j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 2 e5 2 8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of oroxylin A against lipopolysaccharide and/or D-galactosamineeinduced acute liver injury in mice Haiying Huang, MD, Xiaoyu Zhang, MD, and Jingyuan Li, MD* Department of Infectious Diseases, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
article info
abstract
Article history:
Background: Oroxylin A, a natural flavonoid isolated from Scutellariae baicalensis, has been
Received 13 December 2014
reported to possess a wide spectrum of pharmacologic activities. However, the effects of
Received in revised form
oroxylin A on liver injury are poor understood. The purpose of this study was to investigate
13 January 2015
the effects of oroxylin A on acute liver injury in mice induced by lipopolysaccharide and/or
Accepted 26 January 2015
D-galactosamine (LPS and/or D-GalN).
Available online 30 January 2015
Methods: Mice acute liver injury model was induced by LPS (50 mg/kg) and/or GalN (800 mg/ kg). Serum alanine aminotransferase, aspartate aminotransferase, and tumor necrosis
Keywords:
factor-a levels, hepatic tissue histology, malondialdehyde content, and myeloperoxidase
Oroxylin A
activity were analyzed. Meanwhile, nuclear factor kappa B (NF-kB), heme oxygenase-1 (HO-
LPS
1), and nuclear factor erythroid2-related factor 2 (Nrf2) expression were detected by
Nrf2
Western blotting.
TLR4
Results: The results showed that oroxylin A dose-dependently inhibited LPS and/or GalNe
NF-kB
induced serum alanine aminotransferase, aspartate aminotransferase, and tumor necrosis factor-a levels. Hepatic malondialdehyde content and myeloperoxidase activity were also suppressed by oroxylin A. We also found that oroxylin A inhibited LPS and/or GalNe induced toll like receptor 4 (TLR4) expression and NF-kB activation. In addition, oroxylin A upregulated the expression of Nrf2 and HO-1 in a dose-dependent manner. Conclusions: In conclusion, oroxylin A protected against LPS and/or GalNeinduced liver injury through activating Nrf2 and inhibiting TLR4 signaling pathway. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Fulminant hepatic failure is a dramatic clinical syndrome associated with a high rate of mortality [1]. There are no effective preventives and therapies for the disease other than liver transplantation [2]. Lipopolysaccharide (LPS) and galactosamine (GalN)einduced liver injury is a well-known model of human liver injury. It is widely used to investigate the underlying mechanisms and potential therapeutic drugs for clinical fulminant hepatic failure [3,4]. GalN is a hepatotoxic
agent that leads to liver cells necrosis [5]. Stimulating of Kupffer cells by LPS induces TLR4 signaling pathway activation, which subsequently activates nuclear factor kappa B (NFkB) and induces inflammatory cytokines such as tumor necrosis factor (TNF)-a production [6]. Previous studies showed that overproduction of TNF-a led to liver cell necrosis and liver failure [7]. Oroxylin A, a natural flavonoid isolated from Scutellariae baicalensis, is responsible for its pharmacologic activities. Oroxylin A has been reported to have antitumor, antioxidant,
* Corresponding author. Department of Infectious Diseases, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China. Tel.: þ8613809673365; fax: þ8613809673365. E-mail address: [email protected] (J. Li). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.01.047
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 2 e5 2 8
and anti-inflammatory effects [8,9]. Oroxylin A was found to inhibit LPS-induced TNF-a, interleukin-1b, and interleukin-6 production in RAW264.7 cells [10]. Meanwhile, previous studies showed that oroxylin A also inhibited inducible nitric oxide synthase and TNF-a production in LPS-stimulated BV2 cells [11]. In vivo, oroxylin A was found to ameliorate cardiac dysfunction of endotoxemic rats [12]. Furthermore, oroxylin A has been reported to have a protective effect on LPSeinduced acute lung injury in mice [13]. However, the effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury were poorly understood. The purpose of this study was to investigate the effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury in mice.
2.
Materials and methods
2.1.
Materials
Oroxylin A was purchased from the Chinese Institute for Drug and Biological Product Control (Beijing, China). TNF-a enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (Minneapolis, MN). LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma (St. Louis, MO). The myeloperoxidase (MPO) determination kit, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) detection kits were provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Antibodies against NF-kB, inhibitor kappaBa, toll like receptor 4 (TLR4), heme oxygenase-1 (HO-1), nuclear factor erythroid2related factor 2 (Nrf2), and b-actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). All other reagents were of analytical grade.
2.2.
