Article pubs.acs.org/journal/abseba
Targeting TNFα Ameliorated Cationic PAMAM Dendrimer-Induced Hepatotoxicity via Regulating NLRP3 Inflammasomes Pathway Yubin Li,†,‡ Shaofei Wang,† Jiajun Fan,† Xuesai Zhang,§ Xiaolu Qian,∥ Xuyao Zhang,† Jingyun Luan,† Ping Song,† Ziyu Wang,† Qicheng Chen,† and Dianwen Ju*,† †
Department of Microbiological and Biochemical Pharmacy & The Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, and §Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, P.R. China ‡ Department of Dermatology, Perelman School of Medicine, and ∥Center for Advanced Rentinal and Ocular Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States S Supporting Information *
ABSTRACT: Hepatotoxicity of cationic poly amidoamine (PAMAM) dendrimers is one of the most urgent challenges to their medicinal application. Recent studies have indicated that proinflammatory cytokines were critical in nanomaterials-induced toxicity. However, little is known about the roles and underlying regulatory mechanisms of proinflammatory cytokines in cationic PAMAM dendrimer-induced hepatotoxicity. Thus, the aim of the current study was to explore the role of proinflammatory cytokine tumor necrosis factor alpha (TNFα) in cationic PAMAM dendrimer-induced liver injury and its underlying mechanism and develop novel strategies to reduce hepatotoxicity of cationic PAMAM dendrimers through regulating TNFα. In this study, we verified the significant overexpression of TNFα in cationic PAMAM dendrimer-induced hepatotoxicity in mice and found that targeting TNFα by etanercept could protect against cationic PAMAM dendrimer-induced liver injury. Interestingly, etanercept suppressed cationic PAMAM dendrimer-induced inflammasome signaling as demonstrated by reduced activation of NALP3, cleavage of Caspase-1, and maturation of interleukin (IL)-1β. Moreover, suppression of NLRP3 inflammasomes by belnacasan could also protect against cationic PAMAM dendrimer-induced hepatotoxicity and TNFα-induced acute hepatotoxicity. Notably, targeting either TNFα or inflammasomes reduced autophagy activation in hepatotoxicity triggered by cationic PAMAM dendrimers. In general, these findings revealed that targeting TNFα could ameliorate cationic PAMAM dendrimer-induced hepatotoxicity via regulating NLRP3 inflammasome pathway, underscoring that TNFα antagonism by etanercept could be used as an effective pharmacological approach to control hepatotoxicity of cationic PAMAM dendrimers and thus providing novel therapeutic strategies for managing liver toxicity of nanomaterials via regulating inflammatory mediators. KEYWORDS: cationic PAMAM dendrimers, hepatotoxicity, TNFα, NLRP3 inflammasomes
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INTRODUCTION Poly amidoamine (PAMAM) dendrimers, consisting of an ethylenediamine core and repeated amidoamine branches, have been considered to be one of the most hopeful nanomaterials for drug delivery because of their excellent DNA- or proteincontainer properties.1,2 Although PAMAM dendrimers have been widely developed as therapeutic and diagnostic agents for cancer or infectious diseases, the toxicity of PAMAM dendrimers seriously limits their medical applications, especially for high-generation PAMAM dendrimers.3,4 Meanwhile, previous studies have investigated that cationic PAMAM dendrimers could trigger acute lung damage and liver injury, initiate formation of blood clot, break key platelet functions, impair mitochondrial oxidation of brain tissues, and induce central nervous system injury.5,6 Besides, researchers have confirmed that cationic PAMAM dendrimers could exist 24 h to 7 days in vivo after intravenous injection and primarily accumulated in lung, liver and kidney.7 Our previous study have explored the cytotoxic role of autophagy in cationic PAMAM dendrimer-induced liver injury.8 However, the immunological © 2017 American Chemical Society
effects and mechanisms implicated in hepatotoxicity of cationic PAMAM dendrimers are largely unclear, and strategies to ameliorate toxicity of PAMAM dendrimers via regulating immunological signaling factors remain limited. One of the most predominant mechanisms of nanotoxicity is the dysregulated expression of proinflammatory cytokines. Series of literatures have reported that overproduction of inflammatory cytokines triggered by nanomaterials could induce acute damage and apoptosis via influencing inflammatory signaling pathway.9 It is noteworthy that TNFα is proposed to be one of the most dominant cytokines involved in systemic inflammation. For instance, ZnO, silica−titania hollow and poly(L-lysine) nanoparticles could all provoke TNFα overexpression and promote inflammatory responses in epithelial cells or macrophages.10,11 Therefore, increased TNFα was supposed to be the possible mechanism of nanomaterialsReceived: December 19, 2016 Accepted: March 27, 2017 Published: March 27, 2017 843
