International Immunopharmacology 22 (2014) 126–132

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Hepatoprotective effects of Mimic of Manganese superoxide dismutase against carbon tetrachloride-induced hepatic injury Yan-Hong Wang a, Xiang-Jiu Xu b, Hong-Ling Li b,⁎ a b

Department of Pharmacy, Gansu Provincial Hospital, Lanzhou 730000, Gansu, China Division of Oncology, Gansu Provincial Hospital, Lanzhou 730000, Gansu, China

a r t i c l e

i n f o

Article history: Received 18 March 2014 Received in revised form 16 May 2014 Accepted 10 June 2014 Available online 24 June 2014 Keywords: Mimic of Manganese superoxide dismutase Carbon tetrachloride Mice Oxidative stress Pro-inflammatory mediators Nuclear factor-kappa B

a b s t r a c t The aim of this study was to investigate the protective effects of Mimic of Manganese superoxide dismutase (MnSODm) against carbon tetrachloride (CCl4)-induced hepatic injury in mice. Bifendate or MnSODm was intragastrically administered per day for 7 days. On the 8th day, all mice except the normal group were given 0.5% CCl4/peanut oil to induce hepatic injury model by intraperitoneal injection. Mice were sacrificed 24 h after CCl4 treatment. Compared with the CCl4 group, MnSODm significantly decreased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the serum and liver. Moreover, the contents of hepatic and serum malondialdehyde (MDA) and nitric oxide (NO) with inducible nitric oxide synthase (iNOS) activities were reduced. Histological findings also confirmed the antihepatotoxic characterization. In addition, MnSODm inhibited the pro-inflammatory mediators, such as prostaglandin E2 (PGE2), cyclooxygenase-2 (COX2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Further investigation showed that the inhibitory effect of MnSODm on the pro-inflammatory cytokines was associated with the down-regulation of nuclear factor-kappa B (NF-κB). In brief, our results show that the protective effect of MnSODm against CCl4-induced hepatic injury may rely on its ability to reduce oxidative stress and suppress inflammatory responses. © 2014 Published by Elsevier B.V.

1. Introduction The liver plays a pivotal role in metabolism and detoxification of endogenous and exogenous hepatotoxicants in the body, and these metabolic reactions can potentially lead to liver injury [1,2]. With the speeding up of the rhythm of lifestyles and changing diets in recent years, the risk of liver disease has been increasing greatly and is frequently fatal [3]. In recent years, “oxidative stress” and its adverse effects on human health have become a subject of considerable interest in research. It is a well-documented fact that exposure of organisms to the exogenous and endogenous factors generates a wide range of reactive oxygen species (ROS), which results in homeostatic imbalance. Production of free radicals that exceeded, such as superoxide, hydroxyl radicals, hydrogen peroxide and nitric oxide, was certainly found to play multiple important roles in tissue damage and the loss of function [4]. Oxidative stress is one of the major factors in the pathogenesis of liver disease, which can result in various liver diseases ranging from transient elevation of liver enzymes to life-threatening hepatic fibrosis, liver cirrhosis and even hepatocellular carcinoma [5]. Antioxidant enzymes maintain cellular redox homeostasis. Manganese superoxide dismutase (MnSOD) is the key enzyme located in the ⁎ Corresponding author. Tel.: +86 8281563. E-mail address: [email protected] (H.-L. Li).

http://dx.doi.org/10.1016/j.intimp.2014.06.016 1567-5769/© 2014 Published by Elsevier B.V.