Animals
BALB/c mice (6e8-wk-old; 18e22 g) were obtained from the Center of Experimental Animals of Harbin Medical University (HeiLongjiang, China). The mice were housed in an animal room and given a standard diet. The laboratory temperature was 24 1 C, and relative humidity was 40%e80%. All animal experiments were performed according to the guidelines of the Local Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.3.
Experimental design
Sixty mice were randomly divided into five groups as follows: control group, LPS and/or GalN group, oroxylin A (15, 30, and 60 mg/kg) þ LPS and/or GalN groups. Acute liver injury was induced in BALB/c mice with intraperitoneal injection of LPS (50 mg/kg) combined with D-GalN (800 mg/kg). Oroxylin A (15, 30, and 60 mg/kg) was given with an intraperitoneal injection 1 h before LPS and/or GalN treatment. The blood and liver tissues were collected for subsequent analysis.
2.4.
Serum aminotransferase activities
Plasma samples were collected from the mice 8 h after the LPS and/or GalN injection. Serum levels of ALT and AST were
523
tested using test kits (Jiancheng Bioengineering Institute of Nanjing) according to the instructions.
2.5.
ELISA assay
TNF-a level in serum and liver tissues were detected using commercial ELISA kits purchased from R&D Systems according to the manufacturer’s instructions.
2.6.
Quantitative real-time polymerase chain reaction
The messenger RNA (mRNA) expression of TNF-a was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, total RNA from liver tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA) reagent. The RNA was reverse transcribed with M-MuLV Reverse Transcriptase Kit (Fermentas, Lithuania) to obtain complementary DNA. qRTPCR was performed with SYBR green PCR Master Mix, and the relative mRNA concentrations were detected by qRT-PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The sequences for primers are: TNFa (forward 50 -GCCTCCCTCTCATCAGTTCTA-30 and reverse 50 GGCAGCCTTGTCCCTTG-30 ). The comparative cycle threshold (DDCT) method of relative quantification was used to determine the fold-change of expression.
2.7.
Malondialdehyde and MPO activity assay
Liver malondialdehyde (MDA) content and MPO activity were measured by relative test kits (Jiancheng Bioengineering Institute of Nanjing) according to the instructions.
2.8.
Histopathologic examination
Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin and cut into 5-mm sections. After hematoxylin-eosin staining, histopathologic changes were observed using an optical microscope (Olympus Optical Co, Tokyo, Japan).
2.9.
Western blot analysis
Nuclear and cytoplasmic proteins were extracted from the liver tissues using a protein extract kit (Thermo) according to the manufacturer’s protocol. Protein concentrations were determined by the bicinchoninic acid protein assay kit. Equal amounts of protein were fractionated onto 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat milk for 2 h at room temperature. The membranes were incubated with specific antibodies against indicated primary antibodies at 4 C overnight followed by the peroxidase-conjugated secondary antibody at 37 C for 1 h. Protein bands were visualized using enhanced chemiluminescence reagents, and densitometric analysis was performed with the use of a PDI ImageWare System (Bio-Rad, Hercules, CA).
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Fig. 1 e Effects of oroxylin A on histopathologic changes in liver tissues. Representative histologic changes of liver obtained from mice of different groups. (A) Control group, (B) LPS and/or GalN group, (C) LPS and/or GalN D oroxylin A (15 mg/kg) group, (D) LPS and/or GalN D oroxylin A (30 mg/kg) group, and (E) LPS and/or GalN D oroxylin A (60 mg/kg) group (hematoxylin and eosin staining, magnification 3200). (Color version of figure is available online.)
2.10.
Statistical analysis
All data were expressed as means standard deviation Differences between multiple treatment groups were assessed by one-way analysis of variance followed by a Dunnett test. Results are considered significant at P < 0.05 or P < 0.01.
3.
Results
3.1. Effects of oroxylin A on LPS and/or GalNeinduced liver histopathologic changes
GalN group compared with the control group. Treatment of oroxylin A dose-dependently suppressed LPS and/or GalNeinduced serum and hepatic TNF-a levels (Fig. 3A). The expression of TNF-a in the liver tissue was also detected by qRT-PCR and Western blot analysis. The results showed that oroxylin A significantly inhibited TNF-a expression both in mRNA and protein levels (Fig. 3B).
3.4.
Effects of oroxylin A on MDA content
MDA, a marker of oxidative stress, was used to assess the antioxidant effects of oroxylin A. The results showed that
The histologic features of the control group showed normal lobular architecture and cellular structure. LPS and/or GalN treatment group showed significant pathologic changes, including hepatocytes necrosis, destruction of hepatic architecture, and inflammatory cell infiltration. Oroxylin A significantly attenuated these pathologic changes induced by LPS and/or GalN (Fig. 1).
