the rats in Group 1 received an equal volume of saline (0.9% NaCl, w/v) once per day for consecutive 4 days.

2.4.

Blood and liver collection

Before the last injection of saline or ethanol, the animals were fasted overnight with free access to water. On the day of

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sacrifice, 3 h after the last injection, the rats were weighed and anesthetized with 10% aldehyde (3 mL/kg). Blood was drawn from the carotid aorta; 1 mL of the blood was collected into heparin-coated tubes for the determination of hemorheological parameters, the rest of the blood was collected into clean tubes, centrifuged at 3500 rpm at 4 ◦ C for 10 min, then the serum was subjected to analysis of biochemical parameters. After confirmation of euthanasia, the liver was dissected carefully from each animal, dried using filter paper and weighed using an electronic balance (BT 124 S, d = 0.1 mg, Sartorius, Beijing, China). The relative liver weight for each animal was calculated according to Aniagu et al., 2005 as follows: Relative liver weight =

absolute organ weight (g) body weight of rat on sacrifice day (g) × 100.

2.5.

Hemorheological parameters

Whole hemorheological measurements were analyzed using a cone-plate viscometer (Model LBY-N6A, Precil Company, Beijing, China) according to the international guidelines for the measurement of hemorheological parameters (Bull et al., 1986). Briefly, the hematocrit of the blood samples was measured after centrifugation at 5000 rpm for 5 min in a micro-hematocrit tube before blood viscosity measurement. Whole blood viscosity was determined at shear rates of 10, 60 and 150 s−1 using the cone-plate viscometer. Blood was centrifuged at 3000 rpm for 15 min and the plasma was used for plasma viscosity determination. Blood viscosity at corrected hematocrit (45%) at shear rates of 10, 60 and 150/s was also measured. The erythrocyte aggregation index and erythrocyte rigidity index were determined directly using the viscometer. All measurements were strictly performed at 37 ◦ C within 3 h of blood collection.

2.6.

Biochemical assays

Serum MDA levels were measured using the thiobarbituric acid (TBA) method of Buege and Aust, 1978, and expressed as nanomoles per milliliter (nmol/mL). GSH levels were assessed according to the method described by Burtis and Ashwood (1999) and expressed as milligrams per liter (mg/L). Serum SOD activity was determined using a modified photochemical nitroblue tetrazolium (NBT) method utilizing sodium cyanide as a peroxidase inhibitor (Winterbourn et al., 1975), and expressed as units per milliliter (U/mL). Serum gamma-glutamyl transpeptidase activity (GGT) was determined according to the method described by Szasz (1969) using assay kits purchased from Biosino Bio-Technology and Science Incorporation (Beijing, China) in a semi-automatic biochemical analyzer (ECOM-F 6124, Eppendorf Company, Hamburg, Germany), and expressed as units per liter (U/L).

2.7.

Histological analysis

After being weighed, the liver tissues were immediately immersed and fixed in 4% paraformaldehyde in phosphate

Fig. 2 – Effects of Zn(II)–curcumin on whole blood viscosity, plasma viscosity and blood viscosity at corrected hematocrit (45%).

buffer (pH 7.6) for 48 h at 4 ◦ C. Paraffin sections (5 ␮m thick) were prepared and stained with Mayer’s hematoxylin and eosin according to standard procedures. Tissue preparations were observed and microphotographed under a light microscope.

2.8.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) values. Statistical analysis was performed using SPSS 17.0 software (SPSS, Chicago, IL, USA). Multiple comparisons among groups were performed by one-way ANOVA. A value of P < 0.05 was considered statistically significant.

3.

Results

Effects of Zn(II)–curcumin on whole blood 3.1. viscosity, plasma viscosity and blood viscosity at corrected hematocrit (45%) As shown in Fig. 2, whole blood viscosity at shear rates of 10/s, 60/s and 150/s were significantly higher in the model group treated with ethanol (2.5 g/kg) and PVP vehicle (800 mg/kg) than in the control group (P < 0.001 for all parameters). The administration of 50 or 100 mg/kg Zn(II)–curcumin significantly reduced whole blood viscosity, while 25 mg/kg