mitochondria, which protects energy-generating mitochondria from oxidative damage. Superoxide anions play a pro-inflammatory part in many diseases. So, MnSOD is likely to be used as an anti-inflammatory agent because of its superoxide anion scavenging ability. Although many kinds of MnSOD have been found, the intact amino acid sequence analysis has only been completed in less than ten of them. Using natural MnSOD for a long time could induce immunity and allergic reaction because of its big molecular quality. Some researchers have applied molecular engineering methods to molecular modification to mimic MnSOD so that the short half-life and poor stability caused by hydrolysis of the enzyme in natural SODs could be solved [6]. In the present study, the molecular structure and crystallographic parameters of MnSOD mimic (MnSODm, C40H57Mn2N9O21) [7] in Fig. 1 and Table 1 were listed. MnSODm has double active sites which were synthesized by a water soluble aliphatic amine and a biocompatible compound of vanillin. It has higher activity because of the increased concentration of Mn sites. Our previous study has observed that MnSODm clearly inhibited the proliferation of Raji cells and induced apoptosis in vitro [8]. Here, the aim of this study was to investigate the hepatoprotective effects of MnSODm against acute CCl4-induced hepatic injury in mice. The present study is the first report to evaluate the effects of MnSODm on hepatoprotection in mice, and new evidence has been provided to show that MnSODm may be a good hepatoprotective agent against liver damage.

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obtained from Xinchang Pharmaceutical Factory (Zhejiang Medicine Co., Ltd., China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Interleukin-6 (IL6), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Bender (Bender MedSystems, CA, USA). The antibodies against COX-2, p-NF-κBp56 and NF-κBp56 were purchased from Abcam Inc. (USA). Goat anti-rabbit or Goat anti-mouse horseradish peroxidaseconjugated antibody (Santa Cruz Biotechnology, CA, USA) was used as secondary antibody. All other chemicals used were of analytical grade. 2.3. CCl4-induced acute liver injury

Fig. 1. MnSODm molecular structure.

2. Materials and methods 2.1. Animals Male Kunming (KM) mice (18–22 g) were obtained from the Experimental Animal Centre of Lanzhou University. The mice were housed in plastic cages in a room and kept under standardized conditions at a temperature of 22–24 °C, and 20% humidity with a 12 h light/dark cycle, and they had free access to tap water and food throughout the study. They were allowed to acclimatize for 1 week before the experiments were started. Animal experiments were conducted under principles of good laboratory animal care, and approved by the ethical committee for Laboratory Animal Care and Use of Lanzhou University.

The animals were randomly divided into six groups: normal group (n = 10), model group (n = 10), Bifendate group (n = 10) as a positive control and the 3 MnSODm treatment groups (each n = 10), which consisted of low dose (MnSODm-L, 10 mg/kg), medium dose (MnSODm-M, 20 mg/kg), and high dose (MnSODm-H, 40 mg/kg). Low, medium, and high (1, 2, and 4 mg) doses of MnSODm and 5 mg of Bifendate were each suspended in 1 mL of physiological saline. One week after acclimatization, treatment began and continued for 7 days. Mice in the Bifendate and MnSODm groups were intragastrically given Bifendate or MnSODm, respectively. The mice from the normal group and the model group were administrated intragastric physiological saline. On the eighth day, all mice except the normal group mice were given 0.5% CCl4/peanut oil by intraperitoneal injection (i.p., 0.3 mL); whereas the mice of the normal group received peanut oil 0.3 mL only. All the mice were sacrificed after given 0.5% CCl4/peanut oil for 24 h and serum was obtained by centrifugation of the collected blood at 4000 g for 10 min, and stored at 4 °C for the activity of ALT, AST and other biochemical criteria. Liver samples were dissected out and washed immediately with ice-cold saline to remove as much blood as possible. One part of each liver sample was immediately stored at −80 °C until analysis, and another part was excised and fixed in 10% formalin solution for histopathologic analysis. 2.4. Biological assay of serum and liver tissue

2.2. Drugs and reagents Mimic of Manganese superoxide dismutase (MnSODm) was kindly provided by Professor Dou Wei from Lanzhou University (Lanzhou, Gansu, China), the purity of the compound is 98%. MnSODm was dissolved in physiological saline and stored at 4 °C. Bifendate was

Table 1 MnSODm crystallographic parameters. Empirical formula

C40H57Mn2N9O21

Formula weight Temperature Crystal system Space group Unit cell dimensions

1109.83 273(2)K Monoclinic C2/c a = 21.475(14)Ǻ, α = 90° b = 18.203(12)Ǻ, β = 97.110(11°) c = 21.475(14)Ǻ, γ = 90° 5563(6)Ǻ3 4 1.325 Mg/m3 2312 13,216 4610 [R(int) = 0.0632] Semi-empirical from equivalents 1.003 R1 = 0.0753, wR2 = 0.1808 R1 = 0.1619, wR2 = 0.2479

