- Email: [email protected]
d -Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses
Accepted Manuscript Protective effects of alpinetin on lipopolysaccharide/d-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses Tong-gang Liu, Kai-hui Sha, Li-guo Zhang, Xian-xian Liu, Fang Yang, Jin-ying Cheng PII:
S0882-4010(18)30972-0
DOI:
https://doi.org/10.1016/j.micpath.2018.11.007
Reference:
YMPAT 3244
To appear in:
Microbial Pathogenesis
Received Date: 28 May 2018 Revised Date:
6 October 2018
Accepted Date: 5 November 2018
Please cite this article as: Liu T-g, Sha K-h, Zhang L-g, Liu X-x, Yang F, Cheng J-y, Protective effects of alpinetin on lipopolysaccharide/d-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.11.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Protective effects of alpinetin on lipopolysaccharide/D-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses
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Tong-gang Liu1#*, Kai-hui Sha2#, Li-guo Zhang1, Xian-xian Liu1, Fang Yang1, Jin-ying Cheng1 1. Department of infectious diseases, Binzhou Medical University Hospital, Binzhou, Shandong 256603, China. 2. Binzhou Medical University School of Nursing, Binzhou, Shandong 256603, China. # Tong-gang Liu and Kai-hui Sha are co-first authors. *Correspondence to: Tong-gang Liu, Department of infectious diseases, Binzhou Medical University Hospital, Binzhou, Shandong 256603, China. E-mail:[email protected] Abstract
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Alpinetin, a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to have anti-inflammatory effects. The aim of this investigation was designed to reveal the protective effects of alpinetin on Lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced liver injury in mice. Alpinetin (12.5, 25, 50 mg/kg) were given 1 h before LPS and D-Gal treatment. 12 h after LPS and D-Gal treatment, the liver tissues and serum were collected. Our results showed that alpinetin treatment improved liver histology, indicating a marked decrease of inflammatory cell infiltration and restore hepatic lobular architecture. Alpinetin also inhibited liver myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Furthermore, LPS/D-Gal-induced tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In conclusion, these findings suggested that alpinetin inhibited liver injury through inhibiting NF-κB and activating the Nrf2 signaling pathway.
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Keywords: Alpinetin; D-galactosamine; liver injury; Nrf2
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1. Introduction The liver is a substantial organ that takes on the important physiological functions of the human body. Located on the right upper quadrant, the liver is bulky and vulnerable to physical or chemical damage, resulting in liver damage [1]. Liver damage can be caused by surgery, trauma, microbial infections, liver disease, toxic compounds, drug side effects, and so on [2, 3]. These agents stimulate the liver cells, liver tissue and even the entire liver and cause the morphology and pathological changes [4]. LPS is the major factor that leads to liver injury [5]. It can induce the release of hepatic and circulating inflammatory cytokines, which are associated with the development prognosis of liver injury [6, 7]. Suppression of these inflammatory mediators could attenuate the damage of liver tissues [8]. Therefore, the development of new treatment agents that have the ability to inhibit inflammatory cytokines production may be useful for the treatment of liver injury. Alpinetin is a flavonoid isolated Alpinia katsumadai Hayata which has been known to have anti-inflammatory and anti-oxidative properties. A previous study showed that alpinetin had protective effects against DSS-induced colitis in mice [9]. Alpinetin also had the ability to inhibit 1
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LPS-induced mastitis via suppressing inflammatory response [10]. Furthermore, it has been reported that alpinetin had protective effects against LPS-induced lung injury in mice [11]. In addition, alpinetin has been reported to inhibit LPS-induced inflammatory cytokine production in THP-1 cells and RAW264.7 cells [12]. However, whether alpinetin had protective effects against liver injury have not been reported. In the present study, we aimed to clarify the protective effects of alpinetin on liver injury. Our results showed that alpinetin inhibited LPS/D-Gal-induced liver injury through attenuating inflammatory and oxidative responses. 2. Materials and methods 2.1. Animals BALB/c mice, 20-25g, were obtained from the Center of Experimental Animals of Shandong University (Jinan, China). The mice were kept for one week to adapt themselves to the environment. They were housed in a temperature and humidity controlled room and given enough food and water. All the animal experiments were permitted by the Animal Welfare and Research Ethics Committee at Binzhou Medical University. 