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Zn(II)–curcumin only significantly reduced whole blood viscosity at high shear rate (P < 0.05). Compared to curcumin at the same dose, Zn(II)–curcumin (100 mg/kg) had a greater ability to reduce whole blood viscosity at shear rates of 60/s and 150/s (P < 0.05, P < 0.01), indicating curcumin and zinc exerted a synergistic effect. Plasma viscosity was significantly elevated in the model group compared to the control group (1.39 ± 0.08 versus 1.30 ± 0.03 mPa/s, P < 0.001). Zn(II)–curcumin at a dose of 25, 50 or 100 mg/kg significantly reduced plasma viscosity in a dose-dependent manner, to 1.33 ± 0.03, 1.32 ± 0.04 and 1.31 ± 0.04 mPa/s, respectively (P < 0.05, P < 0.01 and P < 0.01, compared to the model group). Blood viscosity at corrected hematocrit (45%) at shear rates of 10/s, 60/s and 150/s were significantly increased in the model group to 25.73 ± 1.27, 13.72 ± 0.85 and 10.28 ± 0.94 mPa/s compared to the control group (all P < 0.001). However, administration of Zn(II)–curcumin (50 or 100 mg/kg) significantly prevented the increases in blood viscosity observed in the model group. Compared to curcumin, Zn(II)–curcumin more potently decreased blood viscosity at corrected hematocrit (45%) at all shear rates (P < 0.01, P < 0.01 and P < 0.001 at 10/s, 60/s and 150/s), suggesting curcumin and zinc exerted a synergistic effect.

3.2. Effects of Zn(II)–curcumin on erythrocyte aggregation index, erythrocyte rigidity index and hematocrit As shown in Fig. 3, administration of ethanol led to an evident increase in the erythrocyte aggregation index, erythrocyte rigidity index and hematocrit in rats (P < 0.001, P < 0.001 and P < 0.01 for groups 3, 4 and 5 versus the control group, respectively). Zn(II)–curcumin (25, 50 and 100 mg/kg) markedly decreased the erythrocyte aggregation index in a dosedependent manner to 2.20 ± 0.15, 2.12 ± 0.12 and 2.11 ± 0.12, respectively, compared to the model group (2.32 ± 0.13; P > 0.05, P < 0.01 and P < 0.01). The erythrocyte rigidity index decreased to 7.26 ± 0.76, 6.75 ± 0.91 and 5.48 ± 0.74 in the groups treated with 25, 50 and 100 mg/kg Zn(II)–curcumin, respectively, compared to the model group (7.87 ± 0.72; P > 0.05, P < 0.01 and P < 0.001). Additionally, the ethanol-induced increase in hematocrit, which indicates an increased volume of packed red blood cells (RBC) in the whole blood sample, was also significantly reduced by 25, 50 and 100 mg/kg Zn(II)–curcumin (42.27 ± 2.48, 39.41 ± 1.77 and 39.06 ± 3.09 versus 45.38 ± 4.48 in the model group; P < 0.05, P < 0.001 and P < 0.001). Curcumin (100 mg/kg) significantly reduced the erythrocyte aggregation index, erythrocyte rigidity index and hematocrit compared to the model group (P < 0.05, P < 0.01 and P < 0.01, respectively). Compared to curcumin at the same dose, Zn(II)–curcumin led to a more significant reduction in erythrocyte rigidity (P < 0.001).

3.3. Effects of Zn(II)–curcumin on serum MDA, GSH content and SOD activity The effects of Zn(II)–curcumin on serum MDA, GSH content and SOD activity are presented in Fig. 4. Intraperitoneal injection of ethanol resulted in a significant increase in serum

Fig. 3 – Effects of Zn(II)–curcumin on erythrocyte aggregation index, erythrocyte rigidity index and hematocrit.

MDA, a marker of lipid peroxidation, compared to the control group (10.55 ± 3.7 nmol/mL versus 7.27 ± 2.32 nmol/mL, P < 0.01). Zn(II)–curcumin (25, 50 and 100 mg/kg) significantly prevented the ethanol-induced increase in the serum MDA level (7.63 ± 2.22, 7.25 ± 1.46 and 7.23 ± 1.82 nmol/mL; P < 0.05, P < 0.01 and P < 0.01 compared with the model group, respectively, Fig. 4A). As shown in Fig. 4B and C, Zn(II)–curcumin at a dose of 25, 50 or 100 mg/kg significantly increased the serum GSH level and SOD activity in a dose-dependent manner. Compared to the model group which had a GSH content of 6.31 ± 3.21, the GSH content increased to 11.36 ± 2.72, 11.63 ± 2.45 and 12.35 ± 2.83 mg/L in the groups receiving 25, 50 and 100 mg/kg Zn(II)–curcumin (P < 0.01, P < 0.01 and P < 0.001, respectively). Zn(II)–curcumin also significantly elevated SOD activity to 233.18 ± 45.25, 235.88 ± 57.88 and 258.59 ± 71.48 U/mL in animals treated with 25, 50 and 100 mg/kg respectively (P < 0.05, P < 0.05 and P < 0.01). Curcumin at a dose of 100 mg/kg significantly prevented the increase in the serum MDA level and decrease in the GSH content observed in the model group (both P < 0.01); however, curcumin did not significantly affect SOD activity (P > 0.05), suggesting that Zn(II)–curcumin more potently increased SOD activity than curcumin alone due to the synergistic effect of curcumin and zinc.

