- Email: [email protected]
Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation pathway and NLRP3 inflammasome
Life Sciences 208 (2018) 26–32
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation pathway and NLRP3 inflammasome Bofeng Sua,1, Hong Yeb,
T
⁎,1
, Xiaohan Youa, Haizhen Nic, Xuduan Chenb, Linlin Lib
a
Department of Nephrology, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou 325000, Zhejiang Province, PR China Department of Nephrology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, PR China c Department of Vascular Surgery, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou 325000, Zhejiang Province, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Icariin Lupus nephritis NF-κB Inflammasome Mice
Aims: Lupus nephritis (LN) is a kidney inflammatory disease caused by systemic lupus erythematosus (SLE). Both NF-κB activation and NLRP3 inflammasome activation are implicated in LN pathogenesis, suggesting they are potential targets for LN treatment. Icariin, which is isolated from Chinese medicine Horny Goat Weed (Ying Yang Huo), has been shown to have anti-inflammation activity, and inhibit activations of both NF-κB and NLRP3 inflammasome. In present study, the effects of icariin on LN were evaluated in MRL/lpr mice. Main methods: We treated MRL/lpr mice with icariin for 8 weeks and then analyzed the renal function and kidney pathology. We monitored the levels of anti-dsDNA antibody and the deposition of immune complex after icariin treatment. We also detected the macrophage infiltration, NF-κB activation, NLRP3 inflammasome activation and inflammatory cytokine TNF-α production in MRL/lpr mice after icariin treatment. Key findings: We found that MRL/lpr mice treated with icariin displayed significantly attenuated the renal disease. Icariin-treated mice showed significantly reduced serum anti-dsDNA antibody level and immune complex deposition. Icariin inhibited NF-κB activation and TNF-α production in MRL/lpr mice. Icariin inhibited CCL2 production and macrophage infiltration in MRL/lpr mice. Finally, icariin suppressed NLRP3 inflammasome activation and IL-1β production in MRL/lpr mice. Significance: Icariin alleviated murine lupus nephritis via inhibiting NF-κB activation and NLRP3 inflammasome activation.
1. Introduction Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease characterized by autoantibodies production, immune complex deposition, leukocyte infiltration, complement activation and inflammatory cell-mediated tissue damage [1]. Lupus nephritis (LN), also known as SLE nephritis, is an inflammation of the kidneys caused by SLE. LN is one of the most serious complications of SLE, and a leading cause of morbidity and mortality in SLE patients. Although immunotherapy has improved the prognosis of SLE patients with renal disease considerably, a large proportion of patients with LN eventually progress to end-stage renal disease [2]. Therefore, elucidating the underlying mechanisms of LN and developing drugs to aim at corresponding targets for LN therapy are in urgent need [3]. It has been reported that nuclear factor-kappa B (NF-κB) signaling
pathway is involved in LN pathogenesis [4]. NF-κB is a key regulator of the expression of numerous proteins involved in inflammation [5]. In steady state, NF-κB is sequestered by its inhibitor of NF-κB (IκB) in cytosol. Upon activation, IκB is phosphorylated by the IκB kinase (IKK) complex and then for degradation, which leads the nuclear translocation of NF-κB and initiation of downstream targets gene transcription including Tumor necrosis factor alpha (TNF-α), interlukine-6 (IL-6) [6]. Patients with LN have upregulated expression and activation of NF-κB in glomerular endothelial and mesangial cells, together with elevated inflammatory cytokines. The activation of NF-κB is well correlated with the LN severity, suggesting the essential role of NF-κB activation in LN pathogenesis [7]. Inhibitions of NF-κB activation have been shown to attenuate LN pathologies [3]. NLRP3 inflammasome has also been implicated in LN and increased expression of inflammasome components including NLRP3 and caspase-
Abbreviations: LN, Lupus nephritis; SLE, Systemic lupus erythematosus; Ying Yang Huo, Horny Goat Weed ⁎ Corresponding author at: Department of Nephrology, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou 350001, Fujian Province, PR China. E-mail address: [email protected] (H. Ye). 1 Contributed equally. https://doi.org/10.1016/j.lfs.2018.07.009 Received 1 June 2018; Received in revised form 28 June 2018; Accepted 5 July 2018 Available online 06 July 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
Life Sciences 208 (2018) 26–32
B. Su et al.
2.5. Immunofluorescence of immune complex deposition
1 has been reported in LN [8]. Activation of NLRP3 inflammasome results in the activation of caspase-1 and release of mature IL-1β and IL18. IL-1β plays essential role in LN pathogenesis and enhanced IL-1β is associated with LN [9]. It has been reported that in macrophages derived from lupus patients, the NLRP3 inflammasome activation was increased, which results in a feed-forward inflammatory loop contributes to disease flares and organ damage [10]. Therefore, NLRP3 inflammasome could be another potential therapy target of LN [3]. Icariin is a bioactive component isolated from Chinese medicine Horny Goat Weed (Ying Yang Huo). Diverse activities of icariin have been reported, including anti-inflammation [11], anti-atherosclerosis [12], anti-tumor [13]. It has also been well demonstrated that icariin inhibited both NF-κB and NLRP3 inflammasome activation [14–17]. For instance, it was found that icariin was able to decrease renal damage through inhibition of NLRP3 inflammasome activation in the rat model of nephropathy [17]. As mentioned above, NLRP3 inflammasome was involved in the pathology of LN, therefore, in current study, we explored the effects of icariin on LN. We demonstrated that icariin inhibited activations of both NF-κB and NLRP3 inflammasome, and attenuated the kidney pathology in MRL/lpr mice.
To evaluate the renal immune complex deposition, frozen kidney sections were stained for mouse immunoglobulins with FITC-conjugated rabbit anti-mouse IgG (Santa Cruz, Dallas, USA) after blocking with 10% fetal bovine serum. The mean intensity of green fluorescence was scored as 0–3 as previously described. For each mouse, 50 glomeruli were analyzed and the sum of each glomeruli mean was used for final analysis. 2.6. ELISA The serum level of anti-DNA IgG antibody was determined by ELISA. Briefly, 96-well ELISA plates were coated with 5 μg/ml calf thymus dsDNA (Sigma-Aldrich, St. Louis, USA). After blocking with 1% BSA, sera were added and incubated at room temperature for 1 h. Normal mouse IgG was used as negative control. Standard curve was prepared by using mouse anti-dsDNA monoclonal antibody (Abcam, Shanghai, China). After washing, peroxidase conjugated goat antimouse IgG antibody (Sigma, USA) was added to detect the bound antidsDNA antibody followed by the peroxidase substrate. The absorbance was measured at 450 nm. The concentration of anti-dsDNA was calculated using the standard curve. Commercial ELISA kits for TNF-α and IL-1β were purchased from R &D systems (Minneapolis, MN, USA) and used for cytokines measurement in kidney homogenates and serum according to manufacturer's instructions.
2. Materials and methods 2.1. Mice MRL/lpr mice which developed the SLE-like phenotype were used in current experiments. Mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China). Wild type C57BL/6 mice with matched sex and age were used as control. The use of animals was followed with National Institutes of Health Guide for Care and Use of Animals and approved by The First Affiliated Hospital of Wenzhou Medical University. Mice were maintained in a constant temperature (23 °C) and 14 h light/10 h dark cycle environment and provided with ad libitum feeding of water and food.