2.2. Materials LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alpinetin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ELISA kits were purchased from R&D (Minneapolis, MN, USA). MPO, MDA, ALT, and AST assay kits were provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). Phospho-NF-κB p65, NF-κB p65, phospho-IκBα, IκBα, Nrf2, HO-1, and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.3. Groups Sixty BALB/c mice were randomly divided into five groups and each group contained twelve mice: (A) the blank control group, in which the mice were received equal amounts of vehicle; (B) LPS/D-Gal group, in which the mice were injected intraperitoneally with LPS (50 mg/kg) and D-GalN (800 mg/kg); (C-E) LPS/D-Gal + alpinetin (12.5, 25, 50 mg/kg) groups, in which the mice were received alpinetin (12.5, 25, 50 mg/kg) intraperitoneally 1 h before LPS/D-gal treatment. 2.4. Serum ALT and AST assay 12 h after LPS and D-Gal treatment, blood samples were collected and centrifuged to collect serum. The levels of ALT and AST in serum were measured by the detection kits following the manufacturer's protocols. 2.5. MPO and MDA assay Liver tissues were collected, homogenized, and centrifuged to obtain the supernatant. The levels of MPO and MDA in the supernatants of liver tissues were measured by detection kits according to the manufacturer's instructions. 2.6. ELISA The liver tissues and serum were collected and the serum and hepatic TNF-α and IL-1β levels were determined by the ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. 2.7. Western blot analysis Liver tissues were homogenized and total proteins were extracted using the protein extraction kit (Thermo). Then, equal amount of proteins were subjected to 12% SDS-PAGE and transferred to 2
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nitrocellulose membranes. The membranes were blocked with 5% skimmed milk. After washing three times with PBST, the membranes were incubated with primary antibodies: Phospho-NF-κB p65, NF-κB p65, phospho-IκBα, IκBα, Nrf2, HO-1, and β-actin overnight at 4 °C. Then, the membranes were washed three times with TBST and incubated with secondary antibodies for 2 h at room temperature. Three times after washing, ECL Western blot detection was used to visualize bands. 2.8. Histopathologic analysis Liver tissues were collected and fixed in 4% paraformaldehyde. Afterwards, the tissues were embedded in paraffin and cut into 5µm thick sections on a rotary microtome. Then, the sections were stained with H&E staining. The extent of histological changes was scored and scores of 0-1 was none; the score of 2-3 was mild; the score of 4-6 was moderate; the score of 7-9 was marked. The following parameters were observed and quantified for liver histopathology score: (1) Lobular
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architecture: Intact-0, Intact with cell swelling-1, Mild disruption-2, Marked disruption-3. (2) Apoptosis: None-0, Occasional-1, Mild-2, Marked-3. (3) Congestion: None-0, Occasional-1, Mild-2, Marked-3.
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2.9. Statistical analysis The data of this study was expressed as the mean ± SEM and analyzed with one-way analysis of variance (ANOVA) followed by Duncan's test using SPSS software. P value < 0.05 was considered to be statistically significant. 3. Results 3.1. Effects of alpinetin on LPS/D-Gal-induced liver histopathologic changes H&E staining was used in this study to detect the protective effects of alpinetin on LPS/D-Gal-induced liver injury. As shown in Fig. 1, liver tissues of the control group showed normal structure of livers. Significant histopathologic changes were observed in the LPS/D-Gal group, including cell necrosis, loss of hepatic architecture and massive inflammatory cells infiltration. These liver histopathologic changes were restored by the treatment of alpinetin. 3.2. Effects of alpinetin on LPS/D-Gal-induced serum ALT and AST levels Serum ALT and AST are important biomarkers of liver injury. As shown in Fig. 2, increased serum ALT and AST were observed in mice of LPS/D-Gal group. And the levels of ALT and AST in LPS/D-Gal + alpinetin group were significantly decreased when compared with LPS/D-Gal group. 3.3. Effects of alpinetin on LPS/D-Gal-induced MPO activity and MDA content MDA is the marker of oxidative stress and MPO is the marker of neutrophils infiltration of tissues. As shown in Fig. 3, increased MDA content and MPO activity of liver tissues were observed in mice of LPS/D-Gal group. And the levels of MDA and MPO in mice of LPS/D-Gal + alpinetin groups were significantly decreased when compared with LPS/D-Gal group (Fig. 3). 3.4. Effects of alpinetin on LPS/D-Gal-induced serum and hepatic TNF-α and IL-1β production As shown in Fig. 4, increased TNF-α and IL-1β levels of serum and liver tissues were observed in mice of LPS/D-Gal group. And the levels of TNF-α and IL-1β in mice of LPS/D-Gal + alpinetin groups were significantly decreased when compared with LPS/D-Gal group (Fig. 4). 3.5. Effects of alpinetin on LPS/D-Gal-induced NF-κB activation As shown in Fig. 5, treatment of LPS/D-Gal obviously increased the levels of p65 and IκBα phosphorylation in the NF-κB signaling pathway. However, pretreatment with alpinetin inhibited the up-regulation of phosphorylation of p65 and IκBα in a dose-dependent manner (Fig. 5). 3
