Nutrition Journal of Parenteral and Enteral

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A New Enteral Diet, MHN-02, Which Contains Abundant Antioxidants and Whey Peptide, Protects Against Carbon Tetrachloride −Induced Hepatitis Takehiko Takayanagi, Hajime Sasaki, Akihiro Kawashima, Yuichiro Mizuochi, Hiroyuki Hirate, Takeshi Sugiura, Takafumi Azami, Kiyofumi Asai and Kazuya Sobue JPEN J Parenter Enteral Nutr 2011 35: 516 DOI: 10.1177/0148607110381599 The online version of this article can be found at: http://pen.sagepub.com/content/35/4/516

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Original Communication

A New Enteral Diet, MHN-02, Which Contains Abundant Antioxidants and Whey Peptide, Protects Against Carbon Tetrachloride–Induced Hepatitis

Journal of Parenteral and Enteral Nutrition Volume 35 Number 4 July 2011 516-522 © 2011 American Society for Parenteral and Enteral Nutrition 10.1177/0148607110381599 http://jpen.sagepub.com hosted at http://online.sagepub.com

Takehiko Takayanagi, MD1; Hajime Sasaki, PhD3; Akihiro Kawashima3; Yuichiro Mizuochi, MD, PhD1; Hiroyuki Hirate, MD1; Takeshi Sugiura, MD, PhD1; Takafumi Azami, MD, PhD1; Kiyofumi Asai, MD, PhD2; and Kazuya Sobue, MD, PhD1 Financial disclosure: none declared.

Background: Inflammatory or oxidative stress is related to various diseases, including not only inflammatory diseases, but also diabetes, cancer, and atherosclerosis. The aim of this study was to evaluate the anti-inflammatory effects of a new enteral diet, MHN-02, which contains abundant antioxidants and whey peptide. The study also investigated the ability of MHN-02 to attenuate lethality, liver injury, the production of inflammatory cytokines, and the production of oxidized products using a carbon tetrachloride–induced rat model of severe fulminant hepatitis. Methods: Male Sprague-Dawley rats were fed either a control diet or the MHN-02 diet for 14 days and injected with 2 mL/kg of carbon tetrachloride. Survival of rats was monitored from day 0 to day 3. To evaluate liver injury, inflammation, and oxidative stress, blood and liver samples were collected, and aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, interleukin 6, tumor necrosis factor–α, and

superoxide dismutase activity as a free radical scavenger were measured. A portion of the liver was evaluated histologically. Results: The survival rates of rats receiving the MHN-02 diet and the control diet were 90% and 55%, respectively. In the MHN-02 diet group, levels of serum liver enzymes and serum cytokines were significantly lower than in the control group. Superoxide dismutase activity in the MHN-02 diet was significantly higher in the MHN-02 group. Pathological lesions were significantly larger in the control group. Conclusion: Supplementation of enteral diets containing whey peptide and antioxidants may protect against severe hepatitis. (JPEN J Parenter Enteral Nutr. 2011;35:516-522)

Clinical Relevancy Statement

reported that reactive oxygen species (ROS) might worsen liver damage and that ROS stimulate tissue macrophages to produce proinflammatory cytokines, which subsequently attract inflammatory cells into the liver and also produce a larger amount of ROS and can contribute to acute hepatitis. Although the protective effects of several agents in the models of acute hepatic injury have been reported, the available drugs are still insufficient clinically. The current practice is to reduce liver damage using this diet including abundant antioxidant substances, ω-3 long-chain polyunsaturated fatty acids (PUFAs), and whey peptide. Although the animals in this study were receiving this diet before the carbon tetrachloride (CCl4) stimulation, this enteral nutrition (EN) will clinically have a substantial impact on severe hepatitis that is critical in illnesses.