2.7. Western blot Total kidney proteins were extracted using cell lysis buffer (Cell Signaling Technology, USA) according to manufacturer's instruction. In some experiment, NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA) were used to isolate nuclear and cytosolic proteins according to manufacturer's instructions. A total of 40 μg of proteins were loaded onto a 12% SDS-PAGE gel. After transfer, membranes were blocked by 5% non-fat milk and incubated with different primary antibodies: Anti-p-IκB (1:1000, Cell Signaling technology, Danvers, MA, USA), Anti-NFκB (1:1000, Santa Cruz, Dallas, TX, USA), Anti-F4/80 (1:1000, Abcam, Cambridge, MA, USA), Anti-CCL2 (1:1000, Abcam, USA), Anti-Capase-1p20 (1:1000, Santa Cruz, USA), Anti-β actin (1:1000, Sigma, USA), Anti-fibrillarin (1:500, Sigma, USA), and anti-caspase-1 p20 (1:1000, Santa Cruz, USA), for overnight at 4 °C. Next day, corresponding HRP-conjugated secondary antibodies were incubated. Peroxidase reaction was visualized by the SuperSignal West Pico Chemiluminenscent Substrate Kit (Thermo Scientific, USA). The bands intensities were quantitated using the Image J software.
2.2. Experimental design Ten weeks old MRL/lpr mice were divided into two groups randomly, with 10 mice in each group. Icariin was purchased from the National Institute for the control of Pharmaceutical and Biological Products (Beijing, China), and was dissolved in saline with ultra-sonication. Group1 was considered as vehicle control group and given saline (vehicle group). Group 2 was the icariin treated group and administrated with icariin (10 mg/kg/day) by gavage every day for total 8 weeks as described before [17]. Wild type C57BL/6 mice were used as non-treated group (control group). 2.3. Renal function evaluation
2.8. Statistical analysis Urine samples were collected every two weeks in metabolic cages with free access to water and standard diet. Urine protein was semiquantified by Multistix 10SG reagent strips and analyzed by Clinitek Sttus analyzer (Bayer Healthcare), and graded on a scale of 0–4, 0 = non, 1 = 30–100 mg/dl, 2 = 100–300 mg/dl, 3 = 300–2000 mg/ dl, or 4 > 2000 mg/dl, as described previously [18]. Blood were obtained from the eyeball and then centrifuged at 3000 rpm for 10 min to separate the serum for further analysis. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using a Cobas®C311 Autoanalyzer (Roche Diagnostics, Indianapolis, USA).
Data were presented as mean ± SD. One-way ANOVA analysis and Tukey's post-hoc test were used. Statistical difference was considered as significant only if p < 0.05. 3. Results 3.1. Icariin attenuated renal lesions in MRL/lpr mice To determine the potential effects of icariin on renal function in MRL/lpr mice, 10 weeks MRL/lpr mice were administrated icariin for 8 weeks and then analyzed for renal function and pathology. As shown in Fig. 1A, in vehicle treated MRL/lpr mice, urine protein scores increased with time increased, which indicated attenuated renal function in MRL/lpr mice. In contrast, MRL/lpr mice treated with icariin had unchanged urine protein scores and the scores on 16 and 18 weeks were significantly lower than the scores in vehicle-treated mice. Thus, this
2.4. Histology analysis For pathology and immunohistochemistry, 10% formalin-fixed and paraffin-embedded renal tissues were cut into 2 μm thickness. After deparaffinization, the slides were stained with hematoxylin (HE) reagents for pathologic evaluation as described previously [19]. 27
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 1. Icariin attenuated renal lesions in MRL/lpr mice. After treatment with icariin for 8 weeks, renal functional and histological parameters were measured. (A) Urine protein excretion was measured every two weeks. (B) Blood urea nitrogen (BUN) levels were measured at 20 weeks of age after Icariin treatment. (C) Serum creatinine levels were measured at 20 weeks of age after Icariin treatment. (D) Kidney histopathological evaluation was measured by H&E staining. (magnification: ×400). nd, not detectable. Scale bar = 50 μm. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
High levels of serum anti-dsDNA antibody were also detected in MRL/ lpr mice while in normal mice the anti-dsDNA antibody was undetectable (Fig. 2C). The levels of anti-dsDNA antibody were significantly decreased in icariin-treated MRL/lpr mice.
result suggested that icariin protected renal function in MRL/lpr mice. Similarly, icariin also significantly decreased serum BUN level (Fig. 1B) and creatine level (Fig. 1C) in MRL/lpr mice. Taking all together, our data suggested that icariin rescued the renal function in MRL/lpr mice. We continued to evaluate the protective effect of icariin on kidney injury at histological level. As shown in Fig. 1D, icariin significantly decreased the glomerular proliferation, glomerular sclerosis and periglomerular inflammation in MRL/lpr mice. Therefore, our data indicated that icariin ameliorated renal pathology in MRL/lpr mice.
3.3. Icariin inhibited NF-κB activation in the kidneys of MRL/lpr mice NF-κB activation has been implicated in the pathogenesis of SLE [21]. Next we explored whether icariin affected NF-κB activation. Consistently, we identified that there was significantly enhanced phosphorylation of IκB in kidneys cytosolic protein of MRL/lpr mice when compared to that of normal mice (Fig. 3A&B). Correspondingly, the nuclear translocation of NF-κBp65 was significantly increased in MRL/lpr mice kidney when compared to normal mice (Fig. 3C&D). These results indicated the robust NF-κB activation in MRL/lpr mice. Once icariin was administrated to MRL/lpr mice, both phosphorylation of IκB (Fig. 3A&B) nuclear translocation of NF-κBp65 (Fig. 3C&D) were significantly decreased. Therefore, our data showed that icariin inhibited NF-κB activation in MRL/lpr mice kidneys.
3.2. Icariin reduced serum anti-dsDNA antibody and renal deposition of immune complex in the MRL/lpr mice Production of anti-dsDNA antibodies and deposition of immune complex in kidney are hallmarks of lupus nephritis [20]. We continued to explore the effects of icariin on anti-dsDNA antibody and immune complex deposition. As shown in Fig. 2A and B, pronounced deposition of IgG was observed in MRL/lpr mice kidney. In contrast, decreased renal deposition of IgG was observed in MRL/lpr mice treated with icariin. After quantitation of fluorescence intensity, we found that icariin significantly prevented renal IgG deposition in MRL/lpr mice. 28
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 2. Icariin reduced serum anti-dsDNA antibody and renal deposition of immune complex in the MRL/lpr mice. (A) Snap-frozen sections were stained with FITC-conjugated antibodies. IgG was significantly decreased after treatment with icariin for 8 weeks. (Magnification: ×400). (B) Fluorescence intensity was subjected to semi-quantitative analysis. (C) Serum anti-dsDNA antibody levels were detected by ELISA. Scale bar = 50 μm. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; # p < 0.05, verses vehicle group.
3.5. Icariin inhibited macrophage infiltration and CCL2 level in the MRL/ lpr mice
3.4. Icariin inhibited TNF-α expression in the MRL/lpr mice Activation of NF-κB resulted in expression of downstream cytokines including TNF-α and in SLE the TNF-α level was increased [22]. Since icariin inhibited NF-κB activation, we detected TNF-α level in both kidney and serum of icariin-treated MRL/lpr mice. As shown in Fig. 4A, compared to normal mice, MRL/lpr mice had significantly increased renal TNF-α level. Icariin treatment significantly decreased the renal level of TNF-α. Similarly, icariin also significantly decreased serum level of TNF-α (Fig. 4B).
