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Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway
Author’s Accepted Manuscript Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway Jie Song, Hao-jun Fan, Hui Li, Hui Ding, Qi Lv, Shi-ke Hou www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(15)30420-9 http://dx.doi.org/10.1016/j.ejphar.2015.12.027 EJP70399
To appear in: European Journal of Pharmacology Received date: 27 November 2015 Accepted date: 11 December 2015 Cite this article as: Jie Song, Hao-jun Fan, Hui Li, Hui Ding, Qi Lv and Shi-ke Hou, Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.027 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 galley proof before it is published in its final citable 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.
Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway Jie Song1,2 , Hao-jun Fan3, Hui Li2, Hui Ding3, Qi Lv3, Shi-ke Hou3* 1 Tianjin Medical University, graduate school 2 Department of Nephrology, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF) 3 Institute for Disaster&Emergency Rescue Medicine, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF) * Corresponding authors: Shi-ke Hou, Institute for Disaster&Emergency Rescue Medicine, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF), Tianjin, 300162, China. E-mail: [email protected].
Abstract Acute kidney injury (AKI) is a serious complication of sepsis. Zingerone, a phenolic alkanone isolated from ginger, has been reported to have anti-inflammatory effect. The aim of this study was to investigate the therapeutic effects of zingerone on lipopolysaccharide (LPS)-induced AKI in mice. Zingerone was administrated 1 h after LPS challenge. The production of blood urea nitrogen (BUN) and creatinine were measured in this study. The expressions of inflammatory cytokines in serum and kidney tissues were detected by ELISA. The expressions of Toll-like receptor 4 (TLR4), MyD88, TRIF, Nuclear factor Kappa B (NF-κB) and IκB were measured by Western blotting. The results showed that zingerone suppressed LPS-induced BUN, creatinine, and inflammatory cytokines TNF-α, IL-6 and IL-1β levels in a dose-dependent manner. Zingerone also attenuated LPS-induced kidney histopathologic changes. Furthermore, zingerone was found to inhibit LPS-induced TLR4, MyD88, TRIF expression and NF-κB activation. In conclusion, the current study demonstrated that zingerone inhibited LPS-induced AKI by suppressing TLR4/NF-κB signaling pathway. Keywords: zingerone; LPS; acute kidney injury; NF-κB; TLR4 1. Introduction Sepsis is a major medical problem that often causes multiple organ failure such as kidney injury (Lowes et al., 2008). Sepsis or acute kidney injury (AKI) is often caused by Gram-negative bacterial infection (Doi et al., 2009a). LPS, the main outer membrane component of Gram-negative bacteria, has been known as the most important factor that causes AKI (Badr et al., 1986; Wang et al., 2009). LPS could activate TLR4 signaling pathway which subsequently induces NF-κB activation and inflammatory mediators production (Fitzgerald et al., 2003; Zhang and Ghosh, 2000). Kidney inflammation is characterized by the production of inflammatory mediators (Akcay et al., 2009; Grigoryev et al., 2008). Up till now, the incidence of AKI keeps rising and the mortality remains high (Ostermann et al., 2008). Therefore, the identification of novel therapeutic drugs to treat LPS-induced AKI is urgently needed. Zingerone, a phenolic alkanone isolated from ginger, has been reported to exhibit various pharmacological activities (Kim et al., 2010; Shin et al., 2005). Zingerone had preventive effects against isoproterenol-induced myocardial infarcted rats (Hemalatha and Prince, 2015). Zingerone
also had the ability to attenuate oxidative perturbations in irritable bowel disorder in rats (Banji et al., 2014). Recently, zingerone had been demonstrated to have anti-inflammatory effects (Hsiang et al., 2013). Zingerone has been reported to inhibit liver inflammation in Pseudomonas aeruginosa peritonitis mouse model. In addition, zingerone was found to inhibit LPS-induced acute lung injury in mice (Xie et al., 2014). Previous studies showed that zingerone exhibited anti-inflammatory effects by inhibiting NF-κB signaling pathway (Hsiang et al., 2015; Xie et al., 2014). In the present study, we investigate the anti-inflammatory effects and mechanism of zingerone on LPS-induced AKI in mice. 2. Materials and methods 2.1. Reagents LPS (Escherichia coli, O111:B4) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Zingerone (purity ≥ 98%) (CAS:122-48-5) were purchased from Changle Pharmaceutical Co., Ltd (Xinxiang, China). TNF-α, IL-6 and IL-1β ELISA kits were purchased from R&D Systems (Minneapolis, MN). Antibodies against NF-κB, IκBα, and β-actin were purchased from Cell Signaling Technology Inc (Beverly, MA). Antibodies against TLR4, MyD88, and TRIF were purchased from Abcam (Cambridge, UK). All other chemicals were of reagent grade. 2.2. Animal model of acute kidney injury Female C57BL/6 mice, 8-12 weeks old, were purchased from the Center of Experimental Animals of Tianjin Medical University (Tianjin, China). The mice were acclimatized for 7 days under standard laboratory conditions before the experiments. The mice were housed in an environmentally controlled room (23 ± 2℃, 40%-80% humidity) with free access to food and water. All mice received human care according to the guidelines of the Local Institutes of Health guide for the care and use of laboratory animals. Eighty-four mice were randomly divided into seven groups and each group contained twelve mice: Control group, Zingerone (40 mg/kg) group, LPS group, LPS + Zingerone (10, 20 and 40 mg/kg) groups, and LPS + VIPER group. VIPER, the TLR4-specific viral inhibitory peptide, has been shown to potentially inhibit TLR4-mediated responses and AKI induced by LPS (Lysakova-Devine et al., 2010; Nair et al., 2014). The doses of zingerone used in this study were based on previous study (Xie et al., 2014). The mice of LPS group were injected intraperitoneally (i.p.) with 10 mg/kg body weight of LPS in 100μL PBS. The mice of LPS + Zingerone (10, 20 and 40 mg/kg) groups were given intraperitoneally (i.p.) of 10, 20 and 40 mg/kg zingerone 1 h after LPS challenge. The mice of LPS+VIPER group were received VIPER (0.1 mg/kg iv) 2 h prior to the LPS injection. The mice of control group were injected with equal amount of PBS. The mice were euthanized and the blood and kidney tissues were collected 24 h after LPS treatment. 