3.2. Oroxylin A inhibits LPS and/or GalNeinduced serum ALT and AST levels The effects of oroxylin A on hepatic failure were detected by measuring serum ALT and AST levels. As shown in Figure 2, compared with the control group, serum ALT and AST levels increased significantly in the LPS and/or GalN group. However, oroxylin A inhibited LPS and/or GalNeinduced ALT and AST levels in a dose-dependent manner (Fig. 2).
3.3. Oroxylin A inhibits serum and hepatic TNF-a production To test the anti-inflammatory effects of oroxylin A, the effects of oroxylin A on LPS and/or GalNeinduced TNF-a expression were detected by ELISA. The results showed that serum and hepatic TNF-a levels increased significantly in LPS and/or
Fig. 2 e Effects of oroxylin A on serum ALT and AST levels. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
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Fig. 3 e (A) Effects of oroxylin A on serum and hepatic TNF-a levels detected by ELISA. (B) Effects of oroxylin A on hepatic TNF-a level detected by qRT-PCR and Western blot analysis. The values presented are the means ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
MDA level significantly increased after LPS and/or GalN treatment and this elevation was attenuated by oroxylin A (Fig. 4A).
3.5.
reported to have anti-inflammatory effects [15]. In this study, we investigated the protective effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury in mice. Our results showed that oroxylin A inhibited serum ALT and AST levels.
Effects of oroxylin A on MPO activity
MPO activity was used to assess the neutrophil accumulation within liver tissues. As shown in Figure 4B, compared with the control group, MPO activity increased significantly in the LPS and/or GalN group. However, this increase was attenuated by oroxylin A in a dose-dependent manner.
3.6. Effect of oroxylin A on TLR4 expression and NF-kB activation TLR4 and NF-kB have been reported to play critical roles in the regulating of inflammatory cytokines production. Thus, the effects of oroxylin A on TLR4 expression and NF-kB activation were determined. The results showed that oroxylin A inhibited LPS and/or GalNeinduced TLR4 expression and NF-kB activation in a dose-dependent manner (Fig. 5).
3.7.
Effects of oroxylin A on Nrf2 and HO-1 expression
The effects of oroxylin A on Nrf2 and HO-1 expression were detected by Western blotting. The results showed that nuclear translocation of Nrf2 and HO-1 expressions were upregulated by LPS and/or GalN. These increases in Nrf2 and HO-1 expression were augmented by oroxylin A (Fig. 6).
4.
Discussion
Fulminant hepatic failure is a life-threatening clinical syndrome associated with a high rate of mortality [14]. Oroxylin A, a natural flavonoid isolated from S baicalensis, has been
Fig. 4 e Effects of oroxylin A on liver MDA level and MPO activity. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
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Fig. 5 e Oroxylin A inhibited LPS and/or GalNeinduced TLR4 expression and NF-kB activation with Western blotting. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05 and **P < 0.01 group versus LPS group.
Meanwhile, histologic analysis demonstrated that oroxylin A had a protective effect on LPS and/or GalNeinduced acute hepatic injury. Oroxylin A may be a potential therapeutic reagent to attenuate hepatic injury.
TLR4, one of the best characterized TLRs, recognizes LPS from gram-negative bacteria [16]. LPS induces TLR4 expression and NF-kB activation, which subsequently leads to inflammatory cytokines production [17,18]. Studies showed that
Fig. 6 e Effects of oroxylin A on the expression of Nrf2 and HO-1. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 group versus LPS group.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 2 e5 2 8
these inflammatory cytokines played important roles in the development of liver failure [19,20]. Among these inflammatory cytokines, TNF-a acted as the crucial mediator in hepatocytic apoptosis and acute liver failure [21]. Elevated TNF-a has been observed in patients with liver failure [22]. Thus, inhibiting of TNF-a production is likely to be therapeutically effective in patients with liver failure. In this study, we found that serum and hepatic TNF-a expression increased significantly and this increase was reduced by oroxylin A. TLR4 and NF-kB are involved in the regulation of TNF-a production [23]. Thus, we tested the effects of oroxylin A on the TLR4 expression and NF-kB activation. Our results showed that oroxylin A inhibited TNF-a production via inhibiting TLR4emediated NFkB activation. MDA, one of the most frequently used indicators of lipid peroxidation, is usually used to assess the oxidative stress of different tissues [24]. To investigate the antioxidant effects of oroxylin A on LPS and/or GalNeinduced acute liver injury, MDA content in liver tissues was detected. Our results showed that oroxylin A decreased oxidative stress induced by LPS and/ or GalN. Nrf2, a redox sensitive transcription factor, was originally known to activate cellular protective pathways against oxidative injury [25,26]. Activating of Nrf2 induces the expression of HO-1, which has been reported to have antioxidant activity [27,28]. Previous studies showed that Nrf2 can be used as an effective therapeutic target in modulating inflammatory liver diseases [29,30]. Thus, the effects of oroxylin A on Nrf2 and HO-1 expression were detected. The results showed that oroxylin A upregulated the expression of Nrf2 and HO-1. These results suggested that oroxylin A exhibited antioxidant effects by inducing oxidative defense system. This study demonstrated that oroxylin A exhibited protective effects on LPS and/or GalNeinduced liver injury. Oroxylin A protected against LPS and/or GalNeinduced liver injury through activating Nrf2 and inhibiting TLR4 signaling pathway. Therefore, oroxylin A can be used as a potential agent for preventing acute liver injury.
