International Immunopharmacology 22 (2014) 492–497
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Potential protection of vitamin C against liver-lesioned mice Min Su a,1, Hongqiu Chen b,1, Chaohe Wei c, Ning Chen d,e, Wei Wu e,⁎ a
Faculty of Basic Medicine, Guilin Medical University, Guilin 541004, PR China Department of Pathology, Guigang City People's Hospital, Guigang, Guangxi 537100, PR China c Central Pharmacy, Guigang City People's Hospital, Guigang, Guangxi 537100, PR China d Guangxi Medical University, Nanning 530021, PR China e Guilin Medical University, Guilin 541004, PR China b
a r t i c l e
i n f o
Article history: Received 30 June 2014 Received in revised form 22 July 2014 Accepted 29 July 2014 Available online 10 August 2014 Keywords: Vitamin C Hepatotoxicity Metabolism Inflammation
a b s t r a c t Pathologically, liver injury can result from sustained trauma to hepatocytes, including acute damage. Thus, attenuation of hepatocellular lesion may help improve liver functions. The purpose of this study was to explore the potential advantages of vitamin C (VC) intake on acutely intralesional liver in carbon tetrachloride (CCl4)-exposed mice. Here our data showed that VC supplementation contributed to ameliorated vital signs of CCl4-lesioned mice, resulting in dose-dependent reduction of hepatomegaly. VC lowered the levels of liver functional enzymes including alanine aminotransferase (ALT) and glutamic-oxaloacetic transaminase (AST) in serum, while concentration of lactic acid concentration in blood plasma was decreased. VC-administered CCl4-lesioned mice manifested increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), while the malondialdehyde (MDA) content was reduced in liver tissue. Moreover, VC consumption attenuated hepatotoxic injuries of CCl 4-lesioned mice, in which the number of TNF-α positive cells was dose-dependently reduced. Furthermore, intrahepatic expression of TRL-4 mRNA, a vital inflammation-regulator, was down-regulated in VC-administered mice. Overall, we conclude that VC has the potentiality of antihepatotoxicity that is capable of ameliorating liver functions, speculating that therapeutic mechanism relates to normalizing metabolism and blocking inflammatory stress in the liver. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Liver disease harbors structural lesions or long-lasting wounds in hepatocytes, due to which the patient with hepatopathy has complications associated with poorly prognostic outcomes [1]. If untreated, the diseased state may gradually develop to advanced events, such as fatty liver, fibrosis, or preneoplasia [2]. Hepatic dysfunction can upset physical signs and metabolic profiles, occurring in digestive disturbances, expellent problems, or immune disorders [3]. There are many categories of liver diseases, which are mainly classified as hepatitis, acute liver injury, hepatic steatosis, and liver cancer [4]. In the clinic, effective methods to manage liver disease include using acceptable medicine plus making lifestyle changes [5]. Unfortunately, widespread use of chemotherapeutics results in adverse impacts, including primary liver impairment, osteoporosis, or allergic response [6]. Therefore, the potential candidate for liver-lesioned management merits further investigation. Vitamin C (VC), known as L-ascorbic acid, is a naturally existent organic substance marked by antioxidant property, and is
also a vital nutrient for humans [7]. Vitamin C is an important cofactor for biosynthesis of intracellular biochemicals as an electron donor, including collagen synthesis reactions. Once avitaminosis occurs, it can suffer from severe scurvy symptoms [8]. In spite of vitamin C's being promoted as a prescription in prophylaxis and therapy for various syndromes, the majority of clinical applications are poorly confirmed by proof with certain contraindications [9]. The reports that VC supplementation averts liver trauma/injury, however, are still not yet understood. It is well established that carbon tetrachloride (CCl4) is scientifically employed as a powerful hepatotoxin to liver cells, whereas it is alternatively used in medical research to assess hepatoprotective agents [10]. This study was conducted to explore the beneficial effects of VC in CCl4-induced liver-lesioned mice, the underlying mechanism would be discussed as well.
2. Materials and methods 2.1. Materials
⁎ Corresponding author at: College of Pharmacy, Guilin Medical University, Huancheng North 2nd 109, Guilin 541004, PR China. Tel.: +86 773 5895812; fax: +86 773 5895810. E-mail address: [email protected] (W. Wu). 1 They contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.07.034 1567-5769/© 2014 Elsevier B.V. All rights reserved.
VC (purity N 95%) was obtained from Succhi Shiqi Pharmaceutical Co. Ltd. (Guangdong, China). Analytically pure grade of carbon tetrachloride (CCl4) was provided by Sigma-Aldrich Company (St. Louis, USA), which
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
was mixed with peanut oil (1:1, v/v) for use. Other materials were labeled hierarchically as follows. 2.2. Animal and drug administration Male Kunming mice (5–6 weeks, 18 ± 2 g) were purchased from the Experimental Animal Centre of Guangxi Medical University (Nanning, China). All mice were housed in a controlled room with temperature of 25 ± 2 °C, relative humidity of 60 ± 10%, room air changes 12–18 times/h, and a 12 h light/dark cycle. All mice had free access to water and standard chow pellets. To induce liver lesions, the mice were fasted for 6 h and intragastrically given 50% peanut oil-dissolved CCl 4 , as described previously [11]. Afterwards, the liver-lesioned mice were randomly divided into three groups and each group contained ten animals. For the VC intake groups (100 mg/kg, 200 mg/kg), the mice were given once daily for 1 month. For the CCl4 control group, the mice were fed with the same amount of CCl 4 mixture (0.5 ml/kg body weight) by oral gavage. Accordingly, healthy mice in the vehicle control group received an equivalent volume of normal saline. This study was approved by the Institutional Animal Care and Use Committee at the Guilin Medical University.