Keywords: immunonutrition; hepatitis; whey peptide; antioxidant effect

Acute severe liver injury results from the massive death of liver cells leading to liver dysfunction. It has been also From the 1Department of Anesthesiology and Medical Crisis Management and 2Molecular Neurobiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; and the 3Food Science Institute Division of Research and Development, Meiji Dairies Corporation, Odawara, Japan. Received for publication February 18, 2010; accepted for publication July 6, 2010. Address correspondence to: Takehiko Takayanagi, Department of Anesthesiology and Medical Crisis Management, Nagoya City University, Graduate School of Medical Sciences, Mizuho-ku, Nagoya 467-8601, Japan; e-mail: [email protected].

516

MHN-02 Protects Against Carbon Tetrachloride–Induced Hepatitis / Takayanagi et al 517

Introduction Severe hepatitis induces acute liver failure (ALF), in which rapid deterioration of liver function results in encephalopathy and coagulopathy requiring a supportive approach, intensive care, and liver transplantation.1,2 In patients with viral hepatitis, survival rates are between 50% and 60%. ALF is often associated with a very high rate of mortality. CCl4, a well-known hepatotoxin, is activated in the liver by the cytochrome P450 system. The initial metabolite is the trichloromethyl free radical, which produces ROS and, ultimately, liver cell necrosis.3,4 In the liver injury induced by CCl4, administration of this toxin has been shown to induce upregulation of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)–α, and interferon-γ, and secondary hepatic injury occurs as a result of the inflammatory processes.5-7 Under physiologic conditions, there is a carefully maintained balance between the formation and the scavenging of ROS by various antioxidants.8 Antioxidative enzymes and low-molecular-weight compounds such as vitamin C and vitamin E are involved in this equilibrium. However, excessive oxidative stress breaks this equilibrium and may lead to disease. The effects on oxidative stress obtained from the administration of various antioxidants have been studied in clinical trials and in experimental animal models. Many individual cytokines are themselves pleiotropic and exert multiple effects.9 Cytokines are important targets in the management of inflammatory conditions,7 and overproduction of cytokines such as TNF-α and IL-6 is known to induce inflammation and lethal shock.10 Although EN diets have only been used for nutrition support under conditions of insufficient oral digestion, several enteral diets have been developed as immunonutrients. The MHN-02 formula, an immunonutrient that contains antioxidants such as vitamins C and E, trace elements such as zinc and selenium, the ω-3 long-chain PUFAs docosahexaenoic acid (DHA; 22:6 ω-3) and eicosapentaenoic acid (EPA; 20:5 ω-3), and hydrolyzed whey peptide (HWP) have strong antioxidant activity and antiinflammatory effects.11-13 In this study, we examined the effects of MHN-02 on lethality, liver injury, the production of inflammatory cytokines, and the production of peroxide compounds using a CCl4-induced rat model of fulminant hepatitis.

Methods Male Sprague-Dawley rats (n = 12; Charles River, Nagoya, Japan) aged 6 to 8 weeks with body weights of approximately 180 g were used to produce this model of CCl4induced hepatitis.14,15 The rats were housed in an air-conditioned room at 23°C ± 1°C and at 50% ± 10% humidity with lights on from 8 am to 8 pm during the

Table 1. Major Nutrients in MHN-02 Diet and Control Diet (per 100 kcal)

Protein g % kcal Sources

Carbohydrate g % kcal Sources

MHN-02

Control

5 20 hydrolyzed whey peptide, fermented milk

4 16 Na caseinates

14.5 53.2 Isomaltulose, dextrin

Lipids g % kcal MCT, g EPA, DHA, g w-6/w-3 ratio Vitamins Vitamin A, mg RE Vitamin E, mg Vitamin C, mg Choline, mg Minerals Zinc, mg Selenium, mg

15.5 59 Dextrin

2.8 25 0.59 0.06 2

2.8 25

150 5 50 9.2

60 3 16 1.7

1 5

0.8 3.5

3.2

DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MCT, medium-chain triglyceride; RE, retinol equivalents.

experiment. The animals were handled according to protocols approved by the Institutional Animal Committee of Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. The experiments were conducted in a manner consistent with the relevant ethical guidelines for animal research. We produced both a control diet and the MHN-02 diet (Meiji Dairies Corporation, Tokyo, Japan). The compositions of each diet are shown in Table 1. The control diet was similar in composition to general enteral diets. The MHN-02 diet was supplemented with increased amounts of vitamin C, vitamin E, trace elements (ie, zinc and selenium), HWP, fermented milk, and isomaltulose.