Acknowledgment Authors’ contributions: H.H.Y. and L.J.Y. contributed to the conception and design. H.H.Y., Z.X.Y., and L.J.Y. did the analysis and interpretation. H.H.Y. did the data collection. H.H.Y. and L.J.Y. wrote the article and did the critical revision of the article.
Disclosure The authors have no conflict of interest to declare.
references
[1] Vaquero J, Polson J, Chung CH, et al. Infection and the progression of hepatic encephalopathy in acute liver failure. Gastroenterology 2003;125:755.
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[2] Farmer DG, Anselmo DM, Ghobrial RM, et al. Liver transplantation for fulminant hepatic failuredexperience with more than 200 patients over a 17-year period. Ann Surg 2003;237:666. [3] Lin X, Zhang S, Huang R, et al. Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/Dgalactosamine induced fulminant hepatic failure in mice. Int Immunopharmacol 2014;19:365. [4] Morita T, Jinno K, Kawagishi H, et al. Hepatoprotective effect of myristicin from nutmeg (Myristica fragrans) on lipopolysaccharide/D-galactosamine-induced liver injury. J Agric Food Chem 2003;51:1560. [5] Wen ZM, Liu Y, Li F, Wen T. Low dose of carbon monoxide intraperitoneal injection provides potent protection against GalN/LPS-induced acute liver injury in mice. J Appl Toxicol 2013;33:1424. [6] Xu FL, You HB, Li XH, et al. Glycine attenuates endotoxininduced liver injury by downregulating TLR4 signaling in Kupffer cells. Am J Surg 2008;196:139. [7] Ding WX, Yin XM. Dissection of the multiple mechanisms of TNF-alpha-induced apoptosis in liver injury. J Cell Mol Med 2004;8:445. [8] Sun Y, Lu N, Ling Y, et al. Oroxylin A suppresses invasion through down-regulating the expression of matrix metalloproteinase-2/9 in MDA-MB-435 human breast cancer cells. Eur J Pharmacol 2009;603:22. [9] Chen Y, Yang L, Lee TJ. Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-kappaB activation. Biochem Pharmacol 2000;59:1445. [10] Wang H, Guo Y, Zhao X, et al. An estrogen receptor dependent mechanism of oroxylin A in the repression of inflammatory response. PLoS One 2013;8:e69555. [11] Liu PW, Chen MF, Tsai AP, Lee TJ. STAT1 mediates oroxylin a inhibition of iNOS and pro-inflammatory cytokines expression in microglial BV-2 cells. Plos One 2012;7:e50363. [12] Liu CH, Chen MF, Tseng TL, et al. Oroxylin a, but not vasopressin, ameliorates cardiac dysfunction of endotoxemic rats. Evid-Based Compl Alt; 2012. [13] Tseng TL, Chen MF, Tsai MJ, et al. Oroxylin-a rescues LPSinduced acute lung injury via regulation of NF-kappa B signaling pathway in rodents. Plos One 2012;7. [14] Canbay A. Acute liver failureda life-threatening disease (vol 108, pg 715, 2011). Dtsch Arztebl Int 2013;110:633. [15] Song XM, Chen Y, Sun YJ, et al. Oroxylin A, a classical natural product, shows a novel inhibitory effect on angiogenesis induced by lipopolysaccharide. Pharmacol Rep 2012;64:1189. [16] Abate W, Alghaithy AA, Parton J, Jones KP, Jackson SK. Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains. J Lipid Res 2010; 51:334. [17] Joh EH, Gu W, Kim DH. Echinocystic acid ameliorates lung inflammation in mice and alveolar macrophages by inhibiting the binding of LPS to TLR4 in NF-kappaB and MAPK pathways. Biochem Pharmacol 2012;84:331. [18] Ha YM, Ham SA, Kim YM, et al. Beta(1)-adrenergic receptormediated HO-1 induction, via PI3K and p38 MAPK, by isoproterenol in RAW 264.7 cells leads to inhibition of HMGB1 release in LPS-activated RAW 264.7 cells and increases in survival rate of CLP-induced septic mice. Biochem Pharmacol 2011;82:769. [19] Streetz KL, Wustefeld T, Klein C, Manns MP, Trautwein C. Mediators of inflammation and acute phase response in the liver. Cell Mol Biol 2001;47:661. [20] Jarrar MH, Baranova A, Collantes R, et al. Adipokines and cytokines in non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2008;27:412.