493
2.8. Pathological screening 5 μm-prepared slices from liver specimens in each group were subjected to routine staining of hematoxylin and eosin (H&E). Histopathological changes were screened and the images were captured (CX41, Olympus, Tokyo, Japan). Meanwhile, the necrotic cells in liver tissue were counted, through calculating a mean from five different optical fields. 2.9. Immunohistochemical assay Deparaffinized liver sections were processed through stepwise concentrations of 80%, 90%, 100% dimethylbenzene and ethanol for 5 min, and then rinsed 3 times with PBS. The sections were added with 3% H2O2 to inactivate endogenous enzymes for 6 min at 37 °C prior to hot-fixed antigen procedure. 5% BSA blocking solution was added, followed by reacting with diluted primary antibody and specific second antibody (TNF-α, 1:500, Boster Bio-engineering Limited Company, Wuhan, China) and coincubating with fresh SABC solution. The subsequent steps were followed by DAB dyeing and hematoxylin counterstaining before dehydrating, transparentizing, mounting procedures, and scanning images by a Leica microsystem.
2.3. Tissue sampling 2.10. Real time-PCR assay for TRL-4 mRNA After the end of the 1 month trial, all mice were subjected to anesthetization with 5% pentobarbital sodium (v/w). Next, serum specimens were collected into heparinized tubes (30 U/mL). Visceral organs were removed and weighted. Liver specimens were surgically dissected and washed with precooled saline to eliminate residual blood. A part of the liver samples were stored at − 80 °C until further analyses. Others were fixed in 4% paraformaldehyde solution, dehydrated and embedded in paraffin for pathological detection. 2.4. Measurement of visceral parameter Liver index ¼ ðliver weight=body weightÞ 100%: 2.5. Measurement of liver functional enzymes Enzymatic levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were calculated using commercial test kits (Nanjing Jiancheng Bioengineering Institute, China). The final data were expressed as U/L. 2.6. Measurement of lactic acid After whole-blood being collected from each mouse, the mixtures were subjected to enzymatic reactions, based on a chemical rationale of NAD+ as a hydrogen acceptor for promoting LDH catalytic dehydrogenation to produce pyruvic acid and then transforming into NADH. The final data represented by a linear relationship between absorbance (530 nm) and lactic acid content (mmol/L). Here, the commercial test kit was provided by Nanjing Jiancheng Bioengineering Institute.
Target RNA was isolated from liver tissue from each group via the Trizol reagent (Life Technologies Corporation, USA). The purity of RNA was confirmed using a spectrophotometer at 260 nm. RNA-to-cDNA transcription was conducted through PCR profiling kit (Life Technologies Corporation, USA) following the manufacturer's procedures. The target primer is shown below: TRL-4 forward primer, 5′ CAT GGA TCA GAA ACT CAG CAA AGT C 3′, antisense primer: 5′ CAT GCC ATG CCT TGT CTT CA 3′(179 bp); β-actin sense primer: 5′ TGT GTC CGT CGT GGA TCT GA 3′, antisense primer: 5′ TTG CTG TTG AAG TCG CAG GAG 3′ (149 bp). Accordingly, PCR circulating process included 30 cycles, marked for denaturation at 92 °C for 30 s, annealing 45 °C for 30 s, and elongation at 65 °C for 2 min. The PCR amplification product was processed in fresh buffer, and loaded in sepharose gels, then electrophoresis was carried out for 30 min. In addition, optical density of gel band from each group was determined via normalizing to β-actin. The final data were expressed as a relative ratio. 2.11. Statistical analysis All data were generated through SPSS 13.0 software (SPSS Inc., USA). Differences between these groups were determined using a one-way analysis of variance (ANOVA) with the Bonferronni post tests for multiple comparisons. Results were expressed as mean ± SE. The level of significance was set at P b 0.05. 3. Results 3.1. The impacts of body weight and liver index in liver-lesioned mice mediated by VC supplementation
2.7. Determination of intrahepatic SOD, GSH-PX, MDA concentrations Fresh mouse liver samples from each group were harvested, and homogenates were prepared in liquid nitrogen. The homogenates were cryogenically centrifuged at 12,000 ×g for 10 min, and then we collected supernatant for SOD, GSH-PX bioactivities and MDA content determinations according to the manufacturer's manuals (Nanjing Jiancheng Bioengineering Institute, China). The final results were expressed as U/mg protein or nmol/mg protein, respectively.
As shown in weight changes of body, CCl 4 -lesioned mice had pronounced body weight loss compared to healthy mice (P b 0.01). After CCl4 exposure plus VC intake for 1 month, the results showed increased body weight at a dose-dependent manner (P b 0.01). Similarly, these CCl4-exposed mice exhibited abnormal enlargement of the liver that was larger than that of normal liver (P b 0.01). Interestingly, one-month supplementation of VC effectively reduced hepatomegaly and liver index in CCl4-lesioned mice (P b 0.01) (Fig. 1).
494
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
Fig. 1. (A) VC supplementation elevated the body weight in liver-lesioned mice. (B) VC supplementation reversed hepatomegaly in liver-lesioned mice. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results were expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
3.2. The impacts of serological parameters in liver-lesioned mice mediated by VC supplementation
signs characterized by elevated SOD, GSH-PX levels, and lowered MDA concentration in liver tissue (P b 0.01).
One month after toxicity to liver tissue, CCl4-exposed mice showed elevated serological levels of ALT and AST, as well as increased lactic acid content (P b 0.01). Compared to the CCl4 controls, the abnormal changes of liver functional enzymes were reversed following the co-administration of VC in CCl4-exposed mice, matched for reductions of ALT, AST and lactic acid concentrations (P b 0.01) (Fig. 2).