2.3. Histological analysis The kidney tissues were harvested 24 h after LPS treatment. The tissues were fixed in 10% formaldehyde, embedded in paraffin, and cut into 5μm sections. The sections were stained with hematoxylin and eosin (H&E). The histological assessments were conducted by two pathologists who were blinded to the treatment. The histological changes in the kidney were scored as previously described. The percentages of tubules that displayed cellular necrosis, loss of brush border, interstitial edema, and tubule dilatation were scored as follows: 0=none, 1=0-20%,
2=20%-50%, 3=50%-70%, 4=more than 70%. 2.4. Measurement of serum creatinine and BUN The levels of serum creatinine and BUN were detected using an AutoAnalyzer (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. 2.5. TNF-α, IL-6, and IL-1β assay The kidney tissues were weighted and homogenized with PBS (1:9, w/v) on ice and centrifuged at 2,000g for 40 min at ZinZin gerone4 ℃. Then lipid was removed and the supernatant was collected. The expressions of TNF-α, IL-6, and IL-1β in the supernatant and serum were measured by using ELISA kits R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. 2.6. Western blot analysis Nuclear and cytoplasmic proteins from kidney tissues were extracted using detecting kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Protein concentration was determined by the Bradford assay (Biorad, Hercules, CA). Equal amount of protein (40μg) were separated by 12% SDS-PAGE and transferred to 0.45μm polyvinylidene fluoride membranes. The membranes were blocked for 2 h with 5% nonfat dry milk at room temperature. Then, the membranes were incubated with a 1:1000 dilution of primary antibody against TLR4, MyD88, TRIF, Nrf2, NF-κB p65, p-IκBα, IκBα, and β-actin overnight at 4 °C. After washing three times, the membranes were probed with the secondary antibody at room temperature for 1 h. Blots were then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK). Band intensities were quantified using UN-SCAN-IT gel analysis software. 2.7. Statistical analysis All values were calculated as means ± S.E.M. Differences within groups were evaluated by one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significantly. 3. Results 3.1. Effects of zingerone on LPS-mediated kidney histopathologic changes Kidney histological changes were detected 24 h after LPS treatment. As shown in Fig. 1, kidney tissues of normal control group and zingerone alone group showed normal kidney histology (Fig. 1A, B). Kidney tissues of LPS group showed tubular epithelial cells sloughing, tubular dilation, and distortion (as shown by the arrows) (Fig. 1C).Treatment of zingerone (10, 20 and 40 mg/kg) or VIPER markedly reduced LPS-induced kidney injury (Fig. 1D, E, F, G). 3.2. Effects of zingerone on LPS-induced renal function To investigate the effects of zingerone on LPS-induced renal function, serum BUN and creatinine levels were measured in this study. As shown in Fig. 2, LPS significantly increased serum BUN and creatinine levels in comparison to control group and zingerone alone group. However, zingerone or VIPER treatment apparently inhibited LPS-induced serum BUN and creatinine production. 3.3. Zingerone inhibits LPS-induced TNF-α, IL-6 and IL-1β production
The effects of zingerone on LPS-induced TNF-α, IL-6 and IL-1β levels were detected by ELISA. As shown in Fig. 3, LPS significantly increased the production of TNF-α, IL-6 and IL-1β both in serum and kidney tissues. However, zingerone dose-dependently inhibited LPS-induced TNF-α, IL-6 and IL-1β production (Fig. 3). VIPER significantly inhibited LPS-induced TNF-α, IL-6 and IL-1β production (Fig. 3) 3.4. Effects of zingerone on LPS-induced NF-κB activation NF-κB has been reported to play important roles in the regulation of inflammatory cytokines production. Thus, the effects of zingerone on LPS-induced NF-κB activation were detected in this study. The results showed that LPS induced NF-κB activation and IκBα degradation. However, treatment of zingerone or VIPER significantly inhibited LPS-induced NF-κB activation and IκBα degradation (Fig. 4). 3.5. Effects of zingerone on LPS-induced TLR4, MyD88, TRIF, and Nrf2 expression TLR4 is the main receptor of LPS. To investigate the anti-inflammatory mechanism of zingerone, the effects of zingerone on LPS-induced TLR4 expression were detected. Our results showed that LPS significantly up-regulated the expression of TLR4, MyD88, and TRIF. However, treatment of zingerone dose-dependently inhibited LPS-induced TLR4, MyD88, and TRIF expression (Fig. 5). Nrf2 was up-regulated by treatment of LPS and treatment of zingerone did not have any effect on Nrf2 expression (Fig. 5).
4. Discussion Zingerone, a phenolic alkanone isolated from ginger, has been reported to have anti-inflammatory effect (Xie et al., 2014). In this study, we investigated the protective effects of zingerone on LPS-induced AKI in mice. The results showed that zingerone inhibited serum BUN and creatinine levels. Meanwhile, histological analysis demonstrated that zingerone had a protective effect on LPS-induced AKI. An increasing body of evidences demonstrated that inflammatory cytokines played vital roles in the pathogenesis of acute kidney injury (Okusa, 2002; Wan et al., 2003). TNF-α is the key mediator of sepsis. It can induce kidney injury by activating TNF receptor (Cunningham et al., 2002). IL-1ß plays a primary role in the inflammatory response by inducing fever and increasing the synthesis of acute-phase proteins (Lachmann et al., 2011). IL-6 also induces the synthesis of acute-phase proteins and stimulates the mesangial proliferation in kidney (Horii et al., 1989). Patients with sepsis or kidney injury showed increased levels of inflammatory cytokines, such as TNF-α, IL-1ß and IL-6, in blood (Chawla et al., 