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which demonstrates that curcumin and zinc exerted a synergistic effect on relative liver weight. As shown in Fig. 6, the serum GGT activity of the control group was 4.4 ± 1.19 U/L, while that of the ethanoltreated model group was 6.5 ± 1.06 U/L (P < 0.01). In rats treated with ethanol and pretreated with 25, 50 or 100 mg/kg Zn(II)–curcumin, serum GGT activity decreased significantly to 4.38 ± 0.56, 4.24 ± 1.3 and 4.24 ± 1.14 U/L (P < 0.01, P < 0.001 and P < 0.001, respectively, compared to the model group).

4.

Fig. 4 – Effects of Zn(II)–curcumin on serum MDA content, GSH level and SOD activity.

3.4. Effects of Zn(II)–curcumin on liver pathological changes, body weight, relative liver weight and serum GGT activity Pathological sections of liver tissue are displayed in Fig. 5. The liver of control group showed distinct hepatic lobules consisting of a central vein surrounded by radiating hepatocytes (Fig. 5A), while the liver of ethanol-treated rats showed significant microvacuolar steatosis and necrosis (Fig. 5B). Zn(II)–curcumin dose-dependently prevented vacuolization and necrosis induced by ethanol (Fig. 5D–F). As shown in Table 1, rats exposed to ethanol showed evident weight loss compared to the control group (238.88 ± 28.09 versus 286.38 ± 16.72 g, P < 0.01). However, pretreatment with Zn(II)–curcumin (25, 50 and 100 mg/kg) significantly prevented the ethanol-induced decrease in body weight in a dose-dependent manner, to 269.88 ± 41.13, 279.13 ± 24.22 and 286.38 ± 12.97 g, respectively, compared to the model group (P < 0.05, P < 0.01 and P < 0.01). Curcumin at a dose of 100 mg/kg had no significant effect on weight loss (P > 0.05). Relative liver weight was significantly elevated in the model group compared to the control group (3.86 ± 0.36 versus 3.02 ± 0.45; P < 0.001). Zn(II)–curcumin significantly reduced relative liver weight to 3.43 ± 0.15, 3.39 ± 0.10 and 3.24 ± 0.09 in the rats pretreated with 25, 50 or 100 mg/kg, respectively, compared to the model group (P < 0.01, P < 0.01 and P < 0.001). Compared to curcumin at the same dose, Zn(II)–curcumin more effectively reduced the relative liver weight (P < 0.05),

Discussion

The etiology of various alcohol-induced biological disorders is mainly attributed to increased oxidative stress (Guo and Ren, 2010). In this study, exposure to ethanol significantly elevated the serum MDA level, an end product of lipid peroxidation of polyunsaturated fatty acids, and also decreased the GSH content and SOD activity; these changes lead to oxidative injury to cells and tissues, especially in the liver, and this damage was reflected by increases in the relative liver weight and GGT activity and the pathological changes of the liver sections including stenosis and necrosis. An increase in the relative liver weight is an important marker of ethanol toxicity, even when significant pathological alterations cannot be detected in the liver by light microscopy (Aguiar et al., 2009). GGT is a glycoprotein enzyme located on the membrane of cells in several tissues. The primary role of GGT is to maintain GSH homeostasis by breaking down extracellular GSH and providing cysteine for intracellular synthesis of GSH. Several investigations have indicated that a higher GGT activity is related to a reduction in hepatic GSH content (Giral et al., 2008; De-Oliveira et al., 1999). These findings were reconfirmed in the present study, as the ethanol-induced increase in GGT activity was accompanied by a decreased GSH content. In addition, serum GGT is also a marker of oxidative stress (Yamada et al., 2006). Determination of serum GGT activity is used widely in the assessment of ethanol-induced liver disease. Individuals with alcoholic liver disease due to a moderate or heavy alcohol intake exhibited higher GGT activities than a non-alcoholic liver disease group and healthy volunteers in response to acute hepatocellular damage (Das and Vasudevan, 2005). In the present study, increased whole blood viscosity, plasma viscosity, blood viscosity at corrected hematocrit (45%), erythrocyte aggregation index, erythrocyte rigidity index and hematocrit were also observed in the model group treated with ethanol. Whole blood viscosity reflects the resistance of blood to flow in vessels, and increased blood viscosity may promote ischemic heart disease and stroke (Becker et al., 2004). Plasma viscosity depends on the content of water and macromolecular components. Elevation of plasma viscosity correlates with the progression of coronary and peripheral artery diseases (Kesmarky et al., 2008). Hematocrit indicates the volume of red blood cells in the total blood volume. Either an increase in the number of erythrocytes or dehydration may lead to elevation of the hematocrit levels. Additionally, it has been demonstrated that a direct relationship exists between the hematocrit level and the risk of venous thrombosis (Schreijer et al., 2010). Furthermore, hematocrit is the major