3.4. Pathological diagnosis
3.3. The impacts of intrahepatic antioxidant parameters in liver-lesioned mice mediated by VC supplementation As reflected in Fig. 3, the lesions of liver tissue in CCl4-exposed mice showed significant decrease in SOD, GSH-PX activities, and elevation of MDA content (P b 0.01). Instead, VC intake mice showed beneficial
To pathologically identify the hepatocellular changes of mice from each group, routine staining (H&E) was carried out. Morphological characteristics of liver cells in healthy mice showed evenly distributed hepatocytes around central veins, integrated hepatic lobule and portal area, marked by fewer necrotic cells or less inflammatory infiltration. Inversely, CCl4-lesioned mice contained impaired liver lobule, hydropic degeneration in hepatocytes along with increased necrotic cell counts, as well as widespread inflammatory infiltration. After one-month supplementation of VC, the hepatotoxic signs in the liver induced by CCl4 exposure had significant improvements, characterized by lowered necrotic cell numbers and cytokine infiltration, and restored cytoskeleton in liver cell/tissue (Fig. 4).
Fig. 2. (A) VC supplementation blocked increased serum level of transaminases (ALT, AST) in liver-lesioned mice. (B) VC supplementation reduced lactic acid concentration in CCl4-exposed mice. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results were expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
495
3.6. The impact of TRL-4 mRNA in the liver of liver-lesioned mice mediated by VC supplementation Further, the inflammation-associated TRL-4 component was investigated in the liver. As revealed in Fig. 6, the data from RT-PCR assay showed that the CCl4-lesioned mice carried upregulated TRL-4 mRNA in the liver when compared to the healthy mice in the vehicle control (P b 0.01). Following the supplementation of VC, elevated TRL-4 mRNA in the liver was blocked, marked by down-regulated expression of TLR-4 mRNA (P b 0.01). 4. Discussion
Fig. 3. (A) VC supplementation elevated intrahepatic activities of SOD, GSH-P X in liver-lesioned mice, while the MDA content was lowered. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
3.5. The impact of TNF-α positive cell in the liver of liver-lesioned mice mediated by VC supplementation In order to explore the anti-inflammatory capability of VC on CCl 4 -lesioned liver, immunohistochemical method was applied in assessing the expression of TNF-α positive cell. Compared to vehicle control, liver-lesioned mice showed excessive expression of TNF-α positive cells in liver tissue (P b 0.01). Attractively, the intrahepatic counts of TNF-α positive cell were effectively attenuated after VC intake in CCl4-lesioned mice (P b 0.01) (Fig. 5).
Together, our present data highlighted that VC supplementation may offer an attractive strategy for liver-lesion control, raising interest for its potential clinical application. Thereby, underlying benefits of VC-mediated hepatoprotection should be discussed, respectively. Generally, liver impairments can undermine the alimental utilization or metabolism, in which hepatocellular dysfunction correlates with immunodeficiency and digestive disturbance [12]. Thus, CCl4-lesioned mice showed significant body weight loss and the marasmic sign with hepatomegaly, indicating that malnourished outcome resulted from liver injury induced by CCl 4-specific toxicity. Interestingly, VC replenishment contributed to averting CCl4-lesioned signs in mice, suggesting that the underlying merits of VC were related to reinforcement of nourishment and metabolic normalization. The changes in hepatic transaminases, including AST and ALT, can be involved in the extent of known liver lesions, following the response to therapy [13]. During normal metabolism, lactic acid is fundamentally produced in the body, where its circulating concentration is elevated after tissue trauma [14]. In this liver-lesioned model, CCl4-exposed mice showed increased transaminase levels and lactic acid content, suggesting that liver injury induced by CCl4 presented in dysmetabolism of lactic acid, thereby inducing lactic acidosis. Instead, this dysregulation in CCl4-lesioned mice was corrected by VC administration, indicating that VC intake showed promise in serological homeostasis. The antioxidant system in the body contains important antioxidant defense enzymes, such as SOD or GSH-PX, which can prevent free radical damage to the cells [15]. In addition, MDA toxicity is a catalyzed product resulting from lipid peroxidation at increased oxidative stress [16]. These results showed that the CCl4 exposure in mice had notably reduced levels of SOD, GSH-PX in liver cell, as well as elevated MDA
Fig. 4. Pathological observations (H&E dyeing, scale bar: 200 μm). Pentagram represents the central vein. Arrows point to necrotic liver cells. Triangle denotes inflammatory emergence. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
496
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