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Meanwhile, LPS/D-Gal lead to the degradation of IκBα and this was prevented by the treatment of alpinetin (Fig. 5). Furthermore, LPS-induced NF-κB p65 nuclear translocation was inhibited by alpinetin (Fig. 6). 3.6. Effects of alpinetin on Nrf2 and HO-1 expression As shown in Fig. 7, treatment of LPS/D-Gal increased the levels of Nrf2 and HO-1 in the NF-κB signaling pathway. Meanwhile, pretreatment with alpinetin up-regulated the expression of Nrf2 and HO-1 in a dose-dependent manner (Fig. 7). 4. Discussion Liver is the major organ of animal or human metabolism. The drugs or toxins can be converted into small molecules to further absorb or can be decomposed into non-toxic or less toxic substances, thereby reducing the damage to the body. Therefore, to attenuate the injury of liver is very important for human health. In this study, using a mouse model of liver injury, we found that alpinetin exhibited a protective effect against LPS/D-Gal-induced liver injury. D-Gal is liver-specific drug that can deplete the liver cells of uracil triphosphate [13]. It can enhance animal sensitivity to LPS [14]. Therefore, it is considered as a good model of liver injury caused by D-Gal and LPS. Meanwhile, it is of great significance to study the mechanism of liver injury and drug evaluation [15]. In the present study, we used this model to detect the protective effects of alpinetin on liver injury. Serum ALT and AST have been known as the major biomarker of liver injury [16]. When epithelial cell necrosis, ALT and AST release into the blood, and serum ALT and AST levels will rise up. The extent of its rise and liver cell damage are consistent. In many liver diseases, serum ALT and AST levels were significantly increased [17]. Our results showed that alpinetin significantly reduced LPS/D-Gal-induced ALT and AST production. Furthermore, histopathological observation showed that alpinetin could attenuate liver histopathologic changes. The above results showed that alpinetin had protective effects against LPS/D-Gal induced liver injury. LPS has the ability to activate the NF-κB signaling pathway, which leads to the release of inflammatory and oxidative mediators [18, 19]. Furthermore, previous studies demonstrated that inflammation and oxidative stress were involved in the development of liver injury [20]. Inhibition of LPS/D-Gal-induced inflammatory and oxidative response could attenuate the damage of liver tissues [21]. In this study, our results showed that alpinetin significantly suppressed LPS/D-Gal-induced inflammatory cytokines production and MDA content. The productions of inflammatory cytokines were regulated by NF-κB activation [22]. We found that LPS/D-Gal-induced NF-κB activation were significantly inhibited by alpinetin. The Nrf2 signaling pathway was involved in the regulation of oxidative response [23]. Activation of Nrf2 could attenuate liver injury induced by various hepatotoxicants [24, 25]. Our results demonstrated that alpinetin significantly increased the expression of Nrf2 and HO-1. In conclusion, our results showed that alpinetin had protective effects against LPS/D-Gal-induced liver injury. Alpinetin protected liver injury through attenuating inflammatory and oxidative response via inhibiting NF-κB and activating the Nrf2 signaling pathways.
Conflict of interest All authors declare that they have no conflict of interest.
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ACCEPTED MANUSCRIPT References [1] McDonald GB, Frieze D. A problem-oriented approach to liver disease in oncology patients. Gut. 2008;57:987-1003. [2] Lunel F, Musset L, Cacoub P, Frangeul L, Cresta P, Perrin M, et al. Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology. 1994;106:1291-300. [3] Andrade RJ, Lucena MI, Fernandez MC, Pelaez G, Pachkoria K, Garcia-Ruiz E, et al. Drug-induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-year period.
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Gastroenterology. 2005;129:512-21.
[4] Michalopoulos G, Pitot HC. Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Experimental cell research. 1975;94:70-8.
[5] Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM. Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci U S A. 1997;94:2557-62. [6] Nowak M, Gaines GC, Rosenberg J, Minter R, Bahjat FR, Rectenwald J, et al. LPS-induced liver injury
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in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor. American journal of physiology Regulatory, integrative and comparative physiology. 2000;278:R1202-9. [7] Hewett JA, Schultze AE, VanCise S, Roth RA. Neutrophil depletion protects against liver injury from
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bacterial endotoxin. Laboratory investigation; a journal of technical methods and pathology. 1992;66:347-61.
[8] Liu Q, Rehman H, Shi Y, Krishnasamy Y, Lemasters JJ, Smith CD, et al. Inhibition of sphingosine kinase-2 suppresses inflammation and attenuates graft injury after liver transplantation in rats. Plos One. 2012;7:e41834.
[9] He X, Wei Z, Wang J, Kou J, Liu W, Fu Y, et al. Alpinetin attenuates inflammatory responses by suppressing TLR4 and NLRP3 signaling pathways in DSS-induced acute colitis. Scientific reports.