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Fig. 5 – Effects of Zn(II)–curcumin on histological changes induced by ethanol.

determinant of whole blood viscosity (Nemeth et al., 2009). Thus blood viscosity at corrected hematocrit (45%) was also determined in this study, in order to eliminate the influence of varied hematocrit levels on the blood viscosity measurements. Our results showed that exposure to ethanol increased blood viscosity at corrected hematocrit (45%) at all shear rates, suggesting that ethanol or its metabolites may alter the properties of erythrocytes, in addition to affecting the hematocrit. The erythrocyte aggregation index is a reflection of the kinetics of rouleau formation and the adhesive strength between erythrocytes (Weng et al., 1998). Increased erythrocyte aggregation can significantly affect in vivo microcirculatory flow dynamics (Baskurt and Meiselman, 2007). Erythrocyte rigidity is an important physiologic factor that affects the delivery of oxygen to the tissues. Oxygen-carrying capacity decreases

as erythrocytes become more rigid, and increased erythrocyte rigidity results in impaired peripheral perfusion and tissue oxygenation (Saldanha et al., 1999; Banerjee, 1998). Pretreatment of rat primary hepatocytes with curcumin inhibited ethanol-induced lipid peroxidation and reduction in GSH content (Bao et al., 2010). Treatment with curcumin also prevented alcohol-induced elevation of GGT activity by decreasing lipid peroxidation and improving the antioxidant status, including GSH content and SOD activity, in Wistar rats (Rukkumani et al., 2004). In addition, curcumin prevented alteration of erythrocyte membrane lipids and reversed erythrocyte deformity and fragility in hypercholesterolemic rats (Kempaiah and Srinivasan, 2005). It has been suggested that curcumin could effectively inhibit free-radical induced oxidative hemolysis of human erythrocytes due to H-atom

Table 1 – Effects of Zn(II)–curcumin on body weight, liver weight and relative liver weight of rats exposed to alcohol. Group

Body weight (g)

1 2 3 4 5 6

286.38 238.88 269.88 279.13 286.38 262.13

± ± ± ± ± ±

16.72 28.09## 41.13* 24.22** 12.97** 44.77

absolute organ weight (g)

Liver weight (g) 8.58 9.23 9.24 9.46 9.28 9.29

± ± ± ± ± ±

0.97 1.49 1.39 0.87 0.36 1.43

Relative liver weight* 3.02 3.86 3.43 3.39 3.24 3.55

± ± ± ± ± ±

0.45 0.36### 0.15** 0.10** 0.09***,  0.19*

Relative liver weight = body weight of rat on sacrifice day (g) × 100. 1: Control group (PVP + saline); 2: Model group (PVP + alcohol); 3: 25 mg/kg Zn(II)–curcumin + alcohol; 4: 50 mg/kg Zn(II)–curcumin + alcohol; 5: 100 mg/kg Zn(II)–curcumin + alcohol; 6: 100 mg/kg curcumin + alcohol. Data are expressed as mean ± S.D. (n = 8 per group). Statistical significance is represented as follows: ## p < 0.01 compared with control group. ### p < 0.001 compared with control group. ∗ p < 0.05 compared with model group. ∗∗ p < 0.01 compared with model group. ∗∗∗ p < 0.001 compared with model group.  p < 0.05 compared with curcumin group.

