autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS.

Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Research Article

The role of Resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS Jun Wu a,n, Xiangyou Li a, Geli Zhu a, Yanxia Zhang a, Min He b, Jian Zhang b a b

Departments of Nephrology, the Third Hospital of Wuhan, Wuhan University, Wuhan, Hubei 430074, China Departments of Nephrology, the Affiliated Yue Bei Hospital of Shantou University Medical College, Shaoguan, Guangdong 512026, China

art ic l e i nf o

a b s t r a c t

Article history: Received 7 October 2015 Received in revised form 10 January 2016 Accepted 23 January 2016

It has been suggested that continuous exposure of peritoneal mesothelial cells (PMCs) to high glucosecontaining peritoneal dialysis (PD) solutions may result in peritoneal inflammatory injury and impairment of local peritoneal host defence. Here, we investigated the effect of glucose-based PD solutions on mitochondrial reactive oxygen species (ROS) and nod-like receptor 3 (NLRP3) inflammasome activation in human PMCs (HPMCs). Exposure of HPMCs to high glucose-based PD solutions resulted in ROS production, which can trigger NLRP3 activation, leading to IL-1β secretion. Additionally, resveratrol (RSV) treatment induced mitophagy/autophagy via adenosine monophosphate-activated protein kinase (AMPK) activation. Increased mitochondrial ROS concentrations and IL-1β upregulation were confirmed following inhibition (siRNA against Beclin1 and ATG5 or autophagy inhibitor 3MA), but not induction (RSV), of mitophagy/autophagy. Furthermore, we observed that ATG5 and Beclin1 downregulation sensitised cells to IL-1β release induced by MSU or nigericin, which is an NLRP3 inflammasome activator. RSV treatment attenuated this effect. Taken together, this study may provide a potential therapeutic strategy for peritoneal inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS. & 2016 Elsevier Inc. All rights reserved.

Keywords: Peritoneal mesothelial cells Mitophagy/autophagy Resveratrol ROS NLRP3

1. Introduction Although peritoneal dialysis (PD) is now considered an established form of renal replacement therapy, its long-term success depends on the structural integrity of the peritoneum, an organ that did not evolve for the purpose of PD. Human peritoneal mesothelial cells (HPMCs) are a critical component of the peritoneal membrane and play a pivotal role in dialysis adequacy. Compelling evidence from in vitro and in vivo studies have highlighted the harmful nature of conventional high glucose-based PD solutions on the structural, functional, and morphologic properties of HPMCs, attributed in part to chronic intraperitoneal inflammation [1,2]. The nod-like receptor 3 (NLRP3) inflammasome is a molecular platform activated upon signs of cellular danger to trigger innate immune defences through the maturation of pro-inflammatory cytokines, such as interleukin (IL)-1β [3]. Strong associations n

Corresponding author. E-mail address: [email protected] (J. Wu).

of a number of human heritable and acquired diseases with dysregulated inflammasome activity highlight the importance of the NLRP inflammasome in regulating immune responses [4]. One model proposed that NLRP3 is activated by a common pathway of reactive oxygen species (ROS) [5]. According to diabetes-related studies, the initial reaction of cells upon exposure to high glucose is an increased production of ROS. ROS form as by-products of oxidative phosphorylation in mitochondria. Mitochondrial ROS overproduction following hyperglycaemia has been postulated to cause redox imbalance, oxidative insults, mitochondrial dysfunction, and cell death [6,7]. Therefore, mitochondria are speculated to be a causal link between conventional high glucose-based PD solutions and HPMC dysfunction, inflammation, and apoptosis. Maintaining a healthy population of mitochondria is thus essential for proper HPMC homoeostasis. Approaches to selective removal of mitochondria by autophagy (termed mitophagy) should be considered. Autophagy is the process of catabolism of cellular components, such as the cytosol, organelles, and protein aggregates, through their encapsulation by a double-membrane structure

http://dx.doi.org/10.1016/j.yexcr.2016.01.014 0014-4827/& 2016 Elsevier Inc. All rights reserved.