Fig. 5. VC supplementation attenuated the counts of TNF-α positive cells in the liver of liver-lesioned mice (immunohistochemistry, scale bar: 200 μm). Pentagram represents the central vein. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
content, implying that antioxidant defense ability and detoxification were disturbed by CCl4. Interestingly, VC supplement showed enhanced the antioxidant capability and attenuated lipotoxicity in CCl4-lesioned liver tissue, reflecting that the VC-specific antioxidant characteristic was implicated in hepatoprotection. Escalating destruction of an organ in the presence of inflammation will jeopardize the function of tissue [17]. Many reports show that tumor necrosis factor (TNF-α), known as an endogenous pyrogen, is
closely involved in systemic inflammation [18]. Dysregulation of endogenous TNF production can result in a series of human diseases, such as neurodegenerative disorder, tumorigenesis, or inflammatory bowel ailment [19]. TLR-4 is a toll-like receptor that activates innate immune response. The signal pathway of toll-like receptors has been shown to interact with inflammatory progression [20]. Therefore, if hepatocellular TNF-α/TLR-4 pathway is inhibited, the benefits will show amelioration of antagonizing liver lesion. Our current data suggested that CCl4-poisoned mice contained outnumbered TNF-α positive cells and over-expression of TLR-4 mRNA in the liver, in which the abnormal phenomena were consistent with the liverlesioned architecture as reflected in the pathological examination. Following one-month of VC supplementation, the unregulated production of TNF-α protein and TLR-4 mRNA was blocked. Taken together, we reasoned that the potential benefits of VC against intrahepatic TNF-α-associated inflammatory lesions in the exposure to CCl4 were related to inactivation of TLR-4 expression in hepatocytes. Thus, VC was superior to inactivating a target of toll-like receptor pathway in liver tissue, characterized by the result of preventing cytoarchitectural lesions and inflammatory stress. Acknowledgments This study was partially supported by the National Natural Science Foundation of China (No. 81260343) and the Science and Technology Research Projects of Guangxi Colleges and Universities (No. YB2014275). References
Fig. 6. VC supplementation down-regulated the mRNA production TRL-4 mRNA in the liver of liver-lesioned mice (PT-PCR assay). Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
[1] Udell JA, Wang CS, Tinmouth J, FitzGerald JM, Ayas NT, Simel DL, et al. Does this patient with liver disease have cirrhosis? JAMA 2012;307(8):832–42. [2] Ahn H, Li CS, Wang W. Sickle cell hepatopathy: clinical presentation, treatment, and outcome in pediatric and adult patients. Pediatr Blood Cancer 2005;45(2):184–90. [3] Verbeeck RK. Pharmacokinetics and dosage adjustment in patients with hepatic dysfunction. Eur J Clin Pharmacol 2008;64(12):1147–61. [4] Cauchy F, Fuks D, Zarzavadjian Le Bian A, Belghiti J, Costi R. Metabolic syndrome and non-alcoholic fatty liver disease in liver surgery: the new scourges? World J Hepatol 2014;6(5):306–14. [5] Su TH, Kao JH, Liu CJ. Molecular mechanism and treatment of viral hepatitis-related liver fibrosis. Int J Mol Sci 2014;15(6):10578–604. [6] Alomar MJ. Factors affecting the development of adverse drug reactions (review article). Saudi Pharm J 2014;22(2):83–94. [7] Lucock M, Yates Z, Boyd L, Naylor C, Choi JH, Ng X, et al. Vitamin C-related nutrientnutrient and nutrient-gene interactions that modify folate status. Eur J Nutr 2013; 52(2):569–82.
M. Su et al. / International Immunopharmacology 22 (2014) 492–497 [8] Ströhle A, Hahn A. Vitamin C and immune function. Med Monatsschr Pharm 2009; 32(2):49–54. [9] Paulsen G, Cumming KT, Holden G, Hallén J, Rønnestad BR, Sveen O, et al. Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial. J Physiol 2014;592(8): 1887–901. [10] Guo C, Xu L, He Q, Liang T, Duan X, Li R. Anti-fibrotic effects of puerarin on CCl4induced hepatic fibrosis in rats possibly through the regulation of PPAR-γ expression and inhibition of PI3K/Akt pathway. Food Chem Toxicol 2013;56:436–42. [11] Li R, Xu L, Liang T, Li Y, Zhang S, Duan X. Puerarin mediates hepatoprotection against CCl4-induced hepatic fibrosis rats through attenuation of inflammation response and amelioration of metabolic function. Food Chem Toxicol 2013;52:69–75. [12] Ramadori G1, Moriconi F, Malik I, Dudas J. Physiology and pathophysiology of liver inflammation, damage and repair. J Physiol Pharmacol 2008;59(1):107–17. [13] Segal I, Rassekh SR, Bond MC, Senger C, Schreiber RA. Abnormal liver transaminases and conjugated hyperbilirubinemia at presentation of acute lymphoblastic leukemia. Pediatr Blood Cancer 2010;55(3):434–9. [14] Zhang G, Sun Y, Wang Y, Li X, Li T, Su S, et al. Local administration of lactic acid and a low dose of the free radical scavenger, edaravone, alleviates myocardial reperfusion injury in rats. J Cardiovasc Pharmacol 2013;62(4):369–78.