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2016;6:28370.
[10] Chen H, Mo X, Yu J, Huang Z. Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2013;17:26-32.
[11] Huo M, Chen N, Chi G, Yuan X, Guan S, Li H, et al. Traditional medicine alpinetin inhibits the
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inflammatory response in Raw 264.7 cells and mouse models. Int Immunopharmacol. 2012;12:241-8. [12] Hu K, Yang Y, Tu Q, Luo Y, Ma R. Alpinetin inhibits LPS-induced inflammatory mediator response by activating PPAR-gamma in THP-1-derived macrophages. European journal of pharmacology.
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2013;721:96-102.
[13] Tran-Thi TA, Phillips J, Falk H, Decker K. Toxicity of D-galactosamine for rat hepatocytes in monolayer culture. Experimental and molecular pathology. 1985;42:89-116. [14] Matsuda H, Ninomiya K, Morikawa T, Yoshikawa M. Inhibitory effect and action mechanism of sesquiterpenes from Zedoariae Rhizoma on D-galactosamine/lipopolysaccharide-induced liver injury. Bioorganic & medicinal chemistry letters. 1998;8:339-44. [15] Xiong Q, Hase K, Tezuka Y, Namba T, Kadota S. Acteoside inhibits apoptosis in D-galactosamine and lipopolysaccharide-induced liver injury. Life sciences. 1999;65:421-30. [16] Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008;245:194-205. [17] Clark JM, Brancati FL, Diehl AM. The prevalence and etiology of elevated aminotransferase levels in the United States. The American journal of gastroenterology. 2003;98:960-7. [18] Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and 5
ACCEPTED MANUSCRIPT progression. Nat Rev Immunol. 2005;5:749-59. [19] Wang J, Guo C, Wei Z, He X, Kou J, Zhou E, et al. Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells. Journal of dairy science. 2016;99:3016-22. [20] Tipoe GL, Leung TM, Liong EC, Lau TY, Fung ML, Nanji AA. Epigallocatechin-3-gallate (EGCG) injury in mice. Toxicology. 2010;273:45-52.
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reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCl4)-induced liver [21] Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts. Journal of gastroenterology and hepatology. 2011;26 Suppl 1:173-9.
[22] Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 2001;107:7-11. [23] Nguyen T, Nioi P, Pickett CB. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem. 2009;284:13291-5.
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[24] Xu W, Hellerbrand C, Kohler UA, Bugnon P, Kan YW, Werner S, et al. The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis. Laboratory investigation; a journal of technical methods and pathology. 2008;88:1068-78.
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[25] Wu KC, Liu JJ, Klaassen CD. Nrf2 activation prevents cadmium-induced acute liver injury. Toxicology and applied pharmacology. 2012;263:14-20.
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Figure Legends Fig. 1 Histopathologic sections of the livers (H&E, ×400). (A) Control group treated with PBS. (B) Group treated with LPS/D-gal. (C) Group pretreated with 12.5 mg/kg alpinetin 1 h before LPS/D-gal administration. (D) Group pretreated with 25 mg/kg alpinetin 1 h before LPS/D-gal administration. (E) Group pretreated with 50 mg/kg alpinetin 1 h before LPS/D-gal administration. F: Liver histopathologic score. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 2 Effects of alpinetin on ALT and AST activities in mice after LPS/D-gal treatment. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 3 Effects of alpinetin on MDA level and MPO activity of liver tissues in LPS/D-gal induced mice. Mice were given an intraperitoneal injection of alpinetin 1 h before LPS/D-gal administration. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 4 Effects of alpinetin on serum and hepatic TNF-α and IL-1β levels in LPS/D-gal induced mice. Mice were given an intraperitoneal injection of alpinetin 1 h before LPS/D-gal administration respectively. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 5 Effects of alpinetin on LPS/D-gal induced NF-κB activation in mice. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 6 Effects of alpinetin on LPS/D-gal induced NF-κB p65 nuclear translocation. Balb/c mice 6
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were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 7 Effects of alpinetin on Nrf2 and HO-1 expression in mice. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group.
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Alpinetin inhibited liver MPO activity and MDA level. LPS/D-Gal-induced TNF-α and IL-1β production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of Nrf2 and HO-1.