Please cite this article as: J. Wu, et al., The role of Resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered..., Exp Cell Res (2016), http://dx.doi.org/10.1016/j.yexcr.2016.01.014i

J. Wu et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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known as the autophagosome [8,9]. A well-studied type of cargo-specific autophagy is mitophagy, which is used to describe the engulfment of mitochondria into vesicles that are coated with the autophagosome marker microtubule-associated protein 1 light-chain 3 (LC3) [10]. Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. They balance the beneficial and detrimental effects of immunity and inflammation and, thereby, may protect against infectious, autoimmune, and inflammatory diseases [11]. Resveratrol (trans-3,4′,5-trihydroxystilbene; RSV), a naturally occurring polyphenolic phytoalexin found in grapes, elicits several beneficial effects in human pathologies through its anti-oxidant, anti-inflammatory, anti-obesity, and anti-cancer properties [12–15]. Interestingly, resveratrol has been reported to induce autophagy in tumour cells, neurons, renal cells, and vascular endothelial cells [16–20]. In this study, we investigated the effect of glucose-based PD on mitochondrial ROS production and following NLRP3 inflammasome activation in HPMCs. We also characterized the molecular mechanism of resveratrol-induced mitophagy/autophagy. In addition, the effects of mitophagy/autophagy regulation on mitochondrial ROS production and IL-1 β expression through NLRP3 inflammasome activation were evaluated. This study may provide a basis for further development of a potential therapeutic strategy for protecting the peritoneum membrane in long-term PD.

2. Materials and methods 2.1. Reagents and antibodies Mitotracker deep red, Mitotracker green, and MitoSOX were obtained from Invitrogen. MSU (monosodium urate crystals) and nigericin were obtained from InvivoGen. Resveratrol, Rapamycin, Rotenone, Thenoyltrifluoroacetone (TTFA), Antimycin A, and 3-methyladenine (3MA) were obtained from Sigma. Anti-LC3 was purchased from Novus Biologicals. Anti-IL-1β , ATG5, Beclin1, phosphor-mammalian target of Rapamycin (mTOR), mTOR, phosphor-liver kinase B1 (LKB1), LKB1, phosphor-adenosine monophosphate-activated protein kinase (AMPK) α 1/2, and AMPK α 1/2 were purchased from Cell Signalling Technology. Other antibodies were obtained from Santa Cruz Biotechnology, Inc. 2.2. Cell culture and assessment of cell viability The SV40 immortalised human peritoneal mesothelial cell line (HMrSV5) has been previously described [21,22]. All experiments on immortalised mesothelial cells were performed between passages 5 and 10. The 3-(4,5)-dimethylthiahiazo (-zy1)-3,5-di-phenytetrazoliumromide assay was used to evaluate cell viability as previously described [22]. 2.3. Flow cytometric analyses Mitochondrial mass was measured using fluorescence levels upon staining with Mitotracker deep red and Mitotracker green (50 nM) for 30 min at 37 °C. Mitochondrial-associated ROS levels were measured by staining cells with MitoSOX (2.5 mM) for 30 min at 37 °C. Mitochondrial membrane potential was measured using the kit from Invitrogen and performed according to the manufacturer's instructions. Cells were then washed with phosphate-buffered saline (PBS) solution and