Macrophage infiltration in kidney was correlated with LN [23]. CCL2, a chemokine also known as MCP-1 (Monocyte chemoattractant protein 1), was used as diagnostic biomarker for active LN and reflected disease activity. In MRL/lpr mice, the level of F4/80, a specific marker of macrophage, was significantly increased (Fig. 5A&B). In contrast, icariin significantly downregulated F4/80 level in the kidney, indicating that icariin inhibited macrophage infiltration in MRL/lpr mice. Fig. 3. Icariin inhibited NF-κB activation in the kidneys of MRL/lpr mice. MRL/lpr mice were treated with vehicle or icariin for 8 weeks. (A) p-IκB was measured by western blot in the cytosol. (B) Protein expression levels were quantified by Image J. (C) NFκB-p65 nuclear translocation was measured by Western blot. (D) Protein expression levels were quantified by Image J. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
29
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 4. Icariin decreased the expression levels of TNF-α in the MRL/lpr mice. Renal (A) and serum (B) TNF-α levels were measured after treatment with icariin. TNFα, tumor necrosis factor-α. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
4. Discussion
Similarly, the CCL2 level was significantly increased in MRL/lpr mice, while icariin treatment inhibited the upregulation of CCL2 in MRL/lpr mice (Fig. 5C&D).
Lupus nephritis (LN) is the leading cause of morbidity and mortality in SLE patients. Searching for new drugs targeting LN is in unmet need [3]. In present study, we demonstrated that icariin treatment ameliorated the severity of LN in MRL/lpr mice. The renal function in MRL/lpr mice was rescued by icariin treatment. The urine protein score, serum BUN and creatinine levels were significantly decreased in icariintreated MRL/lpr mice. Icariin also attenuated the renal pathology in MRL/lpr mice, reduced serum anti-dsDNA antibody and reduced renal deposition of immune complex in MRL/lpr mice. We further explored the underlying mechanisms of the inhibition of LN by icariin and found that icariin treatment significantly inhibited both NF-κB activation and NLRP3 inflammasome activation, associated with reduced production of downstream inflammatory cytokines TNF-α and IL-1β. Icariin treatment also significantly decreased CCL2 expression and macrophage infiltration. NF-κB has been implicated in the pathogenesis of several autoimmune diseases including LN [25]. Elevated expression and activation of NF-κB in glomerular cells and upregulated inflammatory cytokines production are identified in LN patients. Activation of NF-κB requires
3.6. Icariin suppressed NLRP3 inflammasome activation in the kidneys of MRL/lpr mice NLRP3 inflammasome was activated in LN and contributed to the disease pathogenesis [24]. Lastly we explored the effect of icariin on NLRP3 inflammasome activation in MRL/lpr mice. We found significantly increased NLRP3 (Fig. 6A) and caspase-1p20 (Fig. 6B) level in MRL/lpr mice [3], indicating the activation of NLRP3 inflammasome. Correspondingly, there were significantly increased levels of IL-1β in kidney and serum (Fig. 6C&D). In contrast, icariin-treated MRL/lpr mice got significantly decreased levels of NLRP3, caspase-1p20, renal and serum IL-1β, when compared to vehicle treated MRL/lpr mice. Therefore, our data demonstrated that icariin suppressed NLRP3 inflammasome activation.
Fig. 5. Icariin inhibited macrophage infiltration and CCL2 level in the MRL/lpr mice. (A, B) Protein expression level of F4/80 was measured by western blot (A) and quantified by Image J (B). (C, D) Protein expression level of CCL2 was measured by western blot and quantified by Image J. CCL2, C-C motif chemokine ligand 2. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
30
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 6. Icariin suppressed NLRP3 inflammasome activation in the kidneys of MRL/lpr mice. MRL/lpr mice were treated with icariin for 8 weeks and NLRP3 inflammasome activation was examined by western blot analysis. (A) The protein expression levels of NLRP3 were detected by western blot and quantified by Image J. (B) The protein expression levels of Caspase-1 (Casp-1) were detected by western blot and the quantified by Image J. (C, D) Renal and serum IL-1β levels were measured by ELISA. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
addition to NLRP3, the importance of caspase-1 in murine lupus pathogenesis has also been recognized. Caspase-1 deficient mice are resistant to lupus development. Inhibition of P2X7 receptor, which can activate NLRP3 and capase-1, has shown beneficial effects in LN [29]. We showed that icariin treatment significantly inhibited NLRP3 and caspase-1 activation, associated with reduced production of mature IL1β. Therefore, inhibition of inflammasome activation by icariin contributes to the protective effect of icariin on LN. Many studies implicate macrophages in the pathogenesis of LN. Studies by Davidson and colleagues have shown a strong association of activated renal macrophages acting as markers for LN disease [30]. CCL2 (MCP-1) is a chemokine with potent monocyte attracting activity. The receptor for MCP-1, CCR2 in circulating inflammatory monocytes enables them to be rapidly attracted to sites of inflammation, including the kidney during active LN. Targeting of the MCP-1/CCR2 signaling axis in LN allows for the disruption of inflammatory macrophage recruitment to the kidney. Subsequent studies proved that MCP-1 antagonists are suitable for treating disease in MRL/lpr mice [31]. We demonstrated that icariin treatment suppressed CCL2 expression and inhibited macrophage infiltration, which contributed to the protective effect of icariin on LN.
IKKβ activity to phosphorylate IκB. Inhibition of IKKβ attenuates the induction of inflammatory mediator by hypoxia in rat renal tubular cells [26]. The IKK-selective inhibitor Bay11-7082 ameliorates lupus nephritis in mouse by inhibiting NF-κB activation [3]. A20 negatively regulates NF-κB activation and ABIN1, an ubiquitin-binding protein, inhibits NF-κB signaling by facilitating the action of A20 and interfering with signal-induced activation of IKK [27]. A20 deficiency in both human patients and animal models are associated with lupus [28]. Knock-in mice expressing an inactive form of ABIN1 have aberrant activation of NF-κB and develop lupus-like autoimmunity and pathological symptoms resembling human lupus nephritis [4]. Therefore, NFκB signaling pathway should be potential targets for LN treatment. Our data showed that icariin treatment significantly inhibited NF-κB activation, inhibited the production of inflammatory cytokines TNF-α. These results suggest that inhibition of NF-κB activation by icariin contributes to the protective effect of icariin on LN. The NLRP3 inflammasome has received more and more attentions as a contributor to LN in murine model [24]. NZB/NZW F1 mice are used as SLE model. Daily treatment of these mice with epigallacatechin3-gallate, the major bioactive polyphenol in green tea, protects mice from renal pathology by reducing renal inflammation and suppressing NLRP3 activation, IL-1β and IL-18 secretion in the kidneys of these mice. It has also been demonstrated that NF-κB inhibitors inhibited NLRP3 expression and protected MRL/lpr mice from nephritis. In 31