2007; Donnahoo et al., 1999). These inflammatory cytokines initiated and amplified the inflammatory responses and led to the pathogenesis of acute kidney injury (Bonventre and Weinberg, 2003). LPS is the major endotoxin produced by Gram-negative bacteria (Ulevitch and Tobias, 1999). The levels of TNF-α, IL-1ß and IL-6 in serum and kidney tissues increased significantly in LPS-induced AKI in mice (Doi et al., 2009b). Previous studies showed that suppression of these cytokines production could attenuate LPS-induced AKI (Wang et al., 2009; Zager et al., 2006). In the present study, our results showed that zingerone at the dose of 10mg/kg, 20mg/kg and 40 mg/kg significantly inhibited LPS-induced TNF-α, IL-1ß and IL-6 levels both in serum and kidney tissues, suggesting zingerone could attenuate LPS-induced pathogenesis of acute kidney injury. In addition, histological analysis showed that zingerone or VIPER could attenuate LPS-induced kidney injury. Taken together, these
results suggested that zingerone had protective effects against LPS-induced AKI. It is well-known that TLR4 is the main receptor for LPS, the outer membrane component of Gram-negative bacteria (Takeuchi et al., 1999). Studies showed that TLR4 signaling pathway was a major contributor of inflammation in the kidney (Cunningham et al., 2004). The activation of TLR4 by LPS propagates the initial injury through the inducing of inflammatory cytokines production (Chase et al., 2007). NF-κB is a vital transcription factor that regulates the expression of inflammatory cytokines (Blackwell and Christman, 1997). Activation of TLR4 by LPS leads to the activation of NF-κB, which subsequently induces the production of inflammatory cytokines TNF-α, IL-1ß and IL-6 (Covert et al., 2005). In normal conditions, NF-κB is sequestered in the cytoplasm. Once stimulating by LPS, NF-κB p65 translocates into the nucleus to regulate inflammatory cytokines gene transcription (Kim et al., 2015; Sun et al., 2015). Previous studies showed that treatment of zingerone inhibited LPS-induced NF-κB activation in LPS-induced acute lung injury (Xie et al., 2014). This was consistent with our results. Recent studies showed that TLR4 could be a potential therapeutic target for AKI (Leon et al., 2008). To investigate the anti-inflammatory mechanism of zingerone, the effects of zingerone on TLR4 signaling pathway were detected. Our results showed that zingerone at the dose of 10 mg/kg could inhibit LPS-induced TLR4 expression and the inhibition was weak. Zingerone or VIPER significantly inhibited LPS-induced NF-κB activation. These results in the zingerone treated group were consistent with those in the VIPER treated group. These results suggested that the anti-inflammatory effect of zingerone was through inhibiting TLR4-mediated NF-κB activation. In conclusion, our results demonstrated that zingerone had protective effects against LPS-induced AKI. The promising anti-inflammatory mechanism of zingerone is associated with inhibiting TLR4-mediated NF-κB activation and LPS-induced inflammatory response. Acknowledgments This study was supported by Foundation of Key Laboratory Emegerncy and Disaster Medicine in Chinese People's Liberation Army (Item no: JY1404). Conflict of interest
of
The authors have no conflict of interest to declare. References Akcay, A., Nguyen, Q., Edelstein, C.L., 2009. Mediators of inflammation in acute kidney injury. Mediators of inflammation 2009, 137072. Badr, K.F., Kelley, V.E., Rennke, H.G., Brenner, B.M., 1986. Roles for thromboxane A2 and leukotrienes in endotoxin-induced acute renal failure. Kidney Int 30, 474-480. Banji, D., Banji, O.J., Pavani, B., Kranthi Kumar, C., Annamalai, A.R., 2014. Zingerone regulates intestinal transit, attenuates behavioral and oxidative perturbations in irritable bowel disorder in rats. Phytomedicine 21, 423-429. Blackwell, T.S., Christman, J.W., 1997. The role of nuclear factor-kappa B in cytokine gene regulation. American journal of respiratory cell and molecular biology 17, 3-9. Bonventre, J.V., Weinberg, J.M., 2003. Recent advances in the pathophysiology of ischemic acute renal failure. Journal of the American Society of Nephrology : JASN 14, 2199-2210. Chase, M.A., Wheeler, D.S., Lierl, K.M., Hughes, V.S., Wong, H.R., Page, K., 2007. Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappa B-Dependent mechanism. Journal of immunology 179, 6318-6324.
Chawla, L.S., Seneff, M.G., Nelson, D.R., Williams, M., Levy, H., Kimmel, P.L., Macias, W.L., 2007. Elevated plasma concentrations of IL-6 and elevated APACHE II score predict acute kidney injury in patients with severe sepsis. Clinical journal of the American Society of Nephrology : CJASN 2, 22-30. Covert, M.W., Leung, T.H., Gaston, J.E., Baltimore, D., 2005. Achieving stability of lipopolysaccharide-induced NF-kappaB activation. Science 309, 1854-1857. Cunningham, P.N., Dyanov, H.M., Park, P., Wang, J., Newell, K.A., Quigg, R.J., 2002. Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney. Journal of immunology 168, 5817-5823. Cunningham, P.N., Wang, Y., Guo, R., He, G., Quigg, R.J., 2004. Role of Toll-like receptor 4 in endotoxin-induced acute renal failure. Journal of immunology 172, 2629-2635. Doi, K., Leelahavanichkul, A., Yuen, P.S., Star, R.A., 2009a. Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119, 2868-2878. Doi, K., Leelahavanichkul, A., Yuen, P.S.T., Star, R.A., 2009b. Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119, 2868-2878. Donnahoo, K.K., Meng, X., Ayala, A., Cain, M.P., Harken, A.H., Meldrum, D.R., 1999. Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion. The American journal of physiology 277, R922-929. Fitzgerald, K.A., Rowe, D.C., Barnes, B.J., Caffrey, D.R., Visintin, A., Latz, E., Monks, B., Pitha, P.M., Golenbock, D.T., 2003. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 198, 1043-1055. Grigoryev, D.N., Liu, M.C., Hassoun, H.T., Cheadle, C., Barnes, K.C., Rabb, H., 2008. The local and systemic inflammatory transcriptome after acute kidney injury. Journal of the American Society of Nephrology 19, 547-558. Hemalatha, K.L., Prince, P.S., 2015. Preventive effects of zingerone on altered lipid peroxides and nonenzymatic antioxidants in the circulation of isoproterenol-induced myocardial infarcted rats. J Biochem Mol Toxicol 29, 63-69. Horii, Y., Muraguchi, A., Iwano, M., Matsuda, T., Hirayama, T., Yamada, H., Fujii, Y., Dohi, K., Ishikawa, H., Ohmoto, Y., et al., 1989. Involvement of IL-6 in mesangial proliferative glomerulonephritis. Journal of immunology 143, 3949-3955. Hsiang, C.Y., Cheng, H.M., Lo, H.Y., Li, C.C., Chou, P.C., Lee, Y.C., Ho, T.Y., 2015. Ginger and Zingerone Ameliorate Lipopolysaccharide-Induced Acute Systemic Inflammation in Mice, Assessed by Nuclear Factor-kappaB Bioluminescent Imaging. J Agric Food Chem 63, 6051-6058. Hsiang, C.Y., Lo, H.Y., Huang, H.C., Li, C.C., Wu, S.L., Ho, T.Y., 2013. Ginger extract and zingerone ameliorated trinitrobenzene sulphonic acid-induced colitis in mice via modulation of nuclear factor-kappaB activity and interleukin-1beta signalling pathway. Food chemistry 136, 170-177. Kim, M.K., Chung, S.W., Kim, D.H., Kim, J.M., Lee, E.K., Kim, J.Y., Ha, Y.M., Kim, Y.H., No, J.K., Chung, H.S., Park, K.Y., Rhee, S.H., Choi, J.S., Yu, B.P., Yokozawa, T., Kim, Y.J., Chung, H.Y., 2010. Modulation of age-related NF-kappaB activation by dietary zingerone via MAPK pathway. Experimental gerontology 45, 419-426. Kim, S.R., Ha, Y.M., Kim, Y.M., Park, E.J., Kim, J.W., Park, S.W., Kim, H.J., Chung, H.T., Chang, K.C., 2015. Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals. Biochemical pharmacology 95, 279-289. Lachmann, H.J., Quartier, P., So, A., Hawkins, P.N., 2011. The Emerging Role of Interleukin-1 beta in
Autoinflammatory Diseases. Arthritis and rheumatism 63, 314-324. Leon, C.G., Tory, R., Jia, J., Sivak, O., Wasan, K.M., 2008. Discovery and development of toll-like receptor 4 (TLR4) antagonists: a new paradigm for treating sepsis and other diseases. Pharmaceutical research 25, 1751-1761. Lowes, D.A., Thottakam, B.M., Webster, N.R., Murphy, M.P., Galley, H.F., 2008. The mitochondria-targeted
antioxidant
MitoQ
protects
against
organ
damage
in
a
lipopolysaccharide-peptidoglycan model of sepsis. Free radical biology & medicine 45, 1559-1565. Lysakova-Devine, T., Keogh, B., Harrington, B., Nagpal, K., Halle, A., Golenbock, D.T., Monie, T., Bowie, A.G., 2010. Viral inhibitory peptide of TLR4, a peptide derived from vaccinia protein A46, specifically inhibits TLR4 by directly targeting MyD88 adaptor-like and TRIF-related adaptor molecule. Journal of immunology 185, 4261-4271. Nair, A.R., Masson, G.S., Ebenezer, P.J., Del Piero, F., Francis, J., 2014. Role of TLR4 in lipopolysaccharide-induced acute kidney injury: protection by blueberry. Free radical biology & medicine 71, 16-25. Okusa, M.D., 2002. The inflammatory cascade in acute ischemic renal failure. Nephron 90, 133-138. Ostermann, M., Chang, R., Grp, R.I.P.U., 2008. Correlation between the AKI classification and outcome. Crit Care 12. Shin, S.G., Kim, J.Y., Chung, H.Y., Jeong, J.C., 2005. Zingerone as an antioxidant against peroxynitrite. J Agr Food Chem 53, 7617-7622. Sun, Y., Zhao, Y., Yao, J., Zhao, L., Wu, Z., Wang, Y., Pan, D., Miao, H., Guo, Q., Lu, N., 2015. Wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting NF-kappaB and NLRP3 inflammasome activation. Biochemical pharmacology 94, 142-154. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., Akira, S., 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443-451. Ulevitch, R.J., Tobias, P.S., 1999. Recognition of gram-negative bacteria and endotoxin by the innate immune system. Current opinion in immunology 11, 19-22. Wan, L., Bellomo, R., Di Giantomasso, D., Ronco, C., 2003. The pathogenesis of septic acute renal failure. Current opinion in critical care 9, 496-502. Wang, W., Bansal, S., Falk, S., Ljubanovic, D., Schrier, R., 2009. Ghrelin protects mice against endotoxemia-induced acute kidney injury. American journal of physiology. Renal physiology 297, F1032-1037. Xie, X., Sun, S., Zhong, W., Soromou, L.W., Zhou, X., Wei, M., Ren, Y., Ding, Y., 2014. Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice. International immunopharmacology 19, 103-109. Zager, R.A., Johnson, A.C., Lund, S., Hanson, S.Y., Abrass, C.K., 2006. Levosimendan protects against experimental endotoxemic acute renal failure. Am J Physiol-Renal 290, F1453-F1462. Zhang, G.L., Ghosh, S., 2000. Molecular mechanisms of NF-kappa B activation induced by bacterial lipopolysaccharide through Toll-like receptors. Journal of Endotoxin Research 6, 453-457.
Figure Legend Fig. 1 Effects of zingerone on histopathological changes in kidney tissues in LPS-induced AKI mice. Representative histological changes of kidney obtained from mice of different groups. A: Control group, B: zingerone alone group, C: LPS group, D: LPS+ zingerone (10mg/kg) group, E:
LPS+ zingerone (20 mg/kg) group, F: LPS + zingerone (40 mg/kg) group, G: LPS + VIPER group (Hematoxylin and eosin staining, magnification 200×). Fig. 2 Effects of zingerone or VIPER on BUN and creatinine levels in serum. 24 h after LPS treatment, the serum were collected. The levels of serum creatinine and BUN were detected. The values presented are the mean ± S.E.M. (n=12 in each group). P#<0.01 vs. control group, P*<0.05, P**<0.01 vs. LPS group. Fig. 3 Effects of zingerone or VIPER on LPS-induced TNF-α, IL-6 and IL-1β in serum and kidney tissues. 24 h after LPS treatment, the serum and kidney tissues were collected. The levels of TNF-α, IL-6 and IL-1β in serum and kidney tissues were detected. The values presented are mean ± S.E.M. (n=12 in each group). P#<0.01 vs. control group, P*<0.05, P**<0.01 vs. LPS group. Fig. 4 Zingerone and VIPER inhibit LPS-induced NF-κB activation and IκBα degradation. 24 h after LPS treatment, the kidney tissues were collected. The expression of NF-κB and IkBα were detected. The values presented are the means ± S.E.M. (n=12 in each group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS group. Fig. 5 Effects of zingerone on LPS-induced TLR4, MyD88, TRIF, and Nrf2 expression. 24 h after LPS treatment, the kidney tissues were collected. The expression of TLR4, MyD88, TRIF, and Nrf2 were detected. The values presented are the means ± S.E.M. (n=12 in each group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS group.