497
[15] Li R, Zheng N, Liang T, He Q, Xu L. Puerarin attenuates neuronal degeneration and blocks oxidative stress to elicit a neuroprotective effect on substantia nigra injury in 6-OHDA-lesioned rats. Brain Res 2013;1517:28–35. [16] Bird RP, Draper HH, Valli VE. Toxicological evaluation of malonaldehyde: a 12-month study of mice. J Toxicol Environ Health 1982;10(6):897–905. [17] Roth TL, Nayak D, Atanasijevic T, Koretsky AP, Latour LL, McGavern DB. Transcranial amelioration of inflammation and cell death after brain injury. Nature 2014; 505(7482):223–8. [18] Guo C, Li R, Zheng N, Xu L, Liang T, He Q. Anti-diabetic effect of Ramulus mori polysaccharides, isolated from Morus alba L., on STZ-diabetic mice through blocking inflammatory response and attenuating oxidative stress. Int Immunopharmacol 2013;16(1):93–9. [19] Guo C, Liang T, He Q, Wei P, Zheng N, Xu L. Renoprotective effect of Ramulus mori polysaccharides on renal injury in STZ-diabetic mice. Int J Biol Macromol 2013;62: 720–5. [20] Kigerl KA, Lai W, Rivest S, Hart RP, Satoskar AR, Popovich PG. Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury. J Neurochem 2007;102(1):37–50.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Potential protection of vitamin C against liver-lesioned mice Min Su a,1, Hongqiu Chen b,1, Chaohe Wei c, Ning Chen d,e, Wei Wu e,⁎ a
Faculty of Basic Medicine, Guilin Medical University, Guilin 541004, PR China Department of Pathology, Guigang City People's Hospital, Guigang, Guangxi 537100, PR China c Central Pharmacy, Guigang City People's Hospital, Guigang, Guangxi 537100, PR China d Guangxi Medical University, Nanning 530021, PR China e Guilin Medical University, Guilin 541004, PR China b
a r t i c l e
i n f o
Article history: Received 30 June 2014 Received in revised form 22 July 2014 Accepted 29 July 2014 Available online 10 August 2014 Keywords: Vitamin C Hepatotoxicity Metabolism Inflammation
a b s t r a c t Pathologically, liver injury can result from sustained trauma to hepatocytes, including acute damage. Thus, attenuation of hepatocellular lesion may help improve liver functions. The purpose of this study was to explore the potential advantages of vitamin C (VC) intake on acutely intralesional liver in carbon tetrachloride (CCl4)-exposed mice. Here our data showed that VC supplementation contributed to ameliorated vital signs of CCl4-lesioned mice, resulting in dose-dependent reduction of hepatomegaly. VC lowered the levels of liver functional enzymes including alanine aminotransferase (ALT) and glutamic-oxaloacetic transaminase (AST) in serum, while concentration of lactic acid concentration in blood plasma was decreased. VC-administered CCl4-lesioned mice manifested increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), while the malondialdehyde (MDA) content was reduced in liver tissue. Moreover, VC consumption attenuated hepatotoxic injuries of CCl 4-lesioned mice, in which the number of TNF-α positive cells was dose-dependently reduced. Furthermore, intrahepatic expression of TRL-4 mRNA, a vital inflammation-regulator, was down-regulated in VC-administered mice. Overall, we conclude that VC has the potentiality of antihepatotoxicity that is capable of ameliorating liver functions, speculating that therapeutic mechanism relates to normalizing metabolism and blocking inflammatory stress in the liver. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Liver disease harbors structural lesions or long-lasting wounds in hepatocytes, due to which the patient with hepatopathy has complications associated with poorly prognostic outcomes [1]. If untreated, the diseased state may gradually develop to advanced events, such as fatty liver, fibrosis, or preneoplasia [2]. Hepatic dysfunction can upset physical signs and metabolic profiles, occurring in digestive disturbances, expellent problems, or immune disorders [3]. There are many categories of liver diseases, which are mainly classified as hepatitis, acute liver injury, hepatic steatosis, and liver cancer [4]. In the clinic, effective methods to manage liver disease include using acceptable medicine plus making lifestyle changes [5]. Unfortunately, widespread use of chemotherapeutics results in adverse impacts, including primary liver impairment, osteoporosis, or allergic response [6]. Therefore, the potential candidate for liver-lesioned management merits further investigation. Vitamin C (VC), known as L-ascorbic acid, is a naturally existent organic substance marked by antioxidant property, and is
also a vital nutrient for humans [7]. Vitamin C is an important cofactor for biosynthesis of intracellular biochemicals as an electron donor, including collagen synthesis reactions. Once avitaminosis occurs, it can suffer from severe scurvy symptoms [8]. In spite of vitamin C's being promoted as a prescription in prophylaxis and therapy for various syndromes, the majority of clinical applications are poorly confirmed by proof with certain contraindications [9]. The reports that VC supplementation averts liver trauma/injury, however, are still not yet understood. It is well established that carbon tetrachloride (CCl4) is scientifically employed as a powerful hepatotoxin to liver cells, whereas it is alternatively used in medical research to assess hepatoprotective agents [10]. This study was conducted to explore the beneficial effects of VC in CCl4-induced liver-lesioned mice, the underlying mechanism would be discussed as well.
2. Materials and methods 2.1. Materials
⁎ Corresponding author at: College of Pharmacy, Guilin Medical University, Huancheng North 2nd 109, Guilin 541004, PR China. Tel.: +86 773 5895812; fax: +86 773 5895810. E-mail address: [email protected] (W. Wu). 1 They contributed equally to this work.
http://dx.doi.org/10.1016/j.intimp.2014.07.034 1567-5769/© 2014 Elsevier B.V. All rights reserved.
VC (purity N 95%) was obtained from Succhi Shiqi Pharmaceutical Co. Ltd. (Guangdong, China). Analytically pure grade of carbon tetrachloride (CCl4) was provided by Sigma-Aldrich Company (St. Louis, USA), which
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
was mixed with peanut oil (1:1, v/v) for use. Other materials were labeled hierarchically as follows. 2.2. Animal and drug administration Male Kunming mice (5–6 weeks, 18 ± 2 g) were purchased from the Experimental Animal Centre of Guangxi Medical University (Nanning, China). All mice were housed in a controlled room with temperature of 25 ± 2 °C, relative humidity of 60 ± 10%, room air changes 12–18 times/h, and a 12 h light/dark cycle. All mice had free access to water and standard chow pellets. To induce liver lesions, the mice were fasted for 6 h and intragastrically given 50% peanut oil-dissolved CCl 4 , as described previously [11]. Afterwards, the liver-lesioned mice were randomly divided into three groups and each group contained ten animals. For the VC intake groups (100 mg/kg, 200 mg/kg), the mice were given once daily for 1 month. For the CCl4 control group, the mice were fed with the same amount of CCl 4 mixture (0.5 ml/kg body weight) by oral gavage. Accordingly, healthy mice in the vehicle control group received an equivalent volume of normal saline. This study was approved by the Institutional Animal Care and Use Committee at the Guilin Medical University.