Life Sciences 208 (2018) 26–32
B. Su et al.
5. Conclusion
(2011) 890–898. [14] B. Liu, C. Xu, X. Wu, F. Liu, Y. Du, J. Sun, et al., Icariin exerts an antidepressant effect in an unpredictable chronic mild stress model of depression in rats and is associated with the regulation of hippocampal neuroinflammation, Neuroscience 294 (2015) 193–205. [15] C.Q. Xu, B.J. Liu, J.F. Wu, Y.C. Xu, X.H. Duan, Y.X. Cao, et al., Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NFkappaB signaling pathway, Eur. J. Pharmacol. 642 (2010) 146–153. [16] A.A. Basiorka, K.L. McGraw, E.A. Eksioglu, X. Chen, J. Johnson, L. Zhang, et al., The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype, Blood 128 (2016) 2960–2975. [17] L. Zhang, X.Z. Wang, Y.S. Li, L. Zhang, L.R. Hao, Icariin ameliorates IgA nephropathy by inhibition of nuclear factor kappa b/Nlrp3 pathway, FEBS Open Bio 7 (2017) 54–63. [18] G.M. Deng, G.C. Tsokos, Cholera toxin B accelerates disease progression in lupusprone mice by promoting lipid raft aggregation, J. Immunol. 181 (2008) 4019–4026. [19] S.M. Ka, H.K. Sytwu, D.M. Chang, S.L. Hsieh, P.Y. Tsai, A. Chen, Decoy receptor 3 ameliorates an autoimmune crescentic glomerulonephritis model in mice, J. Am. Soc. Nephrol. 18 (2007) 2473–2485. [20] E. Ahlin, L. Mathsson, M.L. Eloranta, T. Jonsdottir, I. Gunnarsson, L. Ronnblom, et al., Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE, Lupus 21 (2012) 586–595. [21] F. Sun, J. Teng, P. Yu, W. Li, J. Chang, H. Xu, Involvement of TWEAK and the NFkappaB signaling pathway in lupus nephritis, Exp. Ther. Med. 15 (2018) 2611–2619. [22] M. Aringer, J.S. Smolen, SLE - complex cytokine effects in a complex autoimmune disease: tumor necrosis factor in systemic lupus erythematosus, Arthritis Res. Ther. 5 (2003) 172–177. [23] G. Flores-Mendoza, S.P. Sanson, S. Rodriguez-Castro, J.C. Crispin, F. Rosetti, Mechanisms of tissue injury in lupus nephritis, Trends Mol. Med. 24 (2018) 364–378. [24] J.M. Kahlenberg, M.J. Kaplan, The inflammasome and lupus: another innate immune mechanism contributing to disease pathogenesis? Curr. Opin. Rheumatol. 26 (2014) 475–481. [25] B. O'Sullivan, A. Thompson, R. Thomas, NF-kappa B as a therapeutic target in autoimmune disease, Expert Opin. Ther. Targets 11 (2007) 111–122. [26] X. Wan, J. Yang, L. Xing, L. Fan, B. Hu, X. Chen, et al., Inhibition of IkappaB kinase beta attenuates hypoxia-induced inflammatory mediators in rat renal tubular cells, Transplant. Proc. 43 (2011) 1503–1510. [27] L. Verstrepen, I. Carpentier, K. Verhelst, R. Beyaert, ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling, Biochem. Pharmacol. 78 (2009) 105–114. [28] A. Ma, B.A. Malynn, A20: linking a complex regulator of ubiquitylation to immunity and human disease, Nat. Rev. Immunol. 12 (2012) 774–785. [29] J. Zhao, H. Wang, C. Dai, H. Wang, H. Zhang, Y. Huang, et al., P2X7 blockade attenuates murine lupus nephritis by inhibiting activation of the NLRP3/ASC/caspase 1 pathway, Arthritis Rheum. 65 (2013) 3176–3185. [30] R. Bethunaickan, R. Sahu, A. Davidson, Analysis of renal mononuclear phagocytes in murine models of SLE, Methods Mol. Biol. 900 (2012) 207–232. [31] O. Kulkarni, D. Eulberg, N. Selve, S. Zollner, R. Allam, R.D. Pawar, et al., Anti-Ccl2 Spiegelmer permits 75% dose reduction of cyclophosphamide to control diffuse proliferative lupus nephritis and pneumonitis in MRL-Fas(lpr) mice, J. Pharmacol. Exp. Ther. 328 (2009) 371–377.
Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation and NLRP3 inflammasome activation. Conflict of interest The authors declare that they have no conflicts of interest to disclose. Acknowledgements None. References [1] H. Bagavant, S.M. Fu, Pathogenesis of kidney disease in systemic lupus erythematosus, Curr. Opin. Rheumatol. 21 (2009) 489–494. [2] M. Faurschou, L. Dreyer, A.L. Kamper, H. Starklint, S. Jacobsen, Long-term mortality and renal outcome in a cohort of 100 patients with lupus nephritis, Arthritis Care Res. 62 (2010) 873–880. [3] J. Zhao, H. Zhang, Y. Huang, H. Wang, S. Wang, C. Zhao, et al., Bay11-7082 attenuates murine lupus nephritis via inhibiting NLRP3 inflammasome and NFkappaB activation, Int. Immunopharmacol. 17 (2013) 116–122. [4] H. Zhang, S.C. Sun, NF-kappaB in inflammation and renal diseases, Cell Biosci. 5 (2015) 63. [5] Q. Li, I.M. Verma, NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [6] M.S. Hayden, S. Ghosh, Signaling to NF-kappaB, Genes Dev. 18 (2004) 2195–2224. [7] L. Zheng, R. Sinniah, S.I. Hsu, Pathogenic role of NF-kappaB activation in tubulointerstitial inflammatory lesions in human lupus nephritis, J. Histochem. Cytochem. 56 (2008) 517–529. [8] J.M. Kahlenberg, S.G. Thacker, C.C. Berthier, C.D. Cohen, M. Kretzler, M.J. Kaplan, Inflammasome activation of IL-18 results in endothelial progenitor cell dysfunction in systemic lupus erythematosus, J. Immunol. 187 (2011) 6143–6156. [9] J.M. Boswell, M.A. Yui, D.W. Burt, V.E. Kelley, Increased tumor necrosis factor and IL-1 beta gene expression in the kidneys of mice with lupus nephritis, J. Immunol. 141 (1988) 3050–3054. [10] J.M. Kahlenberg, C. Carmona-Rivera, C.K. Smith, M.J. Kaplan, Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages, J. Immunol. 190 (2013) 1217–1226. [11] J.F. Wu, J.C. Dong, C.Q. Xu, Effects of icariin on inflammation model stimulated by lipopolysaccharide in vitro and in vivo, Zhongguo Zhong Xi Yi Jie He Za Zhi 29 (2009) 330–334. [12] J. Fang, Y. Zhang, Icariin, an anti-atherosclerotic drug from Chinese medicinal herb horny goat weed, Front. Pharmacol. 8 (2017) 734. [13] J. Zhou, J. Wu, X. Chen, N. Fortenbery, E. Eksioglu, K.N. Kodumudi, et al., Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions, Int. Immunopharmacol. 11
32
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation pathway and NLRP3 inflammasome Bofeng Sua,1, Hong Yeb,
T
⁎,1
, Xiaohan Youa, Haizhen Nic, Xuduan Chenb, Linlin Lib
a
Department of Nephrology, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou 325000, Zhejiang Province, PR China Department of Nephrology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, PR China c Department of Vascular Surgery, The First Affiliated Hospital of Wenzhou Medical University, Nanbaixiang, Ouhai District, Wenzhou 325000, Zhejiang Province, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Icariin Lupus nephritis NF-κB Inflammasome Mice
Aims: Lupus nephritis (LN) is a kidney inflammatory disease caused by systemic lupus erythematosus (SLE). Both NF-κB activation and NLRP3 inflammasome activation are implicated in LN pathogenesis, suggesting they are potential targets for LN treatment. Icariin, which is isolated from Chinese medicine Horny Goat Weed (Ying Yang Huo), has been shown to have anti-inflammation activity, and inhibit activations of both NF-κB and NLRP3 inflammasome. In present study, the effects of icariin on LN were evaluated in MRL/lpr mice. Main methods: We treated MRL/lpr mice with icariin for 8 weeks and then analyzed the renal function and kidney pathology. We monitored the levels of anti-dsDNA antibody and the deposition of immune complex after icariin treatment. We also detected the macrophage infiltration, NF-κB activation, NLRP3 inflammasome activation and inflammatory cytokine TNF-α production in MRL/lpr mice after icariin treatment. Key findings: We found that MRL/lpr mice treated with icariin displayed significantly attenuated the renal disease. Icariin-treated mice showed significantly reduced serum anti-dsDNA antibody level and immune complex deposition. Icariin inhibited NF-κB activation and TNF-α production in MRL/lpr mice. Icariin inhibited CCL2 production and macrophage infiltration in MRL/lpr mice. Finally, icariin suppressed NLRP3 inflammasome activation and IL-1β production in MRL/lpr mice. Significance: Icariin alleviated murine lupus nephritis via inhibiting NF-κB activation and NLRP3 inflammasome activation.