PII: DOI: Reference:
S0014-2999(15)30420-9 http://dx.doi.org/10.1016/j.ejphar.2015.12.027 EJP70399
To appear in: European Journal of Pharmacology Received date: 27 November 2015 Accepted date: 11 December 2015 Cite this article as: Jie Song, Hao-jun Fan, Hui Li, Hui Ding, Qi Lv and Shi-ke Hou, Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.12.027 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 galley proof before it is published in its final citable 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.
Zingerone ameliorates lipopolysaccharide-induced acute kidney injury by inhibiting Toll-like receptor 4 signaling pathway Jie Song1,2 , Hao-jun Fan3, Hui Li2, Hui Ding3, Qi Lv3, Shi-ke Hou3* 1 Tianjin Medical University, graduate school 2 Department of Nephrology, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF) 3 Institute for Disaster&Emergency Rescue Medicine, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF) * Corresponding authors: Shi-ke Hou, Institute for Disaster&Emergency Rescue Medicine, Affiliated Hospital of Logistic University of Chinese People’s Armed Police Force (PAPF), Tianjin, 300162, China. E-mail: [email protected].
Abstract Acute kidney injury (AKI) is a serious complication of sepsis. Zingerone, a phenolic alkanone isolated from ginger, has been reported to have anti-inflammatory effect. The aim of this study was to investigate the therapeutic effects of zingerone on lipopolysaccharide (LPS)-induced AKI in mice. Zingerone was administrated 1 h after LPS challenge. The production of blood urea nitrogen (BUN) and creatinine were measured in this study. The expressions of inflammatory cytokines in serum and kidney tissues were detected by ELISA. The expressions of Toll-like receptor 4 (TLR4), MyD88, TRIF, Nuclear factor Kappa B (NF-κB) and IκB were measured by Western blotting. The results showed that zingerone suppressed LPS-induced BUN, creatinine, and inflammatory cytokines TNF-α, IL-6 and IL-1β levels in a dose-dependent manner. Zingerone also attenuated LPS-induced kidney histopathologic changes. Furthermore, zingerone was found to inhibit LPS-induced TLR4, MyD88, TRIF expression and NF-κB activation. In conclusion, the current study demonstrated that zingerone inhibited LPS-induced AKI by suppressing TLR4/NF-κB signaling pathway. Keywords: zingerone; LPS; acute kidney injury; NF-κB; TLR4 1. Introduction Sepsis is a major medical problem that often causes multiple organ failure such as kidney injury (Lowes et al., 2008). Sepsis or acute kidney injury (AKI) is often caused by Gram-negative bacterial infection (Doi et al., 2009a). LPS, the main outer membrane component of Gram-negative bacteria, has been known as the most important factor that causes AKI (Badr et al., 1986; Wang et al., 2009). LPS could activate TLR4 signaling pathway which subsequently induces NF-κB activation and inflammatory mediators production (Fitzgerald et al., 2003; Zhang and Ghosh, 2000). Kidney inflammation is characterized by the production of inflammatory mediators (Akcay et al., 2009; Grigoryev et al., 2008). Up till now, the incidence of AKI keeps rising and the mortality remains high (Ostermann et al., 2008). Therefore, the identification of novel therapeutic drugs to treat LPS-induced AKI is urgently needed. Zingerone, a phenolic alkanone isolated from ginger, has been reported to exhibit various pharmacological activities (Kim et al., 2010; Shin et al., 2005). Zingerone had preventive effects against isoproterenol-induced myocardial infarcted rats (Hemalatha and Prince, 2015). Zingerone
also had the ability to attenuate oxidative perturbations in irritable bowel disorder in rats (Banji et al., 2014). Recently, zingerone had been demonstrated to have anti-inflammatory effects (Hsiang et al., 2013). Zingerone has been reported to inhibit liver inflammation in Pseudomonas aeruginosa peritonitis mouse model. In addition, zingerone was found to inhibit LPS-induced acute lung injury in mice (Xie et al., 2014). Previous studies showed that zingerone exhibited anti-inflammatory effects by inhibiting NF-κB signaling pathway (Hsiang et al., 2015; Xie et al., 2014). In the present study, we investigate the anti-inflammatory effects and mechanism of zingerone on LPS-induced AKI in mice. 2. Materials and methods 2.1. Reagents LPS (Escherichia coli, O111:B4) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Zingerone (purity ≥ 98%) (CAS:122-48-5) were purchased from Changle Pharmaceutical Co., Ltd (Xinxiang, China). TNF-α, IL-6 and IL-1β ELISA kits were purchased from R&D Systems (Minneapolis, MN). Antibodies against NF-κB, IκBα, and β-actin were purchased from Cell Signaling Technology Inc (Beverly, MA). Antibodies against TLR4, MyD88, and TRIF were purchased from Abcam (Cambridge, UK). All other chemicals were of reagent grade. 2.2. Animal model of acute kidney injury Female C57BL/6 mice, 8-12 weeks old, were purchased from the Center of Experimental Animals of Tianjin Medical University (Tianjin, China). The mice were acclimatized for 7 days under standard laboratory conditions before the experiments. The mice were housed in an environmentally controlled room (23 ± 2℃, 40%-80% humidity) with free access to food and water. All mice received human care according to the guidelines of the Local Institutes of Health guide for the care and use of laboratory animals. Eighty-four mice were randomly divided into seven groups and each group contained twelve mice: Control group, Zingerone (40 mg/kg) group, LPS group, LPS + Zingerone (10, 20 and 40 mg/kg) groups, and LPS + VIPER group. VIPER, the TLR4-specific viral inhibitory peptide, has been shown to potentially inhibit TLR4-mediated responses and AKI induced by LPS (Lysakova-Devine et al., 2010; Nair et al., 2014). The doses of zingerone used in this study were based on previous study (Xie et al., 2014). The mice of LPS group were injected intraperitoneally (i.p.) with 10 mg/kg body weight of LPS in 100μL PBS. The mice of LPS + Zingerone (10, 20 and 40 mg/kg) groups were given intraperitoneally (i.p.) of 10, 20 and 40 mg/kg zingerone 1 h after LPS challenge. The mice of LPS+VIPER group were received VIPER (0.1 mg/kg iv) 2 h prior to the LPS injection. The mice of control group were injected with equal amount of PBS. The mice were euthanized and the blood and kidney tissues were collected 24 h after LPS treatment. 2.3. Histological analysis The kidney tissues were harvested 24 h after LPS treatment. The tissues were fixed in 10% formaldehyde, embedded in paraffin, and cut into 5μm sections. The sections were stained with hematoxylin and eosin (H&E). The histological assessments were conducted by two pathologists who were blinded to the treatment. The histological changes in the kidney were scored as previously described. The percentages of tubules that displayed cellular necrosis, loss of brush border, interstitial edema, and tubule dilatation were scored as follows: 0=none, 1=0-20%,