S0882-4010(18)30972-0
DOI:
https://doi.org/10.1016/j.micpath.2018.11.007
Reference:
YMPAT 3244
To appear in:
Microbial Pathogenesis
Received Date: 28 May 2018 Revised Date:
6 October 2018
Accepted Date: 5 November 2018
Please cite this article as: Liu T-g, Sha K-h, Zhang L-g, Liu X-x, Yang F, Cheng J-y, Protective effects of alpinetin on lipopolysaccharide/d-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/j.micpath.2018.11.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Protective effects of alpinetin on lipopolysaccharide/D-Galactosamine-induced liver injury through inhibiting inflammatory and oxidative responses
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Tong-gang Liu1#*, Kai-hui Sha2#, Li-guo Zhang1, Xian-xian Liu1, Fang Yang1, Jin-ying Cheng1 1. Department of infectious diseases, Binzhou Medical University Hospital, Binzhou, Shandong 256603, China. 2. Binzhou Medical University School of Nursing, Binzhou, Shandong 256603, China. # Tong-gang Liu and Kai-hui Sha are co-first authors. *Correspondence to: Tong-gang Liu, Department of infectious diseases, Binzhou Medical University Hospital, Binzhou, Shandong 256603, China. E-mail:[email protected] Abstract
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Alpinetin, a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, has been reported to have anti-inflammatory effects. The aim of this investigation was designed to reveal the protective effects of alpinetin on Lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced liver injury in mice. Alpinetin (12.5, 25, 50 mg/kg) were given 1 h before LPS and D-Gal treatment. 12 h after LPS and D-Gal treatment, the liver tissues and serum were collected. Our results showed that alpinetin treatment improved liver histology, indicating a marked decrease of inflammatory cell infiltration and restore hepatic lobular architecture. Alpinetin also inhibited liver myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Furthermore, LPS/D-Gal-induced tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β) production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In conclusion, these findings suggested that alpinetin inhibited liver injury through inhibiting NF-κB and activating the Nrf2 signaling pathway.
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Keywords: Alpinetin; D-galactosamine; liver injury; Nrf2
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1. Introduction The liver is a substantial organ that takes on the important physiological functions of the human body. Located on the right upper quadrant, the liver is bulky and vulnerable to physical or chemical damage, resulting in liver damage [1]. Liver damage can be caused by surgery, trauma, microbial infections, liver disease, toxic compounds, drug side effects, and so on [2, 3]. These agents stimulate the liver cells, liver tissue and even the entire liver and cause the morphology and pathological changes [4]. LPS is the major factor that leads to liver injury [5]. It can induce the release of hepatic and circulating inflammatory cytokines, which are associated with the development prognosis of liver injury [6, 7]. Suppression of these inflammatory mediators could attenuate the damage of liver tissues [8]. Therefore, the development of new treatment agents that have the ability to inhibit inflammatory cytokines production may be useful for the treatment of liver injury. Alpinetin is a flavonoid isolated Alpinia katsumadai Hayata which has been known to have anti-inflammatory and anti-oxidative properties. A previous study showed that alpinetin had protective effects against DSS-induced colitis in mice [9]. Alpinetin also had the ability to inhibit 1
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LPS-induced mastitis via suppressing inflammatory response [10]. Furthermore, it has been reported that alpinetin had protective effects against LPS-induced lung injury in mice [11]. In addition, alpinetin has been reported to inhibit LPS-induced inflammatory cytokine production in THP-1 cells and RAW264.7 cells [12]. However, whether alpinetin had protective effects against liver injury have not been reported. In the present study, we aimed to clarify the protective effects of alpinetin on liver injury. Our results showed that alpinetin inhibited LPS/D-Gal-induced liver injury through attenuating inflammatory and oxidative responses. 2. Materials and methods 2.1. Animals BALB/c mice, 20-25g, were obtained from the Center of Experimental Animals of Shandong University (Jinan, China). The mice were kept for one week to adapt themselves to the environment. They were housed in a temperature and humidity controlled room and given enough food and water. All the animal experiments were permitted by the Animal Welfare and Research Ethics Committee at Binzhou Medical University. 2.2. Materials LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alpinetin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). ELISA kits were purchased from R&D (Minneapolis, MN, USA). MPO, MDA, ALT, and AST assay kits were provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). Phospho-NF-κB p65, NF-κB p65, phospho-IκBα, IκBα, Nrf2, HO-1, and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). 