1. Introduction Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease characterized by autoantibodies production, immune complex deposition, leukocyte infiltration, complement activation and inflammatory cell-mediated tissue damage [1]. Lupus nephritis (LN), also known as SLE nephritis, is an inflammation of the kidneys caused by SLE. LN is one of the most serious complications of SLE, and a leading cause of morbidity and mortality in SLE patients. Although immunotherapy has improved the prognosis of SLE patients with renal disease considerably, a large proportion of patients with LN eventually progress to end-stage renal disease [2]. Therefore, elucidating the underlying mechanisms of LN and developing drugs to aim at corresponding targets for LN therapy are in urgent need [3]. It has been reported that nuclear factor-kappa B (NF-κB) signaling
pathway is involved in LN pathogenesis [4]. NF-κB is a key regulator of the expression of numerous proteins involved in inflammation [5]. In steady state, NF-κB is sequestered by its inhibitor of NF-κB (IκB) in cytosol. Upon activation, IκB is phosphorylated by the IκB kinase (IKK) complex and then for degradation, which leads the nuclear translocation of NF-κB and initiation of downstream targets gene transcription including Tumor necrosis factor alpha (TNF-α), interlukine-6 (IL-6) [6]. Patients with LN have upregulated expression and activation of NF-κB in glomerular endothelial and mesangial cells, together with elevated inflammatory cytokines. The activation of NF-κB is well correlated with the LN severity, suggesting the essential role of NF-κB activation in LN pathogenesis [7]. Inhibitions of NF-κB activation have been shown to attenuate LN pathologies [3]. NLRP3 inflammasome has also been implicated in LN and increased expression of inflammasome components including NLRP3 and caspase-
Abbreviations: LN, Lupus nephritis; SLE, Systemic lupus erythematosus; Ying Yang Huo, Horny Goat Weed ⁎ Corresponding author at: Department of Nephrology, Fujian Medical University Union Hospital, 29 Xinquan Road, Fuzhou 350001, Fujian Province, PR China. E-mail address: [email protected] (H. Ye). 1 Contributed equally. https://doi.org/10.1016/j.lfs.2018.07.009 Received 1 June 2018; Received in revised form 28 June 2018; Accepted 5 July 2018 Available online 06 July 2018 0024-3205/ © 2018 Elsevier Inc. All rights reserved.
Life Sciences 208 (2018) 26–32
B. Su et al.
2.5. Immunofluorescence of immune complex deposition
1 has been reported in LN [8]. Activation of NLRP3 inflammasome results in the activation of caspase-1 and release of mature IL-1β and IL18. IL-1β plays essential role in LN pathogenesis and enhanced IL-1β is associated with LN [9]. It has been reported that in macrophages derived from lupus patients, the NLRP3 inflammasome activation was increased, which results in a feed-forward inflammatory loop contributes to disease flares and organ damage [10]. Therefore, NLRP3 inflammasome could be another potential therapy target of LN [3]. Icariin is a bioactive component isolated from Chinese medicine Horny Goat Weed (Ying Yang Huo). Diverse activities of icariin have been reported, including anti-inflammation [11], anti-atherosclerosis [12], anti-tumor [13]. It has also been well demonstrated that icariin inhibited both NF-κB and NLRP3 inflammasome activation [14–17]. For instance, it was found that icariin was able to decrease renal damage through inhibition of NLRP3 inflammasome activation in the rat model of nephropathy [17]. As mentioned above, NLRP3 inflammasome was involved in the pathology of LN, therefore, in current study, we explored the effects of icariin on LN. We demonstrated that icariin inhibited activations of both NF-κB and NLRP3 inflammasome, and attenuated the kidney pathology in MRL/lpr mice.
To evaluate the renal immune complex deposition, frozen kidney sections were stained for mouse immunoglobulins with FITC-conjugated rabbit anti-mouse IgG (Santa Cruz, Dallas, USA) after blocking with 10% fetal bovine serum. The mean intensity of green fluorescence was scored as 0–3 as previously described. For each mouse, 50 glomeruli were analyzed and the sum of each glomeruli mean was used for final analysis. 2.6. ELISA The serum level of anti-DNA IgG antibody was determined by ELISA. Briefly, 96-well ELISA plates were coated with 5 μg/ml calf thymus dsDNA (Sigma-Aldrich, St. Louis, USA). After blocking with 1% BSA, sera were added and incubated at room temperature for 1 h. Normal mouse IgG was used as negative control. Standard curve was prepared by using mouse anti-dsDNA monoclonal antibody (Abcam, Shanghai, China). After washing, peroxidase conjugated goat antimouse IgG antibody (Sigma, USA) was added to detect the bound antidsDNA antibody followed by the peroxidase substrate. The absorbance was measured at 450 nm. The concentration of anti-dsDNA was calculated using the standard curve. Commercial ELISA kits for TNF-α and IL-1β were purchased from R &D systems (Minneapolis, MN, USA) and used for cytokines measurement in kidney homogenates and serum according to manufacturer's instructions.
2. Materials and methods 2.1. Mice MRL/lpr mice which developed the SLE-like phenotype were used in current experiments. Mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China). Wild type C57BL/6 mice with matched sex and age were used as control. The use of animals was followed with National Institutes of Health Guide for Care and Use of Animals and approved by The First Affiliated Hospital of Wenzhou Medical University. Mice were maintained in a constant temperature (23 °C) and 14 h light/10 h dark cycle environment and provided with ad libitum feeding of water and food.