493
2.8. Pathological screening 5 μm-prepared slices from liver specimens in each group were subjected to routine staining of hematoxylin and eosin (H&E). Histopathological changes were screened and the images were captured (CX41, Olympus, Tokyo, Japan). Meanwhile, the necrotic cells in liver tissue were counted, through calculating a mean from five different optical fields. 2.9. Immunohistochemical assay Deparaffinized liver sections were processed through stepwise concentrations of 80%, 90%, 100% dimethylbenzene and ethanol for 5 min, and then rinsed 3 times with PBS. The sections were added with 3% H2O2 to inactivate endogenous enzymes for 6 min at 37 °C prior to hot-fixed antigen procedure. 5% BSA blocking solution was added, followed by reacting with diluted primary antibody and specific second antibody (TNF-α, 1:500, Boster Bio-engineering Limited Company, Wuhan, China) and coincubating with fresh SABC solution. The subsequent steps were followed by DAB dyeing and hematoxylin counterstaining before dehydrating, transparentizing, mounting procedures, and scanning images by a Leica microsystem.
2.3. Tissue sampling 2.10. Real time-PCR assay for TRL-4 mRNA After the end of the 1 month trial, all mice were subjected to anesthetization with 5% pentobarbital sodium (v/w). Next, serum specimens were collected into heparinized tubes (30 U/mL). Visceral organs were removed and weighted. Liver specimens were surgically dissected and washed with precooled saline to eliminate residual blood. A part of the liver samples were stored at − 80 °C until further analyses. Others were fixed in 4% paraformaldehyde solution, dehydrated and embedded in paraffin for pathological detection. 2.4. Measurement of visceral parameter Liver index ¼ ðliver weight=body weightÞ 100%: 2.5. Measurement of liver functional enzymes Enzymatic levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were calculated using commercial test kits (Nanjing Jiancheng Bioengineering Institute, China). The final data were expressed as U/L. 2.6. Measurement of lactic acid After whole-blood being collected from each mouse, the mixtures were subjected to enzymatic reactions, based on a chemical rationale of NAD+ as a hydrogen acceptor for promoting LDH catalytic dehydrogenation to produce pyruvic acid and then transforming into NADH. The final data represented by a linear relationship between absorbance (530 nm) and lactic acid content (mmol/L). Here, the commercial test kit was provided by Nanjing Jiancheng Bioengineering Institute.
Target RNA was isolated from liver tissue from each group via the Trizol reagent (Life Technologies Corporation, USA). The purity of RNA was confirmed using a spectrophotometer at 260 nm. RNA-to-cDNA transcription was conducted through PCR profiling kit (Life Technologies Corporation, USA) following the manufacturer's procedures. The target primer is shown below: TRL-4 forward primer, 5′ CAT GGA TCA GAA ACT CAG CAA AGT C 3′, antisense primer: 5′ CAT GCC ATG CCT TGT CTT CA 3′(179 bp); β-actin sense primer: 5′ TGT GTC CGT CGT GGA TCT GA 3′, antisense primer: 5′ TTG CTG TTG AAG TCG CAG GAG 3′ (149 bp). Accordingly, PCR circulating process included 30 cycles, marked for denaturation at 92 °C for 30 s, annealing 45 °C for 30 s, and elongation at 65 °C for 2 min. The PCR amplification product was processed in fresh buffer, and loaded in sepharose gels, then electrophoresis was carried out for 30 min. In addition, optical density of gel band from each group was determined via normalizing to β-actin. The final data were expressed as a relative ratio. 2.11. Statistical analysis All data were generated through SPSS 13.0 software (SPSS Inc., USA). Differences between these groups were determined using a one-way analysis of variance (ANOVA) with the Bonferronni post tests for multiple comparisons. Results were expressed as mean ± SE. The level of significance was set at P b 0.05. 3. Results 3.1. The impacts of body weight and liver index in liver-lesioned mice mediated by VC supplementation
2.7. Determination of intrahepatic SOD, GSH-PX, MDA concentrations Fresh mouse liver samples from each group were harvested, and homogenates were prepared in liquid nitrogen. The homogenates were cryogenically centrifuged at 12,000 ×g for 10 min, and then we collected supernatant for SOD, GSH-PX bioactivities and MDA content determinations according to the manufacturer's manuals (Nanjing Jiancheng Bioengineering Institute, China). The final results were expressed as U/mg protein or nmol/mg protein, respectively.
As shown in weight changes of body, CCl 4 -lesioned mice had pronounced body weight loss compared to healthy mice (P b 0.01). After CCl4 exposure plus VC intake for 1 month, the results showed increased body weight at a dose-dependent manner (P b 0.01). Similarly, these CCl4-exposed mice exhibited abnormal enlargement of the liver that was larger than that of normal liver (P b 0.01). Interestingly, one-month supplementation of VC effectively reduced hepatomegaly and liver index in CCl4-lesioned mice (P b 0.01) (Fig. 1).
494
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
Fig. 1. (A) VC supplementation elevated the body weight in liver-lesioned mice. (B) VC supplementation reversed hepatomegaly in liver-lesioned mice. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results were expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
3.2. The impacts of serological parameters in liver-lesioned mice mediated by VC supplementation
signs characterized by elevated SOD, GSH-PX levels, and lowered MDA concentration in liver tissue (P b 0.01).
One month after toxicity to liver tissue, CCl4-exposed mice showed elevated serological levels of ALT and AST, as well as increased lactic acid content (P b 0.01). Compared to the CCl4 controls, the abnormal changes of liver functional enzymes were reversed following the co-administration of VC in CCl4-exposed mice, matched for reductions of ALT, AST and lactic acid concentrations (P b 0.01) (Fig. 2).