V Z Density (calculated) F(000) Reflections collected Independent reflections Absorption correction S Final R indices [I N2sigma(I)]

2.4.1. Estimation of the levels of serum marker AST and ALT To assess the liver injury, activities of the serum AST and ALT were assayed using kits obtained from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. 2.4.2. Measurement of MDA level with SOD and GSH-Px activity in serum and liver A region of the inflamed liver was divided and accurately weighed to determine biochemical markers. One gram of liver tissues was homogenized in 9 mL of cold physiological saline. The homogenate was centrifuged at 500 ×g and 4 °C for 15 min and the supernatants were used immediately for the determination of antioxidant status. Activities of antioxidant defense enzymes, SOD and GSH-Px, as well as the level of MDA, as an index of the extent of lipid peroxidation in liver tissue, were determined following the instructions on the kit. In brief, MDA level was determined by the thiobarbituric acid method. Results were expressed as nanomoles of MDA per liter in the blood serum (nmol/L) or per milligram of protein in the wet tissues (nmol/mg) [9]. The assay for SOD was based on its ability to inhibit the oxidation of oxyamine by the xanthine–xanthine oxidase system. The results were expressed as SOD units per milliliter in the blood serum (U/mL) or per milligram of protein in the wet tissues (U/mg). The activity of GSH-Px was measured by the 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) method. The results were expressed as GSH-Px units per liter in the blood serum (U/L) or per milligram of protein in the wet tissues (U/mg). Protein content of the

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homogenates was determined using a standard commercial kit provided by Jiancheng Institute of Biotechnology (Nanjing, China). 2.4.3. Measurement of NO level and iNOS activity in the serum and liver The levels of NO and iNOS activities in the serum and liver homogenates were respectively measured spectrophotometrically at 550 nm and at 530 nm. Results were expressed as nanomoles of NO per liter in the blood serum (nmol/L) or per milligram of protein in the wet tissues (nmol/mg) and as iNOS units per milliliter in the blood serum (U/mL) or per milligram of protein in the wet tissues (U/mg), respectively.

supernatants (nuclear fraction) were saved. The protein concentrations of nuclear fraction were determined by the method of BCA, then aliquoted the fractions, and stocked at −80 °C. Afterwards, the protein expression levels of p-P56 and P56 in the nucleus were determined by using Western blotting. 2.9. Statistical analysis All the experiment data were expressed as mean ± SD. The significant difference from the respective control in all experiments was assessed by one way analysis of variance (ANOVA) using SPSS (version 13). p b 0.05 was considered statistically significant.