2=20%-50%, 3=50%-70%, 4=more than 70%. 2.4. Measurement of serum creatinine and BUN The levels of serum creatinine and BUN were detected using an AutoAnalyzer (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. 2.5. TNF-α, IL-6, and IL-1β assay The kidney tissues were weighted and homogenized with PBS (1:9, w/v) on ice and centrifuged at 2,000g for 40 min at ZinZin gerone4 ℃. Then lipid was removed and the supernatant was collected. The expressions of TNF-α, IL-6, and IL-1β in the supernatant and serum were measured by using ELISA kits R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. 2.6. Western blot analysis Nuclear and cytoplasmic proteins from kidney tissues were extracted using detecting kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. Protein concentration was determined by the Bradford assay (Biorad, Hercules, CA). Equal amount of protein (40μg) were separated by 12% SDS-PAGE and transferred to 0.45μm polyvinylidene fluoride membranes. The membranes were blocked for 2 h with 5% nonfat dry milk at room temperature. Then, the membranes were incubated with a 1:1000 dilution of primary antibody against TLR4, MyD88, TRIF, Nrf2, NF-κB p65, p-IκBα, IκBα, and β-actin overnight at 4 °C. After washing three times, the membranes were probed with the secondary antibody at room temperature for 1 h. Blots were then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK). Band intensities were quantified using UN-SCAN-IT gel analysis software. 2.7. Statistical analysis All values were calculated as means ± S.E.M. Differences within groups were evaluated by one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significantly. 3. Results 3.1. Effects of zingerone on LPS-mediated kidney histopathologic changes Kidney histological changes were detected 24 h after LPS treatment. As shown in Fig. 1, kidney tissues of normal control group and zingerone alone group showed normal kidney histology (Fig. 1A, B). Kidney tissues of LPS group showed tubular epithelial cells sloughing, tubular dilation, and distortion (as shown by the arrows) (Fig. 1C).Treatment of zingerone (10, 20 and 40 mg/kg) or VIPER markedly reduced LPS-induced kidney injury (Fig. 1D, E, F, G). 3.2. Effects of zingerone on LPS-induced renal function To investigate the effects of zingerone on LPS-induced renal function, serum BUN and creatinine levels were measured in this study. As shown in Fig. 2, LPS significantly increased serum BUN and creatinine levels in comparison to control group and zingerone alone group. However, zingerone or VIPER treatment apparently inhibited LPS-induced serum BUN and creatinine production. 3.3. Zingerone inhibits LPS-induced TNF-α, IL-6 and IL-1β production
The effects of zingerone on LPS-induced TNF-α, IL-6 and IL-1β levels were detected by ELISA. As shown in Fig. 3, LPS significantly increased the production of TNF-α, IL-6 and IL-1β both in serum and kidney tissues. However, zingerone dose-dependently inhibited LPS-induced TNF-α, IL-6 and IL-1β production (Fig. 3). VIPER significantly inhibited LPS-induced TNF-α, IL-6 and IL-1β production (Fig. 3) 3.4. Effects of zingerone on LPS-induced NF-κB activation NF-κB has been reported to play important roles in the regulation of inflammatory cytokines production. Thus, the effects of zingerone on LPS-induced NF-κB activation were detected in this study. The results showed that LPS induced NF-κB activation and IκBα degradation. However, treatment of zingerone or VIPER significantly inhibited LPS-induced NF-κB activation and IκBα degradation (Fig. 4). 3.5. Effects of zingerone on LPS-induced TLR4, MyD88, TRIF, and Nrf2 expression TLR4 is the main receptor of LPS. To investigate the anti-inflammatory mechanism of zingerone, the effects of zingerone on LPS-induced TLR4 expression were detected. Our results showed that LPS significantly up-regulated the expression of TLR4, MyD88, and TRIF. However, treatment of zingerone dose-dependently inhibited LPS-induced TLR4, MyD88, and TRIF expression (Fig. 5). Nrf2 was up-regulated by treatment of LPS and treatment of zingerone did not have any effect on Nrf2 expression (Fig. 5).
4. Discussion Zingerone, a phenolic alkanone isolated from ginger, has been reported to have anti-inflammatory effect (Xie et al., 2014). In this study, we investigated the protective effects of zingerone on LPS-induced AKI in mice. The results showed that zingerone inhibited serum BUN and creatinine levels. Meanwhile, histological analysis demonstrated that zingerone had a protective effect on LPS-induced AKI. An increasing body of evidences demonstrated that inflammatory cytokines played vital roles in the pathogenesis of acute kidney injury (Okusa, 2002; Wan et al., 2003). TNF-α is the key mediator of sepsis. It can induce kidney injury by activating TNF receptor (Cunningham et al., 2002). IL-1ß plays a primary role in the inflammatory response by inducing fever and increasing the synthesis of acute-phase proteins (Lachmann et al., 2011). IL-6 also induces the synthesis of acute-phase proteins and stimulates the mesangial proliferation in kidney (Horii et al., 1989). Patients with sepsis or kidney injury showed increased levels of inflammatory cytokines, such as TNF-α, IL-1ß and IL-6, in blood (Chawla et al., 2007; Donnahoo et al., 1999). These inflammatory cytokines initiated and amplified the inflammatory responses and led to the pathogenesis of acute kidney injury (Bonventre and Weinberg, 2003). LPS is the major endotoxin produced by Gram-negative bacteria (Ulevitch and Tobias, 1999). The levels of TNF-α, IL-1ß and IL-6 in serum and kidney tissues increased significantly in LPS-induced AKI in mice (Doi et al., 2009b). Previous studies showed that suppression of these cytokines production could attenuate LPS-induced AKI (Wang et al., 2009; Zager et al., 2006). In the present study, our results showed that zingerone at the dose of 10mg/kg, 20mg/kg and 40 mg/kg significantly inhibited LPS-induced TNF-α, IL-1ß and IL-6 levels both in serum and kidney tissues, suggesting zingerone could attenuate LPS-induced pathogenesis of acute kidney injury. In addition, histological analysis showed that zingerone or VIPER could attenuate LPS-induced kidney injury. Taken together, these