Experimental Design The rats were divided into the following 4 groups: MHN-02 sham group; MHN-02 CCl4-induced hepatitis group (MHN-02 diet group); control diet sham group; and control diet CCl4-induced hepatitis (control diet group). Before the start of the experiment, each group diet was given while drinking water was available ad libitum for 2 weeks. After 2 weeks on the diets, all rats

518 Journal of Parenteral and Enteral Nutrition / Vol. 35, No. 4, July 2011

Whole Blood and Plasma Isolation Immediately after the rats were killed with an overdose of inhaled sevoflurane, blood was collected from the right ventricle of the heart into a syringe that contained a minute amount of heparin and was placed on ice. Serum and plasma were separated by centrifugation at 1,000 g for 20 minutes at 4°C. Samples were kept at –80°C until further analysis.

Superoxide Dismutase Assay The liver was perfused with phosphate-buffered saline (PBS). Samples from the liver and intestines were collected and fixed in 10% formalin for histopathology, and the remaining tissue was immediately snap-frozen in liquid nitrogen and stored at –80°C for analysis of superoxide dismutase (SOD). Liver tissues were homogenized in 0.25 mmol/L sucrose buffer (10 mmol/L pH 7.41 2-amino2-(hydroxymethyl) propane-1,3-diol (Tris)-HCl buffer, 1 mmol/L EDTA) to 10% homogenates using plastic homogenizers and kept on ice. The homogenates were centrifuged at 78,000 g for 60 minutes, and the supernatants were collected. SOD activity was measured by SOD Assay Kit-WST (Dojindo Laboratory, Kumamoto, Japan) using a microplate reader (Dojindo Laboratory, Kumamoto, Japan) at 450 nm.

Plasma Protein Assay Plasma activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured by Fuji Dry Chem (Fuji Film, Tokyo, Japan) at each time point. Plasma TNF-α and IL-6 concentrations were measured using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN)

Histopathologic Examination A small portion of the right lobe of the liver was placed in cassettes and fixed with 10% neutral formalin. The specimens were dehydrated and embedded in paraffin, and tissue sections 10 µm in thickness were stained with hematoxylin and eosin and evaluated using light microscopy. The area of hepatic lesions exhibiting degeneration, infiltration of inflammatory cells, or necrosis was calculated using

Table 2. Changes in Body Weighta

Pre test, g Post test, g

MHN-02 Diet

Control Diet

184.6 ± 3.3 264.0 ± 6.4

185.4 ± 1.4 269.8 ± 4.8

a

Values are given as mean ± standard error of the mean. (%)

Percent survival

consumed their designated diets and had gained weight to the same extent in each group. Hepatitis was induced by intraperitoneal (IP) injection of 2 mL/kg CCl4 (Wako, Osaka, Japan) dissolved in 2 mL/kg corn oil. The sham group was given an IP injection of 2 mL/kg of corn oil. After the injection, each group was also fed its study diet while drinking water was available ad libitum. Animals were euthanized at 0, 4, 8, 12, 24, or 48 hours after injection of CCl4 or corn oil, and blood and tissue samples were collected.

100 90 80 70 60 50 40 30 20 10 0

Sham (control diet) Sham (MHN-02) Control diet MHN-V02 0

10

20

30

40

50

60

70

80 (hr)

Figure 1. Effects of the MHN-02 diet on the survival of carbon tetrachloride (CCl4)–treated rats. Rats were fed the control diet or the MHN-02 diet for 2 weeks and challenged or not with a single intraperitoneal injection of 2 mL/kg CCl4. The survival of rats was monitored for 72 hours. The overall difference in survival rate between the MHN-02 diet group and the control group was statistically significant (P = .011).