2.3. Groups Sixty BALB/c mice were randomly divided into five groups and each group contained twelve mice: (A) the blank control group, in which the mice were received equal amounts of vehicle; (B) LPS/D-Gal group, in which the mice were injected intraperitoneally with LPS (50 mg/kg) and D-GalN (800 mg/kg); (C-E) LPS/D-Gal + alpinetin (12.5, 25, 50 mg/kg) groups, in which the mice were received alpinetin (12.5, 25, 50 mg/kg) intraperitoneally 1 h before LPS/D-gal treatment. 2.4. Serum ALT and AST assay 12 h after LPS and D-Gal treatment, blood samples were collected and centrifuged to collect serum. The levels of ALT and AST in serum were measured by the detection kits following the manufacturer's protocols. 2.5. MPO and MDA assay Liver tissues were collected, homogenized, and centrifuged to obtain the supernatant. The levels of MPO and MDA in the supernatants of liver tissues were measured by detection kits according to the manufacturer's instructions. 2.6. ELISA The liver tissues and serum were collected and the serum and hepatic TNF-α and IL-1β levels were determined by the ELISA kits (R&D, Minneapolis, MN, USA) according to the manufacturer's instructions. 2.7. Western blot analysis Liver tissues were homogenized and total proteins were extracted using the protein extraction kit (Thermo). Then, equal amount of proteins were subjected to 12% SDS-PAGE and transferred to 2
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nitrocellulose membranes. The membranes were blocked with 5% skimmed milk. After washing three times with PBST, the membranes were incubated with primary antibodies: Phospho-NF-κB p65, NF-κB p65, phospho-IκBα, IκBα, Nrf2, HO-1, and β-actin overnight at 4 °C. Then, the membranes were washed three times with TBST and incubated with secondary antibodies for 2 h at room temperature. Three times after washing, ECL Western blot detection was used to visualize bands. 2.8. Histopathologic analysis Liver tissues were collected and fixed in 4% paraformaldehyde. Afterwards, the tissues were embedded in paraffin and cut into 5µm thick sections on a rotary microtome. Then, the sections were stained with H&E staining. The extent of histological changes was scored and scores of 0-1 was none; the score of 2-3 was mild; the score of 4-6 was moderate; the score of 7-9 was marked. The following parameters were observed and quantified for liver histopathology score: (1) Lobular
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architecture: Intact-0, Intact with cell swelling-1, Mild disruption-2, Marked disruption-3. (2) Apoptosis: None-0, Occasional-1, Mild-2, Marked-3. (3) Congestion: None-0, Occasional-1, Mild-2, Marked-3.
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2.9. Statistical analysis The data of this study was expressed as the mean ± SEM and analyzed with one-way analysis of variance (ANOVA) followed by Duncan's test using SPSS software. P value < 0.05 was considered to be statistically significant. 3. Results 3.1. Effects of alpinetin on LPS/D-Gal-induced liver histopathologic changes H&E staining was used in this study to detect the protective effects of alpinetin on LPS/D-Gal-induced liver injury. As shown in Fig. 1, liver tissues of the control group showed normal structure of livers. Significant histopathologic changes were observed in the LPS/D-Gal group, including cell necrosis, loss of hepatic architecture and massive inflammatory cells infiltration. These liver histopathologic changes were restored by the treatment of alpinetin. 3.2. Effects of alpinetin on LPS/D-Gal-induced serum ALT and AST levels Serum ALT and AST are important biomarkers of liver injury. As shown in Fig. 2, increased serum ALT and AST were observed in mice of LPS/D-Gal group. And the levels of ALT and AST in LPS/D-Gal + alpinetin group were significantly decreased when compared with LPS/D-Gal group. 3.3. Effects of alpinetin on LPS/D-Gal-induced MPO activity and MDA content MDA is the marker of oxidative stress and MPO is the marker of neutrophils infiltration of tissues. As shown in Fig. 3, increased MDA content and MPO activity of liver tissues were observed in mice of LPS/D-Gal group. And the levels of MDA and MPO in mice of LPS/D-Gal + alpinetin groups were significantly decreased when compared with LPS/D-Gal group (Fig. 3). 3.4. Effects of alpinetin on LPS/D-Gal-induced serum and hepatic TNF-α and IL-1β production As shown in Fig. 4, increased TNF-α and IL-1β levels of serum and liver tissues were observed in mice of LPS/D-Gal group. And the levels of TNF-α and IL-1β in mice of LPS/D-Gal + alpinetin groups were significantly decreased when compared with LPS/D-Gal group (Fig. 4). 3.5. Effects of alpinetin on LPS/D-Gal-induced NF-κB activation As shown in Fig. 5, treatment of LPS/D-Gal obviously increased the levels of p65 and IκBα phosphorylation in the NF-κB signaling pathway. However, pretreatment with alpinetin inhibited the up-regulation of phosphorylation of p65 and IκBα in a dose-dependent manner (Fig. 5). 3