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Fig. 6 – Effects of Zn(II)–curcumin on serum GGT activity.

abstraction from the phenolic groups (Deng et al., 2006). Curcumin also significantly reduced whole blood viscosity, erythrocyte aggregation index, whole blood viscosity and erythrocyte rigidity in a rat model of cerebral ischemia induced by blood stasis (Miao et al., 2010). Regular ingestion of curcumin (six 150 mg/capsules per day) for 8 weeks significantly increased carotid arterial compliance in postmenopausal woman (Akazawa et al., 2013). Oral administration of surfacecontrolled water-soluble curcumin (30 mg/kg, twice a day over a 28-day cycle) significantly reduced the acetaldehyde plasma concentration after alcohol intake (0.5 mg/kg) in healthy human volunteers (Naksuriya et al., 2014). One of the essential biochemical functions of zinc is to serve as an antioxidant, and one mechanism of its antioxidant activity is that zinc induces metallothioneins, which exert antioxidant effects under a variety of conditions including ochratoxin A-induced toxicity (Zheng et al., 2013). Another study showed that administration of zinc (100 mg/kg) for two months significantly increased the whole blood GSH level and SOD activity in patients undergoing hemodialysis (Mazani et al., 2013). Moreover, a decreased zinc concentration in erythrocyte membranes was related to increased erythrocyte osmotic fragility, which was readily reversed by dietary zinc; and erythrocyte fragility has been studied as an index of zinc status in humans (O’Dell, 2000). In patients with sickle-cell disease, zinc supplementation increased red blood cell numbers and increases hemoglobin, hematocrit, plasma zinc, and antioxidant power, suggesting that zinc might be beneficial for maintaining blood fluidity (Bao et al., 2008). Treatment with zinc chloride strongly reversed cadmium-induced oxidative stressing in rat erythrocytes by increasing SOD and GSH-Px activities (Messaoudi et al., 2010). Accumulating evidence suggests that zinc complexes have a greater biological potency and enhanced bioavailability compared to zinc alone. Our previous investigation also proved that Zn(II)–curcumin significantly increased SOD activity and content of GSH-Px, which represents peroxidases that catalyze the oxidation of glutathione by cumene hydroperoxide, and reduced serum MDA levels in the gastric mucosa of rats exposed to ethanol, indicating that Zn(II)–curcumin acts as an effective antioxidant against ethanol-induced oxidative stress and the subsequent oxidative damage (Mei et al., 2012). In this research, pretreatment with Zn(II)–curcumin evidently inhibited the formation of MDA, restored the GSH level

and increased SOD activity, and prevented the subsequent detrimental alterations including liver injury and hemorheological abnormalities induced by ethanol. Compared to curcumin at the same dose, Zn(II)–curcumin more potently increased SOD activity in rats exposed to ethanol. Additionally, the ethanol-induced increase in relative liver weight was more potently attenuated in the Zn(II)–curcumin group than the curcumin group, which suggests Zn(II)–curcumin has the ability to exert a hepatoprotective effect via an antioxidant mechanism. Additionally, the body weight of rats treated with Zn(II)–curcumin was much closer to normal than that of the curcumin-treated group, which may possibly have helped to reduce the relative liver weight. Furthermore, Zn(II)–curcumin more potently protected against the ethanol-induced increases in whole blood viscosity, blood viscosity at corrected hematocrit (45%) and the erythrocyte rigidity index, which contributed to the maintenance of blood fluidity and normal blood function such as the delivery of oxygen and elimination of toxic substances. Hemorheological alterations are closely related to the oxidative stress induced by ethanol. Thus we propose that Zn(II)–curcumin can protect erythrocytes from ethanol-induced oxidative damage and modifications by reducing oxidative stress, thereby improving blood flow.

5.

Conclusions

Zn(II)–curcumin protected against ethanol-induced increases in whole blood viscosity, plasma viscosity, blood viscosity at corrected hematocrit (45%), erythrocyte aggregation index, erythrocyte rigidity index and hematocrit, and prevented liver injury in a rat model of acute alcoholism via a number of antioxidant mechanisms, including reducing the MDA content, elevating SOD activity and increasing the GSH level. The more potent effects of Zn(II)–curcumin, compared to curcumin alone, are primarily due to the synergistic effect of curcumin and zinc.

Conflict of interest statement The authors declare that no conflicts of interest exist.

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Acknowledgments This work was supported by the National Natural Science Foundation of China; the Program of New Century Excellent Talent of China (no. NCET-04-0808); the project of GuangdongHongkong Key Research Fields (no. 2009A030901005).

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