2.5. Histological analysis of liver 3. Results The liver samples were sectioned and stained with hematoxylin– eosin (H&E) and subsequently examined under a light microscope for general histopathology examination. The extent of hepatic damage was evaluated on H&E slides. The histological changes were scored according to the following criteria: 0, absent; 1, mild; 2, moderate; and 3, severe [10]. 2.6. Determination of the levels of hepatic IL-6, TNF-α and PGE2 One gram of liver tissues was homogenized in 9 mL of cold physiological saline. The homogenate was centrifuged at 12,000 ×g for 10 min; the supernatant was transferred into several new tubes, and stored at − 80 °C until analysis. The concentration of IL-6, TNF-α and PGE2 in liver tissue were quantified by enzyme-linked immunoassay kit and the results were expressed as picograms per gram of wet tissue (pg/g tissue). 2.7. Western blot analysis of hepatic inducible cyclooxygenase-2 (COX-2) The liver tissues were homogenized in RIPA buffer (1% Triton, 0.1% SDS, 0.5% deoxycholate, 1 mM EDTA, 20 mM Tris (pH 7.4), 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)) [11], and 0.1 g of liver tissues was homogenized in 1 mL of cold RIPA buffer. After homogenization and centrifugation (12,000 ×g for 10 min at 4 °C), the supernatant was separated from the pellet and stored in aliquots at −80 °C for the determination of COX-2 expression levels. Protein concentrations were quantified by the method of BCA (the kit of BCA protein assay was obtained from Applygen Technologies Inc., Beijing, China). Samples were diluted to a concentration of 2 mg/mL in SDS-loading buffer and boiled for 5 min. Thirty micrograms of protein from each sample was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Gels were also loaded with colored molecular weight markers to assess electrophoretic transfer and biotinylated protein ladder marker to estimate the molecular weights of bands of interest. The membranes were blocked with 0.5% BSA in TBST (pH 8.0) for 1.5 h and then incubated overnight at 4 °C with suitably diluted primary antibodies against COX-2. The expression of β-action was used to show equal protein loading. The blots were detected using the enhanced chemiluminescence (ECL) reaction. Quantification of protein bands was achieved by densitometric analysis using Image-Pro Plus® software (Media Cybernetics, Inc. USA). All western blot analyses were carried out at least three times. 2.8. Preparation of nuclear fractionation [12] The preparation of nuclear extracts was performed using the Nuclear Extract Kit (Active Motif, Carlsbad, CA). Briefly, 0.1 g of liver tissues was homogenized in 1 mL of cold PBS with phosphatase inhibitors, then lysed in 500 μL hypotonic buffers and centrifuged at 12,000 ×g for 30 s at 4 °C. The supernatant was transferred into fresh 1.5 mL microtubes as cytoplasmic fraction, then pellets were resuspended in 50 μL complete lysis buffer, centrifuged at 12,000 × g for 10 min at 4 °C, and the

3.1. Effects of MnSODm on serum ALT and AST The effects of MnSODm treatment on the CCl4-induced modification in the serum ALT and AST levels were shown in Table 2. Hepatic injury was induced by CCl4 in mice, as indicated by an increase in the serum ALT and AST activities after CCl4 administration. In contrast, the animals treated with MnSODm exhibited a significant decrease in the activities of these serum marker enzymes. Bifendate administration also reversed the alterations of the ALT and AST levels compared with the model group. 3.2. Histopathological examination of mice liver As demonstrated in Fig. 2, liver histopathological studies of hepatocyte morphological changes provided evidence to support the observed biochemical effects of Bifendate or MnSODm. In the normal group, liver slices showed the complete structure of cells with normal cell morphology, well-preserved cytoplasm and a clear plump nucleus (Fig. 2A). As shown in Fig. 2B, significant anomalies of liver cells were observed in CCl4-injured mice, where the cytoplasm was significantly reduced and the nucleus become atrophic, suggesting that CCl4 has induced severe liver cell injury. The pretreatment with Bifendate and MnSODm could effectively protect the liver from acute CCl4-induced hepatocyte morphological damage (Figs. 2C–F, 3). 3.3. Effects of MnSODm on MDA level and SOD, GSH-Px activities in serum and liver homogenates MDA as an indicator of lipid peroxidation, after administration of CCl4 significantly elevated the serum and liver tissue MDA levels in the model group compared to normal mice. Treatment with different doses of MnSODm reversed this biochemical parameter significantly towards normal level in a dose dependent manner (Table 3). The activities of antioxidant enzymes SOD and GSH-Px in serum and liver homogenates were significantly decreased in the liver injury model group when compared to normal control. MnSODm exerted a beneficial effect

Table 2 The effects of MnSODm on serum ALT, AST activity and liver index in CCl4-induced hepatic injury in mice. (x  S). Group

Normal Model Bifendate, 50 mg/kg MnSODm-L, 10 mg/kg MnSODm-M, 20 mg/kg MnSODm-H, 40 mg/kg

Serum ALT activity

Serum AST activity

Liver index

(U/mL)

(U/mL)

(mg/g)

56.87 89.39 68.20 68.49 67.70 58.07

± ± ± ± ± ±

24.77 8.62** 8.53## 6.66## 5.24## 6.15##

13.05 23.07 14.64 12.98 11.42 8.47

± ± ± ± ± ±

5.63 6.46** 7.83# 9.46# 4.93## 5.55##

41.6 64.3 45.6 55.4 50.8 45.8

± ± ± ± ± ±

8.1 7.3** 7.6## 8.2# 7.4## 6.3##

Note: MnSODm, Mimic of Manganese superoxide dismutase. Data are means ± SD, n = 10 per group. **P b 0.01 vs. normal group. #P b 0.05 or ##P b 0.01 vs. model group.