Image J, a free software and public domain Java imageprocessing program inspired by NIH Image.

Statistical Analysis All values are expressed as mean ± standard error of the mean. Statistical analyses were performed on GraphPad Instat3 and Prism software (Graphpad Software, La Jolla, CA) with Student t test and ordinary or repeated-measures analysis of variance. The log-rank test was used for survival analysis. Differences between groups were considered significant at P < .05.

Results Sixty-eight Sprague-Dawley rats (aged 6–8 weeks, weighing approximately 180 g) consumed their diets for 14 consecutive days. Body weight gain was almost identical with the control and MHN-2 diets (Table 2). Kaplan-Meier curves (Figure 1) revealed the absence of mortality after the sham operation (100% survival), whereas survival rate was reduced to 55% in the control diet group 72 hours after IP administration of CCl4 (P < .05 vs sham). The MHN-02 group exhibited a significant improvement in survival rate (90%) throughout the 72-hour observation period (P = .011).


IL -6 AST

IU/L

7,500

(A)

a

a

10,000

a

a

4,000

Control diet MHN-02 Sham (control diet) Sham (MHN-02)

a

5,000

Control diet MHN-02

5,000 pg/mL

(A)

2,500

a

3,000 2,000 1,000

0

0

4

12

(B)

24

(hr)

48

ALT a

10,000

a

5,000

12

24

48

(hr)

(C) LDH a

75,000

a Control diet MHN-02 Sham (control diet) Sham (MHN-02)

IU/L

50,000 a 25,000

0

a

0

4

12

24

48

(hr)

Figure 2. Liver enzyme (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and lactate dehydrogenase [LDH], IU/L) activity significantly increased with hepatocyte injury at 4 hours, 24 hours, and 4 hours, respectively, after carbon tetrachloride injection. aP < .05 compared with control by t test.

48

(hr)

Control diet MHN-02

0

4

8

24

48 (hr)

Figure 3. Effects of the MHN-02 diet on carbon tetrachloride (CCl4)–induced increases in serum levels of (A) interleukin (IL)-6 and (B) tumor necrosis factor (TNF)–α in rats. Samples were obtained before (0 hours) and after an intraperitoneal injection of CCl4 (4 hours, 12 hours, 24 hours, 48 hours, 72 hours). The concentrations of TNF-α and IL-6 in serum were determined with enzyme-linked immunosorbent assay kits. Values are the mean ± standard error of the mean. aP < .05 compared with control by t test. a

SOD activity (IU/mg protein)

40

CCl4 significantly increased (200-fold) the plasma concentrations of the liver enzymes AST and ALT, which are markers of hepatocyte injury. The rats that received the MHN-02 diet were partially but significantly protected against this effect, which peaked at 24 hours (Figure 2). In the control diet group, the concentration of LDH increased significantly (350-fold) after CCl4 injection, peaked at 12 hours, and dropped to near baseline after 48 hours. At 12 hours, the activity of LDH in rats fed the MHN-02 diet was significantly lower than that in rats fed the control diet. CCl4 resulted in significantly more release of inflammatory cytokines in the control diet group than in the MHN-02 diet group (Figure 3). Both TNF-α and IL-6 were barely detectable before CCl4 injection. In the control diet group, TNF-α levels increased significantly after CCl4 injection, peaked at 4 hours, and dropped to near baseline after 48 hours (Figure 3). On the other hand, TNF-α levels peaked at 24 hours and dropped to near baseline after 48 hours in the MHN-02 diet group.

24

pg/mL

4

12

a 110 100 90 80 70 60 50 40 30 20 10 0

2,500

0

4

TNF-α

Control diet MHN-02 Sham (control diet) Sham (MHN-02)

0

0

(B)

7,500 IU/L

0

Control diet MHN-02

a

30 20 10 0

0

24

(hr)

Figure 4. Effects of the MHN-02 diet on carbon tetrachloride (CCl4)–induced increases in liver levels of superoxide dismutase (SOD) in rats. Liver samples were collected before CCl4 injection (0 hours) and 24 hours after injection (24 hours) and were homogenized in 5 volumes of 0.25 mmol/L sucrose buffer. SOD activity in liver was determined by measurement at 450 nm. Values are mean ± standard error of the mean. aP < .05 compared with control by t test.