2.7. Western blot Total kidney proteins were extracted using cell lysis buffer (Cell Signaling Technology, USA) according to manufacturer's instruction. In some experiment, NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA) were used to isolate nuclear and cytosolic proteins according to manufacturer's instructions. A total of 40 μg of proteins were loaded onto a 12% SDS-PAGE gel. After transfer, membranes were blocked by 5% non-fat milk and incubated with different primary antibodies: Anti-p-IκB (1:1000, Cell Signaling technology, Danvers, MA, USA), Anti-NFκB (1:1000, Santa Cruz, Dallas, TX, USA), Anti-F4/80 (1:1000, Abcam, Cambridge, MA, USA), Anti-CCL2 (1:1000, Abcam, USA), Anti-Capase-1p20 (1:1000, Santa Cruz, USA), Anti-β actin (1:1000, Sigma, USA), Anti-fibrillarin (1:500, Sigma, USA), and anti-caspase-1 p20 (1:1000, Santa Cruz, USA), for overnight at 4 °C. Next day, corresponding HRP-conjugated secondary antibodies were incubated. Peroxidase reaction was visualized by the SuperSignal West Pico Chemiluminenscent Substrate Kit (Thermo Scientific, USA). The bands intensities were quantitated using the Image J software.
2.2. Experimental design Ten weeks old MRL/lpr mice were divided into two groups randomly, with 10 mice in each group. Icariin was purchased from the National Institute for the control of Pharmaceutical and Biological Products (Beijing, China), and was dissolved in saline with ultra-sonication. Group1 was considered as vehicle control group and given saline (vehicle group). Group 2 was the icariin treated group and administrated with icariin (10 mg/kg/day) by gavage every day for total 8 weeks as described before [17]. Wild type C57BL/6 mice were used as non-treated group (control group). 2.3. Renal function evaluation
2.8. Statistical analysis Urine samples were collected every two weeks in metabolic cages with free access to water and standard diet. Urine protein was semiquantified by Multistix 10SG reagent strips and analyzed by Clinitek Sttus analyzer (Bayer Healthcare), and graded on a scale of 0–4, 0 = non, 1 = 30–100 mg/dl, 2 = 100–300 mg/dl, 3 = 300–2000 mg/ dl, or 4 > 2000 mg/dl, as described previously [18]. Blood were obtained from the eyeball and then centrifuged at 3000 rpm for 10 min to separate the serum for further analysis. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured using a Cobas®C311 Autoanalyzer (Roche Diagnostics, Indianapolis, USA).
Data were presented as mean ± SD. One-way ANOVA analysis and Tukey's post-hoc test were used. Statistical difference was considered as significant only if p < 0.05. 3. Results 3.1. Icariin attenuated renal lesions in MRL/lpr mice To determine the potential effects of icariin on renal function in MRL/lpr mice, 10 weeks MRL/lpr mice were administrated icariin for 8 weeks and then analyzed for renal function and pathology. As shown in Fig. 1A, in vehicle treated MRL/lpr mice, urine protein scores increased with time increased, which indicated attenuated renal function in MRL/lpr mice. In contrast, MRL/lpr mice treated with icariin had unchanged urine protein scores and the scores on 16 and 18 weeks were significantly lower than the scores in vehicle-treated mice. Thus, this
2.4. Histology analysis For pathology and immunohistochemistry, 10% formalin-fixed and paraffin-embedded renal tissues were cut into 2 μm thickness. After deparaffinization, the slides were stained with hematoxylin (HE) reagents for pathologic evaluation as described previously [19]. 27
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 1. Icariin attenuated renal lesions in MRL/lpr mice. After treatment with icariin for 8 weeks, renal functional and histological parameters were measured. (A) Urine protein excretion was measured every two weeks. (B) Blood urea nitrogen (BUN) levels were measured at 20 weeks of age after Icariin treatment. (C) Serum creatinine levels were measured at 20 weeks of age after Icariin treatment. (D) Kidney histopathological evaluation was measured by H&E staining. (magnification: ×400). nd, not detectable. Scale bar = 50 μm. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
High levels of serum anti-dsDNA antibody were also detected in MRL/ lpr mice while in normal mice the anti-dsDNA antibody was undetectable (Fig. 2C). The levels of anti-dsDNA antibody were significantly decreased in icariin-treated MRL/lpr mice.
result suggested that icariin protected renal function in MRL/lpr mice. Similarly, icariin also significantly decreased serum BUN level (Fig. 1B) and creatine level (Fig. 1C) in MRL/lpr mice. Taking all together, our data suggested that icariin rescued the renal function in MRL/lpr mice. We continued to evaluate the protective effect of icariin on kidney injury at histological level. As shown in Fig. 1D, icariin significantly decreased the glomerular proliferation, glomerular sclerosis and periglomerular inflammation in MRL/lpr mice. Therefore, our data indicated that icariin ameliorated renal pathology in MRL/lpr mice.
3.3. Icariin inhibited NF-κB activation in the kidneys of MRL/lpr mice NF-κB activation has been implicated in the pathogenesis of SLE [21]. Next we explored whether icariin affected NF-κB activation. Consistently, we identified that there was significantly enhanced phosphorylation of IκB in kidneys cytosolic protein of MRL/lpr mice when compared to that of normal mice (Fig. 3A&B). Correspondingly, the nuclear translocation of NF-κBp65 was significantly increased in MRL/lpr mice kidney when compared to normal mice (Fig. 3C&D). These results indicated the robust NF-κB activation in MRL/lpr mice. Once icariin was administrated to MRL/lpr mice, both phosphorylation of IκB (Fig. 3A&B) nuclear translocation of NF-κBp65 (Fig. 3C&D) were significantly decreased. Therefore, our data showed that icariin inhibited NF-κB activation in MRL/lpr mice kidneys.
3.2. Icariin reduced serum anti-dsDNA antibody and renal deposition of immune complex in the MRL/lpr mice Production of anti-dsDNA antibodies and deposition of immune complex in kidney are hallmarks of lupus nephritis [20]. We continued to explore the effects of icariin on anti-dsDNA antibody and immune complex deposition. As shown in Fig. 2A and B, pronounced deposition of IgG was observed in MRL/lpr mice kidney. In contrast, decreased renal deposition of IgG was observed in MRL/lpr mice treated with icariin. After quantitation of fluorescence intensity, we found that icariin significantly prevented renal IgG deposition in MRL/lpr mice. 28
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 2. Icariin reduced serum anti-dsDNA antibody and renal deposition of immune complex in the MRL/lpr mice. (A) Snap-frozen sections were stained with FITC-conjugated antibodies. IgG was significantly decreased after treatment with icariin for 8 weeks. (Magnification: ×400). (B) Fluorescence intensity was subjected to semi-quantitative analysis. (C) Serum anti-dsDNA antibody levels were detected by ELISA. Scale bar = 50 μm. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; # p < 0.05, verses vehicle group.
3.5. Icariin inhibited macrophage infiltration and CCL2 level in the MRL/ lpr mice
3.4. Icariin inhibited TNF-α expression in the MRL/lpr mice Activation of NF-κB resulted in expression of downstream cytokines including TNF-α and in SLE the TNF-α level was increased [22]. Since icariin inhibited NF-κB activation, we detected TNF-α level in both kidney and serum of icariin-treated MRL/lpr mice. As shown in Fig. 4A, compared to normal mice, MRL/lpr mice had significantly increased renal TNF-α level. Icariin treatment significantly decreased the renal level of TNF-α. Similarly, icariin also significantly decreased serum level of TNF-α (Fig. 4B).