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A

B

C

D

E

F

129

Fig. 2. The histologic results of tissues stained with H&E under light microscope (original magnification, ×10). (A) Normal group, liver slices showed complete structure of cells with normal cell morphology, well-preserved cytoplasm and a clear plump nucleus. (B) Model group showed apparently morphological changes including severe hepatocellular hydropic degeneration, many ballooned hepatocytes, and spotty necrosis around the central vein. (C) Bifendate group (50 mg/kg), (D) MnSODm-L (low dose 10 mg/kg), (E) MnSODm-M (medium dose 20 mg/kg) and (F) MnSODm-H (high dose 40 mg/kg) showed moderate hypertrophy of hepatocytes with obviously decreasing the injured area, necrotic cells and ballooning degeneration.

H ist opa t hologic gr a ding ( scor e 0 - 3 )

3.5

**

on antioxidant enzymes since the activities of these enzymes were found to be significantly increased in MnSODm treated groups (Table 3).

3.0 ## 2.5

3.4. Effects of MnSODm on NO level and iNOS activity in the serum and liver

## ##

2.0

## 1.5

1.0

0.5

The levels of NO and the activity of iNOS in the serum and liver were significantly increased in the model group compared with the normal group. Compared with the model group, the levels of NO and the activity of iNOS in the serum and liver were significantly decreased in the MnSODm groups (Table 4). 3.5. Effect of MnSODm on the level of hepatic IL-6, TNF-α and PGE2

0.0 Normal

Model

Bifendate

MnSODm-L MnSODm-M MnSODm-H

Fig. 3. The histological damage scores. The histological changes were scored according to the following criteria: 0, absent; 1, mild; 2, moderate; and 3, severe hepatocellular necrosis and inflammatory infiltration. The results were presented as the means ± SD. **p b 0.01 compared with the normal group, ##p b 0.01 compared with the model group.

The levels of IL-6 and TNF-α in the model group were significantly higher compared to those in the normal control group. Up-regulation was markedly inhibited after treating with MnSODm and Bifendate (Table 5). PGE2 is an important prostanoid that is involved in hepatocyte damage induced by CCl4. The level of PGE2 was low in the normal control animals. However, the level of prostanoid was significantly

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Table 3 The effects of MnSODm on the level of MDA, and the activity of SOD and GSH-Px in serum and liver homogenates in CCl4-induced hepatic injury in mice. (x  S). Group

Normal Model Bifendate, 50 mg/kg MnSODm-L, 10 mg/kg MnSODm-M, 20 mg/kg MnSODm-H, 40 mg/kg

Serum MDA level

Liver MDA level

Serum SOD activity

Liver SOD activity

Serum GSH-Px activity

Liver GSH-Px activity

(nmol/L)

(nmol/mg prot)

(U/mL)

(U/mg prot)

(μmol/mL)

(mg/g prot)

3.36 5.01 4.08 4.21 4.02 3.38

0.177 0.337 0.253 0.301 0.266 0.223

498.8 467.6 508.7 514.3 514.7 528.5

90.75 55.14 72.69 68.28 78.94 81.86

2826 2554 2804 2736 2802 2928

26.31 22.72 27.34 27.24 32.09 32.72

± ± ± ± ± ±

0.35 0.25** 0.52## 0.39## 0.48## 0.42##

± ± ± ± ± ±

0.048 0.061** 0.031## 0.037 0.062# 0.022##

± ± ± ± ± ±

22.4 35.8* 26.9## 15.4## 11.1## 19.2##

± ± ± ± ± ±

14.01 19.61** 8.46 # 13.04# 19.40## 17.74##

± ± ± ± ± ±

107 141** 158## 191# 158## 178##

± ± ± ± ± ±

2.14 3.00* 2.50## 5.27# 3.06## 1.80##

Note: MnSODm, Mimic of Manganese superoxide dismutase. Data are means ± SD, n = 10 per group. *P b 0.05 or **P b 0.01 vs. normal group. #P b 0.05 or ##P b 0.01 vs. model group.

increased in the model animals after the CCl4 treatment. The increase was reduced in the MnSODm and the Bifendate supplemented groups (Table 5).