Peak TNF-α activity in rats fed the MHN-02 diet was significantly less than that in rats fed the control diet. In both groups, IL-6 increased significantly after


Figure 5. Representative hepatic sections stained with hematoxylin and eosin 24 hours after administration of (A) corn oil or (B and C) carbon tetrachloride (CCl4) . (A) Slide from a representative rat injected with corn oil shows normal hepatic structure. (B and C) Hepatic section from a representative rat that consumed the control diet. (B) Twenty-four hours after CCl4 injection shows severe multifocal necrosis, infiltration of inflammatory cells, and necrosis (arrows). (C) The rats with MHN-02 diet exhibit focal necrosis with less severe lesions. Scale bars indicate 200 µm. (D) Regions of liver with hepatocytes exhibiting degradation, necrosis, or infiltration of inflammatory cells. The lesion area of livers in the MHN-02 group was significantly less than that in the control group. Values are the mean ± standard error.a

CCl4 injection, peaked at 8 hours, and dropped to near baseline after 48 hours. At 8 hours, IL-6 activity did not differ significantly between groups (Figure 3). The intrahepatic concentration of the antioxidant SOD increased significantly in rats on the MHN-02 diet compared with those on the control diet (P < .003; Figure 4). SOD levels were dramatically depleted (25%) in rats on the control diet 24 hours after CCl4 injection and notably improved in the animals that had consumed the MHN-02 diet. Histologic analysis revealed intermediate-grade portal fibrosis, signs of focal necrosis, Councilman bodies, and multifocal necrosis and invasion of inflammatory cells such neutrophils and lymphocytes in the control diet group, whereas these changes were less pronounced in the MHN-02 group (Figure 5). Pathologic lesions were significantly larger in the control group than in the MHN-02 group (Figure 5D).

Discussion In rats, the administration of a single dose of CCl4 induced liver injury similar to that seen in human viral

hepatitis and acute self-limiting hepatitis with necrosis, inflammation, and regeneration.16 The present study showed that the MHN-02 diet, supplemented with vitamins C and E, and trace elements (ie, zinc and selenium), the ω-3 long-chain PUFAs DHA (22:6 ω-3) and EPA (20:5 ω-3), and HWP, had higher antioxidant activity and anti-inflammatory effects than the control diet, increased survival rate, and decreased oxidative stress and inflammation in rats injected with CCl4. CCl4-induced liver injury has numerous effects and results in both liver necrosis and apoptosis. CCl4 causes direct injury by altering hepatocyte membrane permeability.17 The toxicity of CCl4 is currently thought to involve a free radical produced by the mixed function oxidase system of the liver endoplasmic reticulum. It also causes indirect injury due to the highly reactive free radicals formed by the metabolism of CCl4. Antioxidant vitamins have been tested for efficacy in suppressing diseases related to ROS. Some reports have demonstrated that vitamin C attenuates ischemia– reperfusion injury18 and cardiovascular disorders.19 Vitamin E inhibits ROS chain reactions and lipid peroxidase.20 In