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[21] Schwabe RF, Brenner DA. Mechanisms of liver injury. I. TNFalpha-induced liver injury: role of IKK, JNK, and ROS pathways. Am J Physiol Gastrointest Liver Physiol 2006;290: G583. [22] Crespo J, Cayon A, Fernandez-Gil P, et al. Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology 2001;34: 1158. [23] Teng X, Xu LF, Zhou P, Sun HW, Sun M. Effects of trefoil peptide 3 on expression of TNF-alpha, TLR4, and NF-kappaB in trinitrobenzene sulphonic acid induced colitis mice. Inflammation 2009;32:120. [24] Jentzsch AM, Bachmann H, Furst P, Biesalski HK. Improved analysis of malondialdehyde in human body fluids. Free Radic Biol Med 1996;20:251. [25] Ishii T, Itoh K, Takahashi S, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 2000;275:16023.
[26] Owuor ED, Kong AN. Antioxidants and oxidants regulated signal transduction pathways. Biochem Pharmacol 2002;64: 765. [27] Reisman SA, Aleksunes LM, Klaassen CD. Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes. Biochem Pharmacol 2009;77:1273. [28] Juan SH, Cheng TH, Lin HC, Chu YL, Lee WS. Mechanism of concentration-dependent induction of heme oxygenase-1 by resveratrol in human aortic smooth muscle cells. Biochem Pharmacol 2005;69:41. [29] Xu W, Hellerbrand C, Kohler UA, et al. The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis. Lab Invest a J Tech Methods Pathol 2008;88:1068. [30] Okada K, Shoda J, Taguchi K, et al. Nrf2 counteracts cholestatic liver injury via stimulation of hepatic defense systems. Biochem biophysical Res Commun 2009; 389:431.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Protective effect of oroxylin A against lipopolysaccharide and/or D-galactosamineeinduced acute liver injury in mice Haiying Huang, MD, Xiaoyu Zhang, MD, and Jingyuan Li, MD* Department of Infectious Diseases, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
article info
abstract
Article history:
Background: Oroxylin A, a natural flavonoid isolated from Scutellariae baicalensis, has been
Received 13 December 2014
reported to possess a wide spectrum of pharmacologic activities. However, the effects of
Received in revised form
oroxylin A on liver injury are poor understood. The purpose of this study was to investigate
13 January 2015
the effects of oroxylin A on acute liver injury in mice induced by lipopolysaccharide and/or
Accepted 26 January 2015
D-galactosamine (LPS and/or D-GalN).
Available online 30 January 2015
Methods: Mice acute liver injury model was induced by LPS (50 mg/kg) and/or GalN (800 mg/ kg). Serum alanine aminotransferase, aspartate aminotransferase, and tumor necrosis
Keywords:
factor-a levels, hepatic tissue histology, malondialdehyde content, and myeloperoxidase
Oroxylin A
activity were analyzed. Meanwhile, nuclear factor kappa B (NF-kB), heme oxygenase-1 (HO-
LPS
1), and nuclear factor erythroid2-related factor 2 (Nrf2) expression were detected by
Nrf2
Western blotting.