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Meanwhile, LPS/D-Gal lead to the degradation of IκBα and this was prevented by the treatment of alpinetin (Fig. 5). Furthermore, LPS-induced NF-κB p65 nuclear translocation was inhibited by alpinetin (Fig. 6). 3.6. Effects of alpinetin on Nrf2 and HO-1 expression As shown in Fig. 7, treatment of LPS/D-Gal increased the levels of Nrf2 and HO-1 in the NF-κB signaling pathway. Meanwhile, pretreatment with alpinetin up-regulated the expression of Nrf2 and HO-1 in a dose-dependent manner (Fig. 7). 4. Discussion Liver is the major organ of animal or human metabolism. The drugs or toxins can be converted into small molecules to further absorb or can be decomposed into non-toxic or less toxic substances, thereby reducing the damage to the body. Therefore, to attenuate the injury of liver is very important for human health. In this study, using a mouse model of liver injury, we found that alpinetin exhibited a protective effect against LPS/D-Gal-induced liver injury. D-Gal is liver-specific drug that can deplete the liver cells of uracil triphosphate [13]. It can enhance animal sensitivity to LPS [14]. Therefore, it is considered as a good model of liver injury caused by D-Gal and LPS. Meanwhile, it is of great significance to study the mechanism of liver injury and drug evaluation [15]. In the present study, we used this model to detect the protective effects of alpinetin on liver injury. Serum ALT and AST have been known as the major biomarker of liver injury [16]. When epithelial cell necrosis, ALT and AST release into the blood, and serum ALT and AST levels will rise up. The extent of its rise and liver cell damage are consistent. In many liver diseases, serum ALT and AST levels were significantly increased [17]. Our results showed that alpinetin significantly reduced LPS/D-Gal-induced ALT and AST production. Furthermore, histopathological observation showed that alpinetin could attenuate liver histopathologic changes. The above results showed that alpinetin had protective effects against LPS/D-Gal induced liver injury. LPS has the ability to activate the NF-κB signaling pathway, which leads to the release of inflammatory and oxidative mediators [18, 19]. Furthermore, previous studies demonstrated that inflammation and oxidative stress were involved in the development of liver injury [20]. Inhibition of LPS/D-Gal-induced inflammatory and oxidative response could attenuate the damage of liver tissues [21]. In this study, our results showed that alpinetin significantly suppressed LPS/D-Gal-induced inflammatory cytokines production and MDA content. The productions of inflammatory cytokines were regulated by NF-κB activation [22]. We found that LPS/D-Gal-induced NF-κB activation were significantly inhibited by alpinetin. The Nrf2 signaling pathway was involved in the regulation of oxidative response [23]. Activation of Nrf2 could attenuate liver injury induced by various hepatotoxicants [24, 25]. Our results demonstrated that alpinetin significantly increased the expression of Nrf2 and HO-1. In conclusion, our results showed that alpinetin had protective effects against LPS/D-Gal-induced liver injury. Alpinetin protected liver injury through attenuating inflammatory and oxidative response via inhibiting NF-κB and activating the Nrf2 signaling pathways.
Conflict of interest All authors declare that they have no conflict of interest.
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ACCEPTED MANUSCRIPT References [1] McDonald GB, Frieze D. A problem-oriented approach to liver disease in oncology patients. Gut. 2008;57:987-1003. [2] Lunel F, Musset L, Cacoub P, Frangeul L, Cresta P, Perrin M, et al. Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology. 1994;106:1291-300. [3] Andrade RJ, Lucena MI, Fernandez MC, Pelaez G, Pachkoria K, Garcia-Ruiz E, et al. Drug-induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-year period.
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Gastroenterology. 2005;129:512-21.
[4] Michalopoulos G, Pitot HC. Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. Experimental cell research. 1975;94:70-8.
[5] Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM. Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci U S A. 1997;94:2557-62. [6] Nowak M, Gaines GC, Rosenberg J, Minter R, Bahjat FR, Rectenwald J, et al. LPS-induced liver injury
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in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor. American journal of physiology Regulatory, integrative and comparative physiology. 2000;278:R1202-9. [7] Hewett JA, Schultze AE, VanCise S, Roth RA. Neutrophil depletion protects against liver injury from
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bacterial endotoxin. Laboratory investigation; a journal of technical methods and pathology. 1992;66:347-61.
[8] Liu Q, Rehman H, Shi Y, Krishnasamy Y, Lemasters JJ, Smith CD, et al. Inhibition of sphingosine kinase-2 suppresses inflammation and attenuates graft injury after liver transplantation in rats. Plos One. 2012;7:e41834.
[9] He X, Wei Z, Wang J, Kou J, Liu W, Fu Y, et al. Alpinetin attenuates inflammatory responses by suppressing TLR4 and NLRP3 signaling pathways in DSS-induced acute colitis. Scientific reports.
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2016;6:28370.
[10] Chen H, Mo X, Yu J, Huang Z. Alpinetin attenuates inflammatory responses by interfering toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced mastitis in mice. Int Immunopharmacol. 2013;17:26-32.