3.4. Pathological diagnosis
3.3. The impacts of intrahepatic antioxidant parameters in liver-lesioned mice mediated by VC supplementation As reflected in Fig. 3, the lesions of liver tissue in CCl4-exposed mice showed significant decrease in SOD, GSH-PX activities, and elevation of MDA content (P b 0.01). Instead, VC intake mice showed beneficial
To pathologically identify the hepatocellular changes of mice from each group, routine staining (H&E) was carried out. Morphological characteristics of liver cells in healthy mice showed evenly distributed hepatocytes around central veins, integrated hepatic lobule and portal area, marked by fewer necrotic cells or less inflammatory infiltration. Inversely, CCl4-lesioned mice contained impaired liver lobule, hydropic degeneration in hepatocytes along with increased necrotic cell counts, as well as widespread inflammatory infiltration. After one-month supplementation of VC, the hepatotoxic signs in the liver induced by CCl4 exposure had significant improvements, characterized by lowered necrotic cell numbers and cytokine infiltration, and restored cytoskeleton in liver cell/tissue (Fig. 4).
Fig. 2. (A) VC supplementation blocked increased serum level of transaminases (ALT, AST) in liver-lesioned mice. (B) VC supplementation reduced lactic acid concentration in CCl4-exposed mice. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results were expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
495
3.6. The impact of TRL-4 mRNA in the liver of liver-lesioned mice mediated by VC supplementation Further, the inflammation-associated TRL-4 component was investigated in the liver. As revealed in Fig. 6, the data from RT-PCR assay showed that the CCl4-lesioned mice carried upregulated TRL-4 mRNA in the liver when compared to the healthy mice in the vehicle control (P b 0.01). Following the supplementation of VC, elevated TRL-4 mRNA in the liver was blocked, marked by down-regulated expression of TLR-4 mRNA (P b 0.01). 4. Discussion
Fig. 3. (A) VC supplementation elevated intrahepatic activities of SOD, GSH-P X in liver-lesioned mice, while the MDA content was lowered. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
3.5. The impact of TNF-α positive cell in the liver of liver-lesioned mice mediated by VC supplementation In order to explore the anti-inflammatory capability of VC on CCl 4 -lesioned liver, immunohistochemical method was applied in assessing the expression of TNF-α positive cell. Compared to vehicle control, liver-lesioned mice showed excessive expression of TNF-α positive cells in liver tissue (P b 0.01). Attractively, the intrahepatic counts of TNF-α positive cell were effectively attenuated after VC intake in CCl4-lesioned mice (P b 0.01) (Fig. 5).
Together, our present data highlighted that VC supplementation may offer an attractive strategy for liver-lesion control, raising interest for its potential clinical application. Thereby, underlying benefits of VC-mediated hepatoprotection should be discussed, respectively. Generally, liver impairments can undermine the alimental utilization or metabolism, in which hepatocellular dysfunction correlates with immunodeficiency and digestive disturbance [12]. Thus, CCl4-lesioned mice showed significant body weight loss and the marasmic sign with hepatomegaly, indicating that malnourished outcome resulted from liver injury induced by CCl 4-specific toxicity. Interestingly, VC replenishment contributed to averting CCl4-lesioned signs in mice, suggesting that the underlying merits of VC were related to reinforcement of nourishment and metabolic normalization. The changes in hepatic transaminases, including AST and ALT, can be involved in the extent of known liver lesions, following the response to therapy [13]. During normal metabolism, lactic acid is fundamentally produced in the body, where its circulating concentration is elevated after tissue trauma [14]. In this liver-lesioned model, CCl4-exposed mice showed increased transaminase levels and lactic acid content, suggesting that liver injury induced by CCl4 presented in dysmetabolism of lactic acid, thereby inducing lactic acidosis. Instead, this dysregulation in CCl4-lesioned mice was corrected by VC administration, indicating that VC intake showed promise in serological homeostasis. The antioxidant system in the body contains important antioxidant defense enzymes, such as SOD or GSH-PX, which can prevent free radical damage to the cells [15]. In addition, MDA toxicity is a catalyzed product resulting from lipid peroxidation at increased oxidative stress [16]. These results showed that the CCl4 exposure in mice had notably reduced levels of SOD, GSH-PX in liver cell, as well as elevated MDA
Fig. 4. Pathological observations (H&E dyeing, scale bar: 200 μm). Pentagram represents the central vein. Arrows point to necrotic liver cells. Triangle denotes inflammatory emergence. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
496
M. Su et al. / International Immunopharmacology 22 (2014) 492–497
Fig. 5. VC supplementation attenuated the counts of TNF-α positive cells in the liver of liver-lesioned mice (immunohistochemistry, scale bar: 200 μm). Pentagram represents the central vein. Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
content, implying that antioxidant defense ability and detoxification were disturbed by CCl4. Interestingly, VC supplement showed enhanced the antioxidant capability and attenuated lipotoxicity in CCl4-lesioned liver tissue, reflecting that the VC-specific antioxidant characteristic was implicated in hepatoprotection. Escalating destruction of an organ in the presence of inflammation will jeopardize the function of tissue [17]. Many reports show that tumor necrosis factor (TNF-α), known as an endogenous pyrogen, is
closely involved in systemic inflammation [18]. Dysregulation of endogenous TNF production can result in a series of human diseases, such as neurodegenerative disorder, tumorigenesis, or inflammatory bowel ailment [19]. TLR-4 is a toll-like receptor that activates innate immune response. The signal pathway of toll-like receptors has been shown to interact with inflammatory progression [20]. Therefore, if hepatocellular TNF-α/TLR-4 pathway is inhibited, the benefits will show amelioration of antagonizing liver lesion. Our current data suggested that CCl4-poisoned mice contained outnumbered TNF-α positive cells and over-expression of TLR-4 mRNA in the liver, in which the abnormal phenomena were consistent with the liverlesioned architecture as reflected in the pathological examination. Following one-month of VC supplementation, the unregulated production of TNF-α protein and TLR-4 mRNA was blocked. Taken together, we reasoned that the potential benefits of VC against intrahepatic TNF-α-associated inflammatory lesions in the exposure to CCl4 were related to inactivation of TLR-4 expression in hepatocytes. Thus, VC was superior to inactivating a target of toll-like receptor pathway in liver tissue, characterized by the result of preventing cytoarchitectural lesions and inflammatory stress. Acknowledgments This study was partially supported by the National Natural Science Foundation of China (No. 81260343) and the Science and Technology Research Projects of Guangxi Colleges and Universities (No. YB2014275). References
Fig. 6. VC supplementation down-regulated the mRNA production TRL-4 mRNA in the liver of liver-lesioned mice (PT-PCR assay). Data were analyzed by one-way ANOVA followed by Bonferroni post tests, and results are expressed as the mean ± SE. Note: vs. vehicle control, aP b 0.01; vs. CCl4-lesioned control, bP b 0.01.