CCl4 in mice directly led to the rise of serum ALT and AST activities. After the pretreatment of MnSODm, the liver was protected, as indicated by the decreased serum enzyme activities of ALT and AST, suggesting that MnSODm may effectively protect hepatocytes against the toxic effects of CCl4. Histological observations of livers also strongly supported the hepatoprotective effect of MnSODm. CCl4 caused various histological changes to the liver, including cell necrosis, fatty metamorphosis in adjacent hepatocytes, ballooning degeneration, and infiltration of lymphocytes and Kupffer cells. These changes were significantly attenuated by MnSODm. The results indicated that MnSODm might have a potential protective effect against CCl4-induced liver injury. It is well recognized that liver injury induced by CCl4 resulted from free radicals which cause lipid peroxidation, leading to hepatic cell damage [19]. MDA is one of the end-products of polyunsaturated fatty acid peroxidation and its tissue level can reflect the extent of lipid peroxidation in hepatocytes [20]. In the present study, the levels of MDA in serum and liver tissues were significantly decreased in the MnSODm groups, indicating its ability to break the chain reaction of lipid peroxidation. In order to further clarify the mechanisms of hepatoprotective activity of MnSODm, its effect on hepatic antioxidant defense system was explored. The two antioxidant enzymes SOD and GSH-Px play critical roles in the cellular defense against the deleterious action of ROS and cellular products of free radical chain reactions [21]. While SOD catalyzes the conversion of superoxide free radical to less toxic hydrogen peroxide, GSH-Px catalyzes the breakdown of hydrogen peroxide into water and oxygen and can directly detoxify lipid peroxides generated by ROS [22]. As SOD and GSH-Px are easily inactivated by ROS or lipid peroxides, this may explain a decrease in activities of these two enzymes observed in serum and liver tissue of CCl4-intoxicated mice in our study. However, our results demonstrated that SOD and GSH-Px were appreciably elevated by MnSODm administration, suggesting that it could restore both enzymes and/or activate enzyme activities in CCl4-damaged liver tissue. Moreover, these findings supported the beneficial effect of MnSODm in maintaining hepatocyte integrity and function. It was conceivable that these effects might be due, at least in part, to its antioxidant activity. As is well known, the inflammation initiated by CCl4-induced hepatotoxicity will release pro-inflammatory mediators, such as iNOS, IL-6, TNF-α, PGE2 and COX-2. Certain evidence has indicated that hepatic

3.6. Effect of MnSODm on the level of COX-2 The protein expression levels of COX-2 in the model group were higher than those of the normal control. In contrast, the increases in COX-2 expression were significantly suppressed by MnSODm (Fig. 4). 3.7. Effect of MnSODm on the translocation of NF-κB NF-κB plays a critical role in inflammatory diseases, and its activation is essential for cytokine production. In this study, we have observed remarkable activation of NF-κBp56 in the model control group compared with the normal control group. Treatment with MnSODm was found to significantly restrict the NF-κBp56 activation (Fig. 5). 4. Discussion In the present study, that MnSODm exerted a beneficial antinflammation effect on CCl4-induced acute hepatic injury in mice was demonstrated for the first time. The results showed that MnSODm is able to protect against oxidation of hepatic cellular membrane damage via scavenging oxygen free radicals and down-regulating the level of pro-inflammatory factors. In addition, the molecular mechanisms related with the therapeutic effects of MnSODm on hepatic injury which involve inhibition of NF-κB translocated to the nucleus was shown. CCl4 as a well-known hepatotoxin was used to induce the acute liver damage in mice liver injury. It is reported that CCl4 generates two active microsomal radicals or peroxides (•CCl3 or CCl3OO•) by cytochrome P450 during the toxic metabolic process of CCl4 in the liver [13,14]. These substances may cause lipid peroxidation with liver cell membranes and sub-cellular structures, and undermine the integrity of the cell membrane structure [15], which eventually may lead to liver cell death [16]. It is also well known that the increased ALT enzyme activity is an indicator of the degree of liver cell membrane damage, and elevated AST activity is another indicator of liver mitochondrial damage, which are therefore the most important and effective index for evaluating liver cell damage [17,18]. Our results showed that the injection of