DOI: 10.1021/acsbiomaterials.6b00790 ACS Biomater. Sci. Eng. 2017, 3, 843−853
Article
ACS Biomaterials Science & Engineering
Figure 1. Targeting TNFa ameliorated cationic PAMAM dendrimer-induced hepatotoxicity. BALB/c mice were injected daily with or without etanercept (8 mg/kg) followed by G5-NH2 PAMAM dendrimers (100 mg/kg) injection for 9 consecutive days. All the mice were sacrificed after the experiment, and the serum samples were isolated. (A) TNFa level in the serum samples were detected by mTNFa ELISA kit. The data were presented as means ± SEM (n = 3). *P < 0.05 and **P < 0.01. (B) Changes of body weight in each group were recorded, and the data were presented as means ± SEM (n = 8). *P < 0.05. (C) Changes of liver index were recorded and compared. The data were presented as means ± SEM (n = 8), **P < 0.01. (D) Representative photographs of HE straining. (E) Whole protein of liver tissues were isolated and extracted. Changes in Cyclin D1, C-Myc, Bax, Bcl-2, cleaved Caspase-9, and cleaved Caspase-3 in each group were analyzed by Western blot.
dependent release of these cytokines.13 It has been reported that carbon black, TiO2 and SiO2 nanoparticles could induce pyroptosis, an inflammasome-dependent mode of cell death via activating NLRP3 inflammasomes.14,15 Thus, we further conjectured that NLRP3 inflammasomes might be activated and involved in cationic PAMAM dendrimer-induced hepatotoxicity. In the current study, TNFα antagonist etanercept, a dimeric soluble form of TNFα receptor that could interfere with
induced inflammatory damage and we hypothesized that TNFα might contribute to cationic PAMAM dendrimer-induced hepatotoxicity. As a component of the inflammatory process, NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasomes played critical roles in maturation and secretion of IL-1β and IL-18 from macrophages.12 Exposure of nanoparticles to human monocytes and epithelial cells could predominantly induce NLRP3 inflammasome844
DOI: 10.1021/acsbiomaterials.6b00790 ACS Biomater. Sci. Eng. 2017, 3, 843−853
Article
ACS Biomaterials Science & Engineering
Drug Administration. Etanercept dissolved in 200 μL of saline solution was intraperitoneally injected to the mice (8 mg/kg BW). Belnacasan dissolved in 200 μL of 20% β-cyclodextrin in saline solution was injected intraperitoneally into the mice (40 mg/kg BW) daily. The vehicle group was treated with 200 μL of saline solution or 20% β-cyclodextrin in saline solution. Histopathology. Liver tissues fixed in 4% paraformaldehyde were processed for light microscopy assay. All sections of liver tissues were stained with hematoxylin and eosin (HE) and further evaluated in a blinded fashion. Serum Biochemical Parameters Assay. Biochemical analysis of serum samples was detected with a spectrophotometer using appropriate kits. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and total cholesterol (TCHOL) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α and IL-1β Cytokine Measurement. The serum from mice in each group was harvested and used for cytokine measurement. The quantities of TNF-α and IL-1β were detected by using enzyme-linked immunosorbent assay (ELISA) kit (Shanghai, China) following manufacturer’s instructions. Western Blot Analysis. Liver tissues were washed with cold phosphate-buffered saline, and homogenized in RIPA lysis buffer (Haimen, China). The lysates were centrifuged for the supernatants collection. Equivalent amounts of protein were separated and analyzed by Western blot following the previous instructions.18 The band densities were measured with ImageJ software (National Institutes of Health, Bethesda, MD). Statistical Analysis. Graphpad Prism 6 (San Diego, CA) was used for statistical analysis. All data were presented as means ± standard error of mean (SEM). Comparisons between two groups were evaluated with unpaired two-tailed Student’s t test. Multiple comparisons between the groups were performed by one-way Anova and analyzed by the Student−Newman−Keuls posthoc method. P values