addition, vitamin E functions in a synergistic fashion with vitamin C.21 Vitamin E is transformed to vitamin radicals when there is a reduction in lipid peroxidase. However, vitamin C reduces the oxidized form of vitamin E radicals and restores its antioxidant function.22 Thus, vitamin C and vitamin E may have synergistic effects. It has been reported that early administration of vitamins C and E to critically ill patients reduced organ damage and shortened the duration of hospital stay.22 Trace elements, including zinc and selenium, have various protective effects against the metabolism of ROS. Trace elements maintain and enhance the activity of antioxidant enzymes, attenuate oxidative stress, and decrease the severity of various conditions such as ischemia–reperfusion injury.23 In critically ill patients, the administration of selenium, zinc, and vitamin E suppressed decreases in antioxidant enzymes.24 A 14-day prefeeding period with the MHN-02 diet containing antioxidants such as vitamin C, vitamin E, and trace elements increased serum antioxidative activity, and the activity was evaluated by SOD. SOD plays an important role in the antioxidant defense system against superoxide anion (O2−) generated in vivo and is involved in defense against many diseases.25-27 Our study found that SOD activity was higher in the MHN-02 diet group than in the control diet group at 24 hours after CCl4 injection and thus improved survival. The mechanism by which antioxidants improve endothelial function probably involves direct scavenging for free radicals. The ingested antioxidants were enterally absorbed and thereby increased individual antioxidant levels in the liver and may have enhanced antioxidant capacity. CCl4 is known to induce hepatitis through high levels of TNF-α and IL-6, which enhance injury and promote apoptotic death of hepatocytes.17,28-30 TNF-α and IL-6 concentrations were also decreased in the MHN-02 diet group. The serum concentrations of TNF-α and IL-6 dramatically increase in experimental models, and overproduction of these cytokines is induced in inflammation and lethal shock.10 Generally, neutrophils and macrophages are activated, and inflammatory cytokines are produced with overproduction of ROS in critical illnesses. In this fashion, inflammatory and oxidative stresses are closely related and increase the severity of CCl4-induced hepatitis. The amount of lipids and profiles of the diets used in this study differed, with an ω-6:ω-3 ratio in the control diet nearly 1.6 times that in the MHN-02 diet. This ratio is recommended by the Institute of Medicine guidelines for the general population.31,32 The MHN-02 formula contained the ω-3 long-chain PUFAs DHA (22:6 ω-3) and EPA (20:5 ω-3), which have been associated with reduced production of inflammatory eicosanoids; this diet was therefore anticipated to have anti-inflammatory effects. The ω-3 and ω-6 PUFAs are incorporated into the cell

membrane’s physical and biochemical signaling pathways.33 The most studied mechanisms center around the observation that shifts in dietary consumption of ω-6 and ω-3 PUFAs lead to alterations in the quantities of ω-6– derived and ω-3–derived eicosanoids produced in animals and humans, which disturb the balance of lipid-based pro inflammatory and anti-inflammatory mediators produced at sites of inflammation. For example, the 4-series leukotrienes (LTs) (eg, LTB4) produced by 5-lipoxygenase or the ω-6 PUFA arachidonic acid (20:4) are highly inflammatory, whereas the 5-series LTs (eg, LTB5) produced from the ω-3 PUFA EPA are 10-fold to 100-fold less active.34 Although the roles played by EPA and DHA in our study cannot be directly determined, EPA and DHA probably contributed to the protective effects of the MHN-02 diet in this model. Our findings suggest that the MHN-02 diet may have attenuated not only oxidative stress, but also excessive inflammatory reaction in hepatitis. However, we found that the time to peak value differed between cytokine and death rate in the control diet group. Effects of cytokines at 12 hours may have influenced the mortality rate. The MHN-02 diet contains a large amount of HWP, which is reported to have anti-inflammatory effects in inflammatory models in rats.12,13 Although its mechanism of action is unclear, the α-lactoalbumin included in whey protein was recently found to have anti-inflammatory effects. It has also been reported to inhibit cyclooxygenase to induce prostaglandin mediators of inflammation.35,36 Our findings suggest the possibility that the HWP in the MHN-02 diet indirectly attenuated excessive inflammatory reaction in hepatitis. The present study revealed that an enteral diet containing abundant antioxidant substances, ω-3 long-chain PUFAs, and HWP exhibited high antioxidant and anti-inflammatory activities in vivo. Oral intake of this diet enhanced the capacity for high antioxidant and anti-inflammatory activities and protected against CCl4-induced hepatitis by counteracting inflammatory and oxidative stress. Supplementation of this type of enteral diet may protect against severe hepatitis, which is critical in illnesses related to oxidative stress and inflammation in the clinical setting.

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