TLR4
Results: The results showed that oroxylin A dose-dependently inhibited LPS and/or GalNe
NF-kB
induced serum alanine aminotransferase, aspartate aminotransferase, and tumor necrosis factor-a levels. Hepatic malondialdehyde content and myeloperoxidase activity were also suppressed by oroxylin A. We also found that oroxylin A inhibited LPS and/or GalNe induced toll like receptor 4 (TLR4) expression and NF-kB activation. In addition, oroxylin A upregulated the expression of Nrf2 and HO-1 in a dose-dependent manner. Conclusions: In conclusion, oroxylin A protected against LPS and/or GalNeinduced liver injury through activating Nrf2 and inhibiting TLR4 signaling pathway. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
Fulminant hepatic failure is a dramatic clinical syndrome associated with a high rate of mortality [1]. There are no effective preventives and therapies for the disease other than liver transplantation [2]. Lipopolysaccharide (LPS) and galactosamine (GalN)einduced liver injury is a well-known model of human liver injury. It is widely used to investigate the underlying mechanisms and potential therapeutic drugs for clinical fulminant hepatic failure [3,4]. GalN is a hepatotoxic
agent that leads to liver cells necrosis [5]. Stimulating of Kupffer cells by LPS induces TLR4 signaling pathway activation, which subsequently activates nuclear factor kappa B (NFkB) and induces inflammatory cytokines such as tumor necrosis factor (TNF)-a production [6]. Previous studies showed that overproduction of TNF-a led to liver cell necrosis and liver failure [7]. Oroxylin A, a natural flavonoid isolated from Scutellariae baicalensis, is responsible for its pharmacologic activities. Oroxylin A has been reported to have antitumor, antioxidant,
* Corresponding author. Department of Infectious Diseases, The Second Affiliated Hospital of Harbin Medical University, Harbin 150081, China. Tel.: þ8613809673365; fax: þ8613809673365. E-mail address: [email protected] (J. Li). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2015.01.047
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 5 ( 2 0 1 5 ) 5 2 2 e5 2 8
and anti-inflammatory effects [8,9]. Oroxylin A was found to inhibit LPS-induced TNF-a, interleukin-1b, and interleukin-6 production in RAW264.7 cells [10]. Meanwhile, previous studies showed that oroxylin A also inhibited inducible nitric oxide synthase and TNF-a production in LPS-stimulated BV2 cells [11]. In vivo, oroxylin A was found to ameliorate cardiac dysfunction of endotoxemic rats [12]. Furthermore, oroxylin A has been reported to have a protective effect on LPSeinduced acute lung injury in mice [13]. However, the effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury were poorly understood. The purpose of this study was to investigate the effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury in mice.
2.
Materials and methods
2.1.
Materials
Oroxylin A was purchased from the Chinese Institute for Drug and Biological Product Control (Beijing, China). TNF-a enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (Minneapolis, MN). LPS (Escherichia coli, O55:B5) and D-galactosamine were purchased from Sigma (St. Louis, MO). The myeloperoxidase (MPO) determination kit, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) detection kits were provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). Antibodies against NF-kB, inhibitor kappaBa, toll like receptor 4 (TLR4), heme oxygenase-1 (HO-1), nuclear factor erythroid2related factor 2 (Nrf2), and b-actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). All other reagents were of analytical grade.
2.2.
Animals
BALB/c mice (6e8-wk-old; 18e22 g) were obtained from the Center of Experimental Animals of Harbin Medical University (HeiLongjiang, China). The mice were housed in an animal room and given a standard diet. The laboratory temperature was 24 1 C, and relative humidity was 40%e80%. All animal experiments were performed according to the guidelines of the Local Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.3.
Experimental design
Sixty mice were randomly divided into five groups as follows: control group, LPS and/or GalN group, oroxylin A (15, 30, and 60 mg/kg) þ LPS and/or GalN groups. Acute liver injury was induced in BALB/c mice with intraperitoneal injection of LPS (50 mg/kg) combined with D-GalN (800 mg/kg). Oroxylin A (15, 30, and 60 mg/kg) was given with an intraperitoneal injection 1 h before LPS and/or GalN treatment. The blood and liver tissues were collected for subsequent analysis.
2.4.
Serum aminotransferase activities
Plasma samples were collected from the mice 8 h after the LPS and/or GalN injection. Serum levels of ALT and AST were
523
tested using test kits (Jiancheng Bioengineering Institute of Nanjing) according to the instructions.
2.5.
ELISA assay
TNF-a level in serum and liver tissues were detected using commercial ELISA kits purchased from R&D Systems according to the manufacturer’s instructions.
2.6.
Quantitative real-time polymerase chain reaction
The messenger RNA (mRNA) expression of TNF-a was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Briefly, total RNA from liver tissues was extracted using TRIzol (Invitrogen, Carlsbad, CA) reagent. The RNA was reverse transcribed with M-MuLV Reverse Transcriptase Kit (Fermentas, Lithuania) to obtain complementary DNA. qRTPCR was performed with SYBR green PCR Master Mix, and the relative mRNA concentrations were detected by qRT-PCR using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The sequences for primers are: TNFa (forward 50 -GCCTCCCTCTCATCAGTTCTA-30 and reverse 50 GGCAGCCTTGTCCCTTG-30 ). The comparative cycle threshold (DDCT) method of relative quantification was used to determine the fold-change of expression.