[11] Huo M, Chen N, Chi G, Yuan X, Guan S, Li H, et al. Traditional medicine alpinetin inhibits the
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inflammatory response in Raw 264.7 cells and mouse models. Int Immunopharmacol. 2012;12:241-8. [12] Hu K, Yang Y, Tu Q, Luo Y, Ma R. Alpinetin inhibits LPS-induced inflammatory mediator response by activating PPAR-gamma in THP-1-derived macrophages. European journal of pharmacology.
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2013;721:96-102.
[13] Tran-Thi TA, Phillips J, Falk H, Decker K. Toxicity of D-galactosamine for rat hepatocytes in monolayer culture. Experimental and molecular pathology. 1985;42:89-116. [14] Matsuda H, Ninomiya K, Morikawa T, Yoshikawa M. Inhibitory effect and action mechanism of sesquiterpenes from Zedoariae Rhizoma on D-galactosamine/lipopolysaccharide-induced liver injury. Bioorganic & medicinal chemistry letters. 1998;8:339-44. [15] Xiong Q, Hase K, Tezuka Y, Namba T, Kadota S. Acteoside inhibits apoptosis in D-galactosamine and lipopolysaccharide-induced liver injury. Life sciences. 1999;65:421-30. [16] Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008;245:194-205. [17] Clark JM, Brancati FL, Diehl AM. The prevalence and etiology of elevated aminotransferase levels in the United States. The American journal of gastroenterology. 2003;98:960-7. [18] Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and 5
ACCEPTED MANUSCRIPT progression. Nat Rev Immunol. 2005;5:749-59. [19] Wang J, Guo C, Wei Z, He X, Kou J, Zhou E, et al. Morin suppresses inflammatory cytokine expression by downregulation of nuclear factor-kappaB and mitogen-activated protein kinase (MAPK) signaling pathways in lipopolysaccharide-stimulated primary bovine mammary epithelial cells. Journal of dairy science. 2016;99:3016-22. [20] Tipoe GL, Leung TM, Liong EC, Lau TY, Fung ML, Nanji AA. Epigallocatechin-3-gallate (EGCG) injury in mice. Toxicology. 2010;273:45-52.
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reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCl4)-induced liver [21] Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver injury: Present concepts. Journal of gastroenterology and hepatology. 2011;26 Suppl 1:173-9.
[22] Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J Clin Invest. 2001;107:7-11. [23] Nguyen T, Nioi P, Pickett CB. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. J Biol Chem. 2009;284:13291-5.
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[24] Xu W, Hellerbrand C, Kohler UA, Bugnon P, Kan YW, Werner S, et al. The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis. Laboratory investigation; a journal of technical methods and pathology. 2008;88:1068-78.
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[25] Wu KC, Liu JJ, Klaassen CD. Nrf2 activation prevents cadmium-induced acute liver injury. Toxicology and applied pharmacology. 2012;263:14-20.
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Figure Legends Fig. 1 Histopathologic sections of the livers (H&E, ×400). (A) Control group treated with PBS. (B) Group treated with LPS/D-gal. (C) Group pretreated with 12.5 mg/kg alpinetin 1 h before LPS/D-gal administration. (D) Group pretreated with 25 mg/kg alpinetin 1 h before LPS/D-gal administration. (E) Group pretreated with 50 mg/kg alpinetin 1 h before LPS/D-gal administration. F: Liver histopathologic score. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 2 Effects of alpinetin on ALT and AST activities in mice after LPS/D-gal treatment. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 3 Effects of alpinetin on MDA level and MPO activity of liver tissues in LPS/D-gal induced mice. Mice were given an intraperitoneal injection of alpinetin 1 h before LPS/D-gal administration. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 4 Effects of alpinetin on serum and hepatic TNF-α and IL-1β levels in LPS/D-gal induced mice. Mice were given an intraperitoneal injection of alpinetin 1 h before LPS/D-gal administration respectively. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 5 Effects of alpinetin on LPS/D-gal induced NF-κB activation in mice. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 6 Effects of alpinetin on LPS/D-gal induced NF-κB p65 nuclear translocation. Balb/c mice 6
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were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group. Fig. 7 Effects of alpinetin on Nrf2 and HO-1 expression in mice. Balb/c mice were pretreated intraperitoneally with alpinetin 1 h before LPS/D-gal administration. β-Actin was used as a control. The values presented are the mean ± SEM of three independent experiments. p#<0.01 vs. control group, p*<0.05, p**<0.01 vs. LPS/D-gal group.
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Alpinetin inhibited liver MPO activity and MDA level. LPS/D-Gal-induced TNF-α and IL-1β production were dose-dependently inhibited by alpinetin. Alpinetin also attenuated LPS/D-Gal-induced expression of phospho-NF-κB p65 and phospho-IκBα. In addition, alpinetin was found to increase the expression of Nrf2 and HO-1.