Macrophage infiltration in kidney was correlated with LN [23]. CCL2, a chemokine also known as MCP-1 (Monocyte chemoattractant protein 1), was used as diagnostic biomarker for active LN and reflected disease activity. In MRL/lpr mice, the level of F4/80, a specific marker of macrophage, was significantly increased (Fig. 5A&B). In contrast, icariin significantly downregulated F4/80 level in the kidney, indicating that icariin inhibited macrophage infiltration in MRL/lpr mice. Fig. 3. Icariin inhibited NF-κB activation in the kidneys of MRL/lpr mice. MRL/lpr mice were treated with vehicle or icariin for 8 weeks. (A) p-IκB was measured by western blot in the cytosol. (B) Protein expression levels were quantified by Image J. (C) NFκB-p65 nuclear translocation was measured by Western blot. (D) Protein expression levels were quantified by Image J. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
29
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 4. Icariin decreased the expression levels of TNF-α in the MRL/lpr mice. Renal (A) and serum (B) TNF-α levels were measured after treatment with icariin. TNFα, tumor necrosis factor-α. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
4. Discussion
Similarly, the CCL2 level was significantly increased in MRL/lpr mice, while icariin treatment inhibited the upregulation of CCL2 in MRL/lpr mice (Fig. 5C&D).
Lupus nephritis (LN) is the leading cause of morbidity and mortality in SLE patients. Searching for new drugs targeting LN is in unmet need [3]. In present study, we demonstrated that icariin treatment ameliorated the severity of LN in MRL/lpr mice. The renal function in MRL/lpr mice was rescued by icariin treatment. The urine protein score, serum BUN and creatinine levels were significantly decreased in icariintreated MRL/lpr mice. Icariin also attenuated the renal pathology in MRL/lpr mice, reduced serum anti-dsDNA antibody and reduced renal deposition of immune complex in MRL/lpr mice. We further explored the underlying mechanisms of the inhibition of LN by icariin and found that icariin treatment significantly inhibited both NF-κB activation and NLRP3 inflammasome activation, associated with reduced production of downstream inflammatory cytokines TNF-α and IL-1β. Icariin treatment also significantly decreased CCL2 expression and macrophage infiltration. NF-κB has been implicated in the pathogenesis of several autoimmune diseases including LN [25]. Elevated expression and activation of NF-κB in glomerular cells and upregulated inflammatory cytokines production are identified in LN patients. Activation of NF-κB requires
3.6. Icariin suppressed NLRP3 inflammasome activation in the kidneys of MRL/lpr mice NLRP3 inflammasome was activated in LN and contributed to the disease pathogenesis [24]. Lastly we explored the effect of icariin on NLRP3 inflammasome activation in MRL/lpr mice. We found significantly increased NLRP3 (Fig. 6A) and caspase-1p20 (Fig. 6B) level in MRL/lpr mice [3], indicating the activation of NLRP3 inflammasome. Correspondingly, there were significantly increased levels of IL-1β in kidney and serum (Fig. 6C&D). In contrast, icariin-treated MRL/lpr mice got significantly decreased levels of NLRP3, caspase-1p20, renal and serum IL-1β, when compared to vehicle treated MRL/lpr mice. Therefore, our data demonstrated that icariin suppressed NLRP3 inflammasome activation.
Fig. 5. Icariin inhibited macrophage infiltration and CCL2 level in the MRL/lpr mice. (A, B) Protein expression level of F4/80 was measured by western blot (A) and quantified by Image J (B). (C, D) Protein expression level of CCL2 was measured by western blot and quantified by Image J. CCL2, C-C motif chemokine ligand 2. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
30
Life Sciences 208 (2018) 26–32
B. Su et al.
Fig. 6. Icariin suppressed NLRP3 inflammasome activation in the kidneys of MRL/lpr mice. MRL/lpr mice were treated with icariin for 8 weeks and NLRP3 inflammasome activation was examined by western blot analysis. (A) The protein expression levels of NLRP3 were detected by western blot and quantified by Image J. (B) The protein expression levels of Caspase-1 (Casp-1) were detected by western blot and the quantified by Image J. (C, D) Renal and serum IL-1β levels were measured by ELISA. n = 10 in each experimental group. ⁎p < 0.05, verses normal group; #p < 0.05, verses vehicle group.
addition to NLRP3, the importance of caspase-1 in murine lupus pathogenesis has also been recognized. Caspase-1 deficient mice are resistant to lupus development. Inhibition of P2X7 receptor, which can activate NLRP3 and capase-1, has shown beneficial effects in LN [29]. We showed that icariin treatment significantly inhibited NLRP3 and caspase-1 activation, associated with reduced production of mature IL1β. Therefore, inhibition of inflammasome activation by icariin contributes to the protective effect of icariin on LN. Many studies implicate macrophages in the pathogenesis of LN. Studies by Davidson and colleagues have shown a strong association of activated renal macrophages acting as markers for LN disease [30]. CCL2 (MCP-1) is a chemokine with potent monocyte attracting activity. The receptor for MCP-1, CCR2 in circulating inflammatory monocytes enables them to be rapidly attracted to sites of inflammation, including the kidney during active LN. Targeting of the MCP-1/CCR2 signaling axis in LN allows for the disruption of inflammatory macrophage recruitment to the kidney. Subsequent studies proved that MCP-1 antagonists are suitable for treating disease in MRL/lpr mice [31]. We demonstrated that icariin treatment suppressed CCL2 expression and inhibited macrophage infiltration, which contributed to the protective effect of icariin on LN.
IKKβ activity to phosphorylate IκB. Inhibition of IKKβ attenuates the induction of inflammatory mediator by hypoxia in rat renal tubular cells [26]. The IKK-selective inhibitor Bay11-7082 ameliorates lupus nephritis in mouse by inhibiting NF-κB activation [3]. A20 negatively regulates NF-κB activation and ABIN1, an ubiquitin-binding protein, inhibits NF-κB signaling by facilitating the action of A20 and interfering with signal-induced activation of IKK [27]. A20 deficiency in both human patients and animal models are associated with lupus [28]. Knock-in mice expressing an inactive form of ABIN1 have aberrant activation of NF-κB and develop lupus-like autoimmunity and pathological symptoms resembling human lupus nephritis [4]. Therefore, NFκB signaling pathway should be potential targets for LN treatment. Our data showed that icariin treatment significantly inhibited NF-κB activation, inhibited the production of inflammatory cytokines TNF-α. These results suggest that inhibition of NF-κB activation by icariin contributes to the protective effect of icariin on LN. The NLRP3 inflammasome has received more and more attentions as a contributor to LN in murine model [24]. NZB/NZW F1 mice are used as SLE model. Daily treatment of these mice with epigallacatechin3-gallate, the major bioactive polyphenol in green tea, protects mice from renal pathology by reducing renal inflammation and suppressing NLRP3 activation, IL-1β and IL-18 secretion in the kidneys of these mice. It has also been demonstrated that NF-κB inhibitors inhibited NLRP3 expression and protected MRL/lpr mice from nephritis. In 31