Table 4 The effects of MnSODm on NO level and iNOS activity in serum and liver homogenates in CCl4-induced hepatic injury in mice. (x  S). Group

Normal Model Bifendate, 50 mg/kg MnSODm-L, 10 mg/kg MnSODm-M, 20 mg/kg MnSODm-H, 40 mg/kg

Serum NO level

Liver NO level

Serum iNOS activity

Liver iNOS activity

(μmol/L)

(μmol/g prot)

(U/mL)

(U/mg prot)

1.01 2.68 1.39 1.44 1.36 1.26

9.58 13.90 11.42 12.29 11.89 10.01

10.00 19.39 13.14 14.31 13.53 11.75

± ± ± ± ± ±

2.52 3.53** 3.21## 3.40## 1.92## 2.69##

± ± ± ± ± ±

0.26 0.33** 0.52## 0.26## 0.37## 0.39##

± ± ± ± ± ±

1.54 1.67** 1.54## 1.73## 1.22## 1.05##

2.01 3.69 2.70 2.86 2.42 2.08

± ± ± ± ± ±

0.22 0.37** 0.28## 0.34## 0.26## 0.39##

Note: MnSODm, Mimic of Manganese superoxide dismutase. Data are means ± SD, n = 10 per group. *P b 0.05 or **P b 0.01 vs. normal group. ##P b 0.01 vs. model group.

Y.-H. Wang et al. / International Immunopharmacology 22 (2014) 126–132 Table 5 The effects of MnSODm on in the level of IL-6, TNF-α and PGE2 in liver homogenate in CCl4-induced hepatic injury in mice. (x  S). Group

Normal Model Bifendate, 50 mg/kg MnSODm-L, 10 mg/kg MnSODm-M, 20 mg/kg MnSODm-H, 40 mg/kg

IL-6

TNF-α

PGE2

(pg/g tissue)

(pg/g tissue)

(pg/g tissue)

316 464 417 373 354 316

1233 1921 1582 1603 1533 1455

212 366 241 294 252 218

± ± ± ± ± ±

67 62** 87 77# 68## 61##

± ± ± ± ± ±

168 225** 201## 173## 187## 192##

± ± ± ± ± ±

33 35** 28## 36## 26## 39##

p-P65

65 KD

P65

65 KD

Lamin B

67 KD

1.8

COX-2

74 KD

β-actin

42 KD

2.0

**

1.8 ##

COX-2/ β-actin levels

1.6

## ##

1.4

##

1.2 1.0 0.8 0.6 0.4 0.2 0.0 Normal

Model

Bifendate

MnSODm-L MnSODm-M MnSODm-H

Fig. 4. Effect of 3 doses of MnSODm on COX-2 level in liver tissue of CCl4-induced hepatic injury in mice revealed by western blot. Parallel blotting was performed with β-action antibody. Data are means ± SD, n = 3 per group. **P b 0.01 vs. normal group. ##P b 0.01 vs. model group. MnSODm, Mimic of Manganese superoxide dismutase at MnSODm-L 10 mg/kg, MnSODm-M 20 mg/kg, or MnSODm-H 40 mg/kg.

**

1.6

Note: MnSODm, Mimic of Manganese superoxide dismutase. Data are means ± SD, n = 10 per group. **P b 0.01 vs. normal group. #P b 0.05 or ##P b 0.01 vs. model group.