2.7.
Malondialdehyde and MPO activity assay
Liver malondialdehyde (MDA) content and MPO activity were measured by relative test kits (Jiancheng Bioengineering Institute of Nanjing) according to the instructions.
2.8.
Histopathologic examination
Liver tissues were fixed in 4% paraformaldehyde and embedded in paraffin and cut into 5-mm sections. After hematoxylin-eosin staining, histopathologic changes were observed using an optical microscope (Olympus Optical Co, Tokyo, Japan).
2.9.
Western blot analysis
Nuclear and cytoplasmic proteins were extracted from the liver tissues using a protein extract kit (Thermo) according to the manufacturer’s protocol. Protein concentrations were determined by the bicinchoninic acid protein assay kit. Equal amounts of protein were fractionated onto 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat milk for 2 h at room temperature. The membranes were incubated with specific antibodies against indicated primary antibodies at 4 C overnight followed by the peroxidase-conjugated secondary antibody at 37 C for 1 h. Protein bands were visualized using enhanced chemiluminescence reagents, and densitometric analysis was performed with the use of a PDI ImageWare System (Bio-Rad, Hercules, CA).
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Fig. 1 e Effects of oroxylin A on histopathologic changes in liver tissues. Representative histologic changes of liver obtained from mice of different groups. (A) Control group, (B) LPS and/or GalN group, (C) LPS and/or GalN D oroxylin A (15 mg/kg) group, (D) LPS and/or GalN D oroxylin A (30 mg/kg) group, and (E) LPS and/or GalN D oroxylin A (60 mg/kg) group (hematoxylin and eosin staining, magnification 3200). (Color version of figure is available online.)
2.10.
Statistical analysis
All data were expressed as means standard deviation Differences between multiple treatment groups were assessed by one-way analysis of variance followed by a Dunnett test. Results are considered significant at P < 0.05 or P < 0.01.
3.
Results
3.1. Effects of oroxylin A on LPS and/or GalNeinduced liver histopathologic changes
GalN group compared with the control group. Treatment of oroxylin A dose-dependently suppressed LPS and/or GalNeinduced serum and hepatic TNF-a levels (Fig. 3A). The expression of TNF-a in the liver tissue was also detected by qRT-PCR and Western blot analysis. The results showed that oroxylin A significantly inhibited TNF-a expression both in mRNA and protein levels (Fig. 3B).
3.4.
Effects of oroxylin A on MDA content
MDA, a marker of oxidative stress, was used to assess the antioxidant effects of oroxylin A. The results showed that
The histologic features of the control group showed normal lobular architecture and cellular structure. LPS and/or GalN treatment group showed significant pathologic changes, including hepatocytes necrosis, destruction of hepatic architecture, and inflammatory cell infiltration. Oroxylin A significantly attenuated these pathologic changes induced by LPS and/or GalN (Fig. 1).
3.2. Oroxylin A inhibits LPS and/or GalNeinduced serum ALT and AST levels The effects of oroxylin A on hepatic failure were detected by measuring serum ALT and AST levels. As shown in Figure 2, compared with the control group, serum ALT and AST levels increased significantly in the LPS and/or GalN group. However, oroxylin A inhibited LPS and/or GalNeinduced ALT and AST levels in a dose-dependent manner (Fig. 2).
3.3. Oroxylin A inhibits serum and hepatic TNF-a production To test the anti-inflammatory effects of oroxylin A, the effects of oroxylin A on LPS and/or GalNeinduced TNF-a expression were detected by ELISA. The results showed that serum and hepatic TNF-a levels increased significantly in LPS and/or
Fig. 2 e Effects of oroxylin A on serum ALT and AST levels. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
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Fig. 3 e (A) Effects of oroxylin A on serum and hepatic TNF-a levels detected by ELISA. (B) Effects of oroxylin A on hepatic TNF-a level detected by qRT-PCR and Western blot analysis. The values presented are the means ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
MDA level significantly increased after LPS and/or GalN treatment and this elevation was attenuated by oroxylin A (Fig. 4A).
3.5.
reported to have anti-inflammatory effects [15]. In this study, we investigated the protective effects of oroxylin A on LPS and/or GalNeinduced acute hepatic injury in mice. Our results showed that oroxylin A inhibited serum ALT and AST levels.