Life Sciences 208 (2018) 26–32
B. Su et al.
5. Conclusion
(2011) 890–898. [14] B. Liu, C. Xu, X. Wu, F. Liu, Y. Du, J. Sun, et al., Icariin exerts an antidepressant effect in an unpredictable chronic mild stress model of depression in rats and is associated with the regulation of hippocampal neuroinflammation, Neuroscience 294 (2015) 193–205. [15] C.Q. Xu, B.J. Liu, J.F. Wu, Y.C. Xu, X.H. Duan, Y.X. Cao, et al., Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NFkappaB signaling pathway, Eur. J. Pharmacol. 642 (2010) 146–153. [16] A.A. Basiorka, K.L. McGraw, E.A. Eksioglu, X. Chen, J. Johnson, L. Zhang, et al., The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype, Blood 128 (2016) 2960–2975. [17] L. Zhang, X.Z. Wang, Y.S. Li, L. Zhang, L.R. Hao, Icariin ameliorates IgA nephropathy by inhibition of nuclear factor kappa b/Nlrp3 pathway, FEBS Open Bio 7 (2017) 54–63. [18] G.M. Deng, G.C. Tsokos, Cholera toxin B accelerates disease progression in lupusprone mice by promoting lipid raft aggregation, J. Immunol. 181 (2008) 4019–4026. [19] S.M. Ka, H.K. Sytwu, D.M. Chang, S.L. Hsieh, P.Y. Tsai, A. Chen, Decoy receptor 3 ameliorates an autoimmune crescentic glomerulonephritis model in mice, J. Am. Soc. Nephrol. 18 (2007) 2473–2485. [20] E. Ahlin, L. Mathsson, M.L. Eloranta, T. Jonsdottir, I. Gunnarsson, L. Ronnblom, et al., Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE, Lupus 21 (2012) 586–595. [21] F. Sun, J. Teng, P. Yu, W. Li, J. Chang, H. Xu, Involvement of TWEAK and the NFkappaB signaling pathway in lupus nephritis, Exp. Ther. Med. 15 (2018) 2611–2619. [22] M. Aringer, J.S. Smolen, SLE - complex cytokine effects in a complex autoimmune disease: tumor necrosis factor in systemic lupus erythematosus, Arthritis Res. Ther. 5 (2003) 172–177. [23] G. Flores-Mendoza, S.P. Sanson, S. Rodriguez-Castro, J.C. Crispin, F. Rosetti, Mechanisms of tissue injury in lupus nephritis, Trends Mol. Med. 24 (2018) 364–378. [24] J.M. Kahlenberg, M.J. Kaplan, The inflammasome and lupus: another innate immune mechanism contributing to disease pathogenesis? Curr. Opin. Rheumatol. 26 (2014) 475–481. [25] B. O'Sullivan, A. Thompson, R. Thomas, NF-kappa B as a therapeutic target in autoimmune disease, Expert Opin. Ther. Targets 11 (2007) 111–122. [26] X. Wan, J. Yang, L. Xing, L. Fan, B. Hu, X. Chen, et al., Inhibition of IkappaB kinase beta attenuates hypoxia-induced inflammatory mediators in rat renal tubular cells, Transplant. Proc. 43 (2011) 1503–1510. [27] L. Verstrepen, I. Carpentier, K. Verhelst, R. Beyaert, ABINs: A20 binding inhibitors of NF-kappa B and apoptosis signaling, Biochem. Pharmacol. 78 (2009) 105–114. [28] A. Ma, B.A. Malynn, A20: linking a complex regulator of ubiquitylation to immunity and human disease, Nat. Rev. Immunol. 12 (2012) 774–785. [29] J. Zhao, H. Wang, C. Dai, H. Wang, H. Zhang, Y. Huang, et al., P2X7 blockade attenuates murine lupus nephritis by inhibiting activation of the NLRP3/ASC/caspase 1 pathway, Arthritis Rheum. 65 (2013) 3176–3185. [30] R. Bethunaickan, R. Sahu, A. Davidson, Analysis of renal mononuclear phagocytes in murine models of SLE, Methods Mol. Biol. 900 (2012) 207–232. [31] O. Kulkarni, D. Eulberg, N. Selve, S. Zollner, R. Allam, R.D. Pawar, et al., Anti-Ccl2 Spiegelmer permits 75% dose reduction of cyclophosphamide to control diffuse proliferative lupus nephritis and pneumonitis in MRL-Fas(lpr) mice, J. Pharmacol. Exp. Ther. 328 (2009) 371–377.
Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation and NLRP3 inflammasome activation. Conflict of interest The authors declare that they have no conflicts of interest to disclose. Acknowledgements None. References [1] H. Bagavant, S.M. Fu, Pathogenesis of kidney disease in systemic lupus erythematosus, Curr. Opin. Rheumatol. 21 (2009) 489–494. [2] M. Faurschou, L. Dreyer, A.L. Kamper, H. Starklint, S. Jacobsen, Long-term mortality and renal outcome in a cohort of 100 patients with lupus nephritis, Arthritis Care Res. 62 (2010) 873–880. [3] J. Zhao, H. Zhang, Y. Huang, H. Wang, S. Wang, C. Zhao, et al., Bay11-7082 attenuates murine lupus nephritis via inhibiting NLRP3 inflammasome and NFkappaB activation, Int. Immunopharmacol. 17 (2013) 116–122. [4] H. Zhang, S.C. Sun, NF-kappaB in inflammation and renal diseases, Cell Biosci. 5 (2015) 63. [5] Q. Li, I.M. Verma, NF-kappaB regulation in the immune system, Nat. Rev. Immunol. 2 (2002) 725–734. [6] M.S. Hayden, S. Ghosh, Signaling to NF-kappaB, Genes Dev. 18 (2004) 2195–2224. [7] L. Zheng, R. Sinniah, S.I. Hsu, Pathogenic role of NF-kappaB activation in tubulointerstitial inflammatory lesions in human lupus nephritis, J. Histochem. Cytochem. 56 (2008) 517–529. [8] J.M. Kahlenberg, S.G. Thacker, C.C. Berthier, C.D. Cohen, M. Kretzler, M.J. Kaplan, Inflammasome activation of IL-18 results in endothelial progenitor cell dysfunction in systemic lupus erythematosus, J. Immunol. 187 (2011) 6143–6156. [9] J.M. Boswell, M.A. Yui, D.W. Burt, V.E. Kelley, Increased tumor necrosis factor and IL-1 beta gene expression in the kidneys of mice with lupus nephritis, J. Immunol. 141 (1988) 3050–3054. [10] J.M. Kahlenberg, C. Carmona-Rivera, C.K. Smith, M.J. Kaplan, Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages, J. Immunol. 190 (2013) 1217–1226. [11] J.F. Wu, J.C. Dong, C.Q. Xu, Effects of icariin on inflammation model stimulated by lipopolysaccharide in vitro and in vivo, Zhongguo Zhong Xi Yi Jie He Za Zhi 29 (2009) 330–334. [12] J. Fang, Y. Zhang, Icariin, an anti-atherosclerotic drug from Chinese medicinal herb horny goat weed, Front. Pharmacol. 8 (2017) 734. [13] J. Zhou, J. Wu, X. Chen, N. Fortenbery, E. Eksioglu, K.N. Kodumudi, et al., Icariin and its derivative, ICT, exert anti-inflammatory, anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions, Int. Immunopharmacol. 11
32