[1] Udell JA, Wang CS, Tinmouth J, FitzGerald JM, Ayas NT, Simel DL, et al. Does this patient with liver disease have cirrhosis? JAMA 2012;307(8):832–42. [2] Ahn H, Li CS, Wang W. Sickle cell hepatopathy: clinical presentation, treatment, and outcome in pediatric and adult patients. Pediatr Blood Cancer 2005;45(2):184–90. [3] Verbeeck RK. Pharmacokinetics and dosage adjustment in patients with hepatic dysfunction. Eur J Clin Pharmacol 2008;64(12):1147–61. [4] Cauchy F, Fuks D, Zarzavadjian Le Bian A, Belghiti J, Costi R. Metabolic syndrome and non-alcoholic fatty liver disease in liver surgery: the new scourges? World J Hepatol 2014;6(5):306–14. [5] Su TH, Kao JH, Liu CJ. Molecular mechanism and treatment of viral hepatitis-related liver fibrosis. Int J Mol Sci 2014;15(6):10578–604. [6] Alomar MJ. Factors affecting the development of adverse drug reactions (review article). Saudi Pharm J 2014;22(2):83–94. [7] Lucock M, Yates Z, Boyd L, Naylor C, Choi JH, Ng X, et al. Vitamin C-related nutrientnutrient and nutrient-gene interactions that modify folate status. Eur J Nutr 2013; 52(2):569–82.
M. Su et al. / International Immunopharmacology 22 (2014) 492–497 [8] Ströhle A, Hahn A. Vitamin C and immune function. Med Monatsschr Pharm 2009; 32(2):49–54. [9] Paulsen G, Cumming KT, Holden G, Hallén J, Rønnestad BR, Sveen O, et al. Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial. J Physiol 2014;592(8): 1887–901. [10] Guo C, Xu L, He Q, Liang T, Duan X, Li R. Anti-fibrotic effects of puerarin on CCl4induced hepatic fibrosis in rats possibly through the regulation of PPAR-γ expression and inhibition of PI3K/Akt pathway. Food Chem Toxicol 2013;56:436–42. [11] Li R, Xu L, Liang T, Li Y, Zhang S, Duan X. Puerarin mediates hepatoprotection against CCl4-induced hepatic fibrosis rats through attenuation of inflammation response and amelioration of metabolic function. Food Chem Toxicol 2013;52:69–75. [12] Ramadori G1, Moriconi F, Malik I, Dudas J. Physiology and pathophysiology of liver inflammation, damage and repair. J Physiol Pharmacol 2008;59(1):107–17. [13] Segal I, Rassekh SR, Bond MC, Senger C, Schreiber RA. Abnormal liver transaminases and conjugated hyperbilirubinemia at presentation of acute lymphoblastic leukemia. Pediatr Blood Cancer 2010;55(3):434–9. [14] Zhang G, Sun Y, Wang Y, Li X, Li T, Su S, et al. Local administration of lactic acid and a low dose of the free radical scavenger, edaravone, alleviates myocardial reperfusion injury in rats. J Cardiovasc Pharmacol 2013;62(4):369–78.
497
[15] Li R, Zheng N, Liang T, He Q, Xu L. Puerarin attenuates neuronal degeneration and blocks oxidative stress to elicit a neuroprotective effect on substantia nigra injury in 6-OHDA-lesioned rats. Brain Res 2013;1517:28–35. [16] Bird RP, Draper HH, Valli VE. Toxicological evaluation of malonaldehyde: a 12-month study of mice. J Toxicol Environ Health 1982;10(6):897–905. [17] Roth TL, Nayak D, Atanasijevic T, Koretsky AP, Latour LL, McGavern DB. Transcranial amelioration of inflammation and cell death after brain injury. Nature 2014; 505(7482):223–8. [18] Guo C, Li R, Zheng N, Xu L, Liang T, He Q. Anti-diabetic effect of Ramulus mori polysaccharides, isolated from Morus alba L., on STZ-diabetic mice through blocking inflammatory response and attenuating oxidative stress. Int Immunopharmacol 2013;16(1):93–9. [19] Guo C, Liang T, He Q, Wei P, Zheng N, Xu L. Renoprotective effect of Ramulus mori polysaccharides on renal injury in STZ-diabetic mice. Int J Biol Macromol 2013;62: 720–5. [20] Kigerl KA, Lai W, Rivest S, Hart RP, Satoskar AR, Popovich PG. Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury. J Neurochem 2007;102(1):37–50.