results suggested that zingerone had protective effects against LPS-induced AKI. It is well-known that TLR4 is the main receptor for LPS, the outer membrane component of Gram-negative bacteria (Takeuchi et al., 1999). Studies showed that TLR4 signaling pathway was a major contributor of inflammation in the kidney (Cunningham et al., 2004). The activation of TLR4 by LPS propagates the initial injury through the inducing of inflammatory cytokines production (Chase et al., 2007). NF-κB is a vital transcription factor that regulates the expression of inflammatory cytokines (Blackwell and Christman, 1997). Activation of TLR4 by LPS leads to the activation of NF-κB, which subsequently induces the production of inflammatory cytokines TNF-α, IL-1ß and IL-6 (Covert et al., 2005). In normal conditions, NF-κB is sequestered in the cytoplasm. Once stimulating by LPS, NF-κB p65 translocates into the nucleus to regulate inflammatory cytokines gene transcription (Kim et al., 2015; Sun et al., 2015). Previous studies showed that treatment of zingerone inhibited LPS-induced NF-κB activation in LPS-induced acute lung injury (Xie et al., 2014). This was consistent with our results. Recent studies showed that TLR4 could be a potential therapeutic target for AKI (Leon et al., 2008). To investigate the anti-inflammatory mechanism of zingerone, the effects of zingerone on TLR4 signaling pathway were detected. Our results showed that zingerone at the dose of 10 mg/kg could inhibit LPS-induced TLR4 expression and the inhibition was weak. Zingerone or VIPER significantly inhibited LPS-induced NF-κB activation. These results in the zingerone treated group were consistent with those in the VIPER treated group. These results suggested that the anti-inflammatory effect of zingerone was through inhibiting TLR4-mediated NF-κB activation. In conclusion, our results demonstrated that zingerone had protective effects against LPS-induced AKI. The promising anti-inflammatory mechanism of zingerone is associated with inhibiting TLR4-mediated NF-κB activation and LPS-induced inflammatory response. Acknowledgments This study was supported by Foundation of Key Laboratory Emegerncy and Disaster Medicine in Chinese People's Liberation Army (Item no: JY1404). Conflict of interest
of
The authors have no conflict of interest to declare. References Akcay, A., Nguyen, Q., Edelstein, C.L., 2009. Mediators of inflammation in acute kidney injury. Mediators of inflammation 2009, 137072. Badr, K.F., Kelley, V.E., Rennke, H.G., Brenner, B.M., 1986. Roles for thromboxane A2 and leukotrienes in endotoxin-induced acute renal failure. Kidney Int 30, 474-480. Banji, D., Banji, O.J., Pavani, B., Kranthi Kumar, C., Annamalai, A.R., 2014. Zingerone regulates intestinal transit, attenuates behavioral and oxidative perturbations in irritable bowel disorder in rats. Phytomedicine 21, 423-429. Blackwell, T.S., Christman, J.W., 1997. The role of nuclear factor-kappa B in cytokine gene regulation. American journal of respiratory cell and molecular biology 17, 3-9. Bonventre, J.V., Weinberg, J.M., 2003. Recent advances in the pathophysiology of ischemic acute renal failure. Journal of the American Society of Nephrology : JASN 14, 2199-2210. Chase, M.A., Wheeler, D.S., Lierl, K.M., Hughes, V.S., Wong, H.R., Page, K., 2007. Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappa B-Dependent mechanism. Journal of immunology 179, 6318-6324.
Chawla, L.S., Seneff, M.G., Nelson, D.R., Williams, M., Levy, H., Kimmel, P.L., Macias, W.L., 2007. Elevated plasma concentrations of IL-6 and elevated APACHE II score predict acute kidney injury in patients with severe sepsis. Clinical journal of the American Society of Nephrology : CJASN 2, 22-30. Covert, M.W., Leung, T.H., Gaston, J.E., Baltimore, D., 2005. Achieving stability of lipopolysaccharide-induced NF-kappaB activation. Science 309, 1854-1857. Cunningham, P.N., Dyanov, H.M., Park, P., Wang, J., Newell, K.A., Quigg, R.J., 2002. Acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney. Journal of immunology 168, 5817-5823. Cunningham, P.N., Wang, Y., Guo, R., He, G., Quigg, R.J., 2004. Role of Toll-like receptor 4 in endotoxin-induced acute renal failure. Journal of immunology 172, 2629-2635. Doi, K., Leelahavanichkul, A., Yuen, P.S., Star, R.A., 2009a. Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119, 2868-2878. Doi, K., Leelahavanichkul, A., Yuen, P.S.T., Star, R.A., 2009b. Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119, 2868-2878. Donnahoo, K.K., Meng, X., Ayala, A., Cain, M.P., Harken, A.H., Meldrum, D.R., 1999. Early kidney TNF-alpha expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion. The American journal of physiology 277, R922-929. Fitzgerald, K.A., Rowe, D.C., Barnes, B.J., Caffrey, D.R., Visintin, A., Latz, E., Monks, B., Pitha, P.M., Golenbock, D.T., 2003. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 198, 1043-1055. Grigoryev, D.N., Liu, M.C., Hassoun, H.T., Cheadle, C., Barnes, K.C., Rabb, H., 2008. The local and systemic inflammatory transcriptome after acute kidney injury. Journal of the American Society of Nephrology 19, 547-558. Hemalatha, K.L., Prince, P.S., 2015. Preventive effects of zingerone on altered lipid peroxides and nonenzymatic antioxidants in the circulation of isoproterenol-induced myocardial infarcted rats. J Biochem Mol Toxicol 29, 63-69. Horii, Y., Muraguchi, A., Iwano, M., Matsuda, T., Hirayama, T., Yamada, H., Fujii, Y., Dohi, K., Ishikawa, H., Ohmoto, Y., et al., 1989. Involvement of IL-6 in mesangial proliferative glomerulonephritis. Journal of immunology 143, 3949-3955. Hsiang, C.Y., Cheng, H.M., Lo, H.Y., Li, C.C., Chou, P.C., Lee, Y.C., Ho, T.Y., 2015. Ginger and Zingerone Ameliorate Lipopolysaccharide-Induced Acute Systemic Inflammation in Mice, Assessed by Nuclear Factor-kappaB Bioluminescent Imaging. J Agric Food Chem 63, 6051-6058. Hsiang, C.Y., Lo, H.Y., Huang, H.C., Li, C.C., Wu, S.L., Ho, T.Y., 2013. Ginger extract and zingerone ameliorated trinitrobenzene sulphonic acid-induced colitis in mice via modulation of nuclear factor-kappaB activity and interleukin-1beta signalling pathway. Food chemistry 136, 170-177. Kim, M.K., Chung, S.W., Kim, D.H., Kim, J.M., Lee, E.K., Kim, J.Y., Ha, Y.M., Kim, Y.H., No, J.K., Chung, H.S., Park, K.Y., Rhee, S.H., Choi, J.S., Yu, B.P., Yokozawa, T., Kim, Y.J., Chung, H.Y., 2010. Modulation of age-related NF-kappaB activation by dietary zingerone via MAPK pathway. Experimental gerontology 45, 419-426. Kim, S.R., Ha, Y.M., Kim, Y.M., Park, E.J., Kim, J.W., Park, S.W., Kim, H.J., Chung, H.T., Chang, K.C., 2015. Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals. Biochemical pharmacology 95, 279-289. Lachmann, H.J., Quartier, P., So, A., Hawkins, P.N., 2011. The Emerging Role of Interleukin-1 beta in
Autoinflammatory Diseases. Arthritis and rheumatism 63, 314-324. Leon, C.G., Tory, R., Jia, J., Sivak, O., Wasan, K.M., 2008. Discovery and development of toll-like receptor 4 (TLR4) antagonists: a new paradigm for treating sepsis and other diseases. Pharmaceutical research 25, 1751-1761. Lowes, D.A., Thottakam, B.M., Webster, N.R., Murphy, M.P., Galley, H.F., 2008. The mitochondria-targeted
antioxidant
MitoQ
protects
against
organ
damage
in
a
lipopolysaccharide-peptidoglycan model of sepsis. Free radical biology & medicine 45, 1559-1565. Lysakova-Devine, T., Keogh, B., Harrington, B., Nagpal, K., Halle, A., Golenbock, D.T., Monie, T., Bowie, A.G., 2010. Viral inhibitory peptide of TLR4, a peptide derived from vaccinia protein A46, specifically inhibits TLR4 by directly targeting MyD88 adaptor-like and TRIF-related adaptor molecule. Journal of immunology 185, 4261-4271. Nair, A.R., Masson, G.S., Ebenezer, P.J., Del Piero, F., Francis, J., 2014. Role of TLR4 in lipopolysaccharide-induced acute kidney injury: protection by blueberry. Free radical biology & medicine 71, 16-25. Okusa, M.D., 2002. The inflammatory cascade in acute ischemic renal failure. Nephron 90, 133-138. Ostermann, M., Chang, R., Grp, R.I.P.U., 2008. Correlation between the AKI classification and outcome. Crit Care 12. Shin, S.G., Kim, J.Y., Chung, H.Y., Jeong, J.C., 2005. Zingerone as an antioxidant against peroxynitrite. J Agr Food Chem 53, 7617-7622. Sun, Y., Zhao, Y., Yao, J., Zhao, L., Wu, Z., Wang, Y., Pan, D., Miao, H., Guo, Q., Lu, N., 2015. Wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting NF-kappaB and NLRP3 inflammasome activation. Biochemical pharmacology 94, 142-154. Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K., Akira, S., 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443-451. Ulevitch, R.J., Tobias, P.S., 1999. Recognition of gram-negative bacteria and endotoxin by the innate immune system. Current opinion in immunology 11, 19-22. Wan, L., Bellomo, R., Di Giantomasso, D., Ronco, C., 2003. The pathogenesis of septic acute renal failure. Current opinion in critical care 9, 496-502. Wang, W., Bansal, S., Falk, S., Ljubanovic, D., Schrier, R., 2009. Ghrelin protects mice against endotoxemia-induced acute kidney injury. American journal of physiology. Renal physiology 297, F1032-1037. Xie, X., Sun, S., Zhong, W., Soromou, L.W., Zhou, X., Wei, M., Ren, Y., Ding, Y., 2014. Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice. International immunopharmacology 19, 103-109. Zager, R.A., Johnson, A.C., Lund, S., Hanson, S.Y., Abrass, C.K., 2006. Levosimendan protects against experimental endotoxemic acute renal failure. Am J Physiol-Renal 290, F1453-F1462. Zhang, G.L., Ghosh, S., 2000. Molecular mechanisms of NF-kappa B activation induced by bacterial lipopolysaccharide through Toll-like receptors. Journal of Endotoxin Research 6, 453-457.
Figure Legend Fig. 1 Effects of zingerone on histopathological changes in kidney tissues in LPS-induced AKI mice. Representative histological changes of kidney obtained from mice of different groups. A: Control group, B: zingerone alone group, C: LPS group, D: LPS+ zingerone (10mg/kg) group, E:
LPS+ zingerone (20 mg/kg) group, F: LPS + zingerone (40 mg/kg) group, G: LPS + VIPER group (Hematoxylin and eosin staining, magnification 200×). Fig. 2 Effects of zingerone or VIPER on BUN and creatinine levels in serum. 24 h after LPS treatment, the serum were collected. The levels of serum creatinine and BUN were detected. The values presented are the mean ± S.E.M. (n=12 in each group). P#<0.01 vs. control group, P*<0.05, P**<0.01 vs. LPS group. Fig. 3 Effects of zingerone or VIPER on LPS-induced TNF-α, IL-6 and IL-1β in serum and kidney tissues. 24 h after LPS treatment, the serum and kidney tissues were collected. The levels of TNF-α, IL-6 and IL-1β in serum and kidney tissues were detected. The values presented are mean ± S.E.M. (n=12 in each group). P#<0.01 vs. control group, P*<0.05, P**<0.01 vs. LPS group. Fig. 4 Zingerone and VIPER inhibit LPS-induced NF-κB activation and IκBα degradation. 24 h after LPS treatment, the kidney tissues were collected. The expression of NF-κB and IkBα were detected. The values presented are the means ± S.E.M. (n=12 in each group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS group. Fig. 5 Effects of zingerone on LPS-induced TLR4, MyD88, TRIF, and Nrf2 expression. 24 h after LPS treatment, the kidney tissues were collected. The expression of TLR4, MyD88, TRIF, and Nrf2 were detected. The values presented are the means ± S.E.M. (n=12 in each group). #P<0.01 vs. control group, *P<0.05 and **P<0.01 vs. LPS group.