Effects of oroxylin A on MPO activity
MPO activity was used to assess the neutrophil accumulation within liver tissues. As shown in Figure 4B, compared with the control group, MPO activity increased significantly in the LPS and/or GalN group. However, this increase was attenuated by oroxylin A in a dose-dependent manner.
3.6. Effect of oroxylin A on TLR4 expression and NF-kB activation TLR4 and NF-kB have been reported to play critical roles in the regulating of inflammatory cytokines production. Thus, the effects of oroxylin A on TLR4 expression and NF-kB activation were determined. The results showed that oroxylin A inhibited LPS and/or GalNeinduced TLR4 expression and NF-kB activation in a dose-dependent manner (Fig. 5).
3.7.
Effects of oroxylin A on Nrf2 and HO-1 expression
The effects of oroxylin A on Nrf2 and HO-1 expression were detected by Western blotting. The results showed that nuclear translocation of Nrf2 and HO-1 expressions were upregulated by LPS and/or GalN. These increases in Nrf2 and HO-1 expression were augmented by oroxylin A (Fig. 6).
4.
Discussion
Fulminant hepatic failure is a life-threatening clinical syndrome associated with a high rate of mortality [14]. Oroxylin A, a natural flavonoid isolated from S baicalensis, has been
Fig. 4 e Effects of oroxylin A on liver MDA level and MPO activity. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS and/or GalN group.
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Fig. 5 e Oroxylin A inhibited LPS and/or GalNeinduced TLR4 expression and NF-kB activation with Western blotting. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05 and **P < 0.01 group versus LPS group.
Meanwhile, histologic analysis demonstrated that oroxylin A had a protective effect on LPS and/or GalNeinduced acute hepatic injury. Oroxylin A may be a potential therapeutic reagent to attenuate hepatic injury.
TLR4, one of the best characterized TLRs, recognizes LPS from gram-negative bacteria [16]. LPS induces TLR4 expression and NF-kB activation, which subsequently leads to inflammatory cytokines production [17,18]. Studies showed that
Fig. 6 e Effects of oroxylin A on the expression of Nrf2 and HO-1. The values presented are the mean ± standard deviation (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 group versus LPS group.
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these inflammatory cytokines played important roles in the development of liver failure [19,20]. Among these inflammatory cytokines, TNF-a acted as the crucial mediator in hepatocytic apoptosis and acute liver failure [21]. Elevated TNF-a has been observed in patients with liver failure [22]. Thus, inhibiting of TNF-a production is likely to be therapeutically effective in patients with liver failure. In this study, we found that serum and hepatic TNF-a expression increased significantly and this increase was reduced by oroxylin A. TLR4 and NF-kB are involved in the regulation of TNF-a production [23]. Thus, we tested the effects of oroxylin A on the TLR4 expression and NF-kB activation. Our results showed that oroxylin A inhibited TNF-a production via inhibiting TLR4emediated NFkB activation. MDA, one of the most frequently used indicators of lipid peroxidation, is usually used to assess the oxidative stress of different tissues [24]. To investigate the antioxidant effects of oroxylin A on LPS and/or GalNeinduced acute liver injury, MDA content in liver tissues was detected. Our results showed that oroxylin A decreased oxidative stress induced by LPS and/ or GalN. Nrf2, a redox sensitive transcription factor, was originally known to activate cellular protective pathways against oxidative injury [25,26]. Activating of Nrf2 induces the expression of HO-1, which has been reported to have antioxidant activity [27,28]. Previous studies showed that Nrf2 can be used as an effective therapeutic target in modulating inflammatory liver diseases [29,30]. Thus, the effects of oroxylin A on Nrf2 and HO-1 expression were detected. The results showed that oroxylin A upregulated the expression of Nrf2 and HO-1. These results suggested that oroxylin A exhibited antioxidant effects by inducing oxidative defense system. This study demonstrated that oroxylin A exhibited protective effects on LPS and/or GalNeinduced liver injury. Oroxylin A protected against LPS and/or GalNeinduced liver injury through activating Nrf2 and inhibiting TLR4 signaling pathway. Therefore, oroxylin A can be used as a potential agent for preventing acute liver injury.
Acknowledgment Authors’ contributions: H.H.Y. and L.J.Y. contributed to the conception and design. H.H.Y., Z.X.Y., and L.J.Y. did the analysis and interpretation. H.H.Y. did the data collection. H.H.Y. and L.J.Y. wrote the article and did the critical revision of the article.
Disclosure The authors have no conflict of interest to declare.
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