##

1.4 p-P65/p65 levels

damage may be caused by excessive nitric oxide production through iNOS [23]. Several studies demonstrated that the excessive accumulation of NO would cause damage to the liver and that its inhibition can reduce inflammatory damage. NO, as a gas that represents one of the smallest biologically active molecules synthesized by mammalian cells could interact rapidly with oxygen radicals to yield a variety of cytotoxic nitrogen species that induce lipid peroxidation and other cellular oxidative stress. The present study showed that MnSODm could not only remarkably decrease the elevated contents of NO, but also evidently decrease the iNOS activity in liver tissues and serum of mice with CCl4-induced hepatic injury. Therefore, the anti-inflammatory effect of MnSODm may decrease the production of NO through inhibiting the expression of iNOS. IL-6 and TNF-α, are pleiotropic pro-inflammatory cytokines that are rapidly produced by macrophages in response to tissue damage [24], which conversely stimulate the release of cytokines from macrophages and promote oxidative metabolism and NO production of the phagocyte. Additional factors closely related to NO production included COX-2 activity and the subsequent generation of PGE2 [25]. COX-2 is the rate-limiting enzyme and is responsible for the catalysis of PGE2 from arachidonic acid [26]. The overproduction of PGE2 mediated by COX-2 has been linked to the development of inflammation and carcinogenesis [27]. Being consistent with the previous studies,

131

## ##

##

1.2 1.0 0.8 0.6 0.4 0.2 0.0 Normal

Model

Bifendate

MnSODm-L MnSODm-M MnSODm-H

Fig. 5. Effect of 3 doses of MnSODm on the translocation of NF-κB in liver tissue of CCl4induced hepatic injury in mice revealed by western blot. Parallel blotting was performed with Lamin B antibody. Data are means ± SD, n = 3 per group. **P b 0.01 vs. normal group. ##P b 0.01 vs. model group. MnSODm, Mimic of Manganese superoxide dismutase at MnSODm-L 10 mg/kg, MnSODm-M 20 mg/kg, or MnSODm-H 40 mg/kg.

there were significant increases in COX-2 and PGE2 productions as well as TNF-α and IL-6 levels after administration of CCl4 in this study, confirming that an inflammatory response was elicited at the site of injury. In contrast, the expression levels of these pro-inflammatory factors were down-regulated after treatment with MnSODm, which may suppress the secretion and/or enhance the degradation of these proteins. Nuclear factor (NF)-κB is a transcription factor that is important for the activation of many inflammatory mediators. Further exploration of the underlying mechanism of MnSODm on the pro-inflammatory mediators showed that MnSODm played a crucial role in the reduction of NFκB activation. NF-κB exists mainly as a heterodimer composed of subunits of the Rel family, p50 and p65. In the resting state, NF-κB normally localizes to the cytoplasm, where it is bound by the inhibitory IκB proteins. However, following inflammatory stimulus, IκB is phosphorylated by IκB kinase (IKK) and is subsequently degraded by the proteasome. NF-κB is then released and translocates to the nucleus, where it triggers the transcription of multiple genes through the cis-acting κB element. NF-κB promotes the expression of various pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α [28,29]. Oxygen free radicals and proinflammatory cytokines released during inflammation could activate NF-κB, and then activate the subsequent inflammatory cascade in CCl4-induced hepatic injury. Activation of NF-κB may be a pivotal event in pro-inflammatory signal transduction [30,31]. Our results showed that CCl4 significantly increased the NF-κBp65 activation. However, treatment of mice with MnSODm suppressed the NF-κBp65 activation significantly towards normal level. This result indicated that MnSODm inhibited inflammatory responses, in large part, via the down-regulation of NF-κB. In conclusion, our study demonstrated that MnSODm was significantly beneficial in the prevention of CCl4-induced liver toxicity, possibly by scavenging reactive free radicals, boosting the endogenous antioxidant system, and inhibiting pro-inflammatory cytokines via the down-regulation of NF-κB. Further studies elucidating these findings

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Hepatoprotective effects of Mimic of Manganese superoxide dismutase against carbon tetrachloride-induced hepatic injury.

The aim of this study was to investigate the protective effects of Mimic of Manganese superoxide dismutase (MnSODm) against carbon tetrachloride (CCl(...
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