resuspended in cold PBS solution containing 1% foetal bovine serum for FACS analysis. 2.4. Western blotting assay Western blot experiments have been described in detail previously [22]. Briefly, protein concentrations were measured using the Bradford method, and prepared protein was analysed by 10% gradient sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and electro-transferred to nitrocellulose membranes. Nonspecific protein binding was blocked with blocking solution (Tris-buffered saline Tween-20 and 5% non-fat dried milk). Specific antibodies were applied to the membrane and incubated overnight at 4 °C. After rinsing with 1 Tris-buffered saline Tween-20, diluted secondary antibodies were added for 60 min at room temperature. The detection of specific signals was performed using the enhanced chemiluminescence system. 2.5. Knockdown by small interfering RNA Small interfering RNA (siRNA) targeting NLRP3 was purchased from Dharmacon. ATG5, Beclin1, mTOR, LKB1, and AMPK α 1/2 were purchased from Invitrogen. The experiments were conducted according to the manufacturer's instructions. 2.6. Transduction of GFP-LC3 in HMrSV5 cells The pSELECT-GFP-LC3 plasmid containing the human LC3 gene fused at the 5′ end to the gene encoding green fluorescent protein (GFP) was purchased from InvivoGen. HMrSV5 cells were plated at a density of 2 10 5 on a coverslip and cultured up to 60% confluence. Transfection experiments were performed according to the manufacturer's instructions. The pSELECT-GFP-LC3 plasmid was selectable in HMrSV5 cells with Zeocin (400 μ g/ml, Invitrogen) on day 3 after plasmid transfection. 2.7. Confocal microscopy HMrSV5 cells were seeded into a slide chamber and incubated with fresh FCS-free DMEM for 24 h and then stimulated or stained with Mitotracker (50 nm). After washing two times with cold PBS, the cells were fixed in fresh 100% methanol for 15 min at 20 °C and then rinsed with PBS. After permeabilisation with Triton X-100 and blocking with 5% bovine serum albumin in PBS for 30 min at room temperature, cells were incubated with primary antibodies in 5% bovine serum albumin in PBS at 4 °C overnight. Cells were then rinsed with PBS, incubated with secondary antibodies for 60 min, and rinsed again with PBS. Confocal microscopy analyses were performed using a Zeiss LSM510. The images were obtained with LSM image browser. 2.8. Transmission electron microscopy HMrSV5 cells pellets were collected, fixed with 2% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in an alcohol series, and embedded in epoxy resin. Thin sections were contrasted with uranyl acetate and lead citrate. Preparations were observed with a Jeol 1400 mounted with CCD cameras (Morada, Olympus SIS). 2.9. Statistical analysis Data are expressed as mean 7SD of three determinations.



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3. Results

labels that distinguish respiring (Mitotracker deep red), total (Mitotracker green), and ROS-generating mitochondria (MitoSOX) (Fig. 1). We also investigated the effect of blocking key enzymes of the mitochondria respiratory chain of ROS production. ROS-generating mitochondria were observed when complex I and complex III were inhibited by Rotenone and antimycin A, respectively, whereas the complex II inhibitor TTFA had only a minor effect (Fig. 1).

3.1. The effect of glucose-based PD on mitochondrial mass and mitochondrial ROS production in HMrSV5 cells

3.2. Mitochondrial ROS upregulated IL-1β expression through NLRP3 inflammasome activation

Treatment with glucose-based PD in HMrSV5 cells resulted in increased total mitochondrial and ROS production, which were determined using three types of mitochondrial-specific

We investigated the effect of glucose-based PD and mitochondria respiratory chain key enzyme complex I, II, and III inhibitors on IL-1β expression in HMrSV5 cells. IL-1β expression was

Statistical differences among groups were assessed by one-way analysis of variance (ANOVA), followed by a Bonferroni (post hoc) test for continuous variables distributed normally and by Mann– Whitney test for continuous variables without a normal distribution. A P value of o0.05 was considered statistically significant.

Fig. 1. Effect of glucose-based peritoneal dialysis on mitochondrial mass and mitochondrial ROS production in HMrSV5 cells. HMrSV5 cells were treated with 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, Rotenone (10 μM), Antimycin A (50 μg/ml), or TTFA (10 μM) for 12 h and then stained with Mitotracker green and Mitotracker deep red (A) or MitoSOX (B) for 30 min and analysed by flow cytometry. D1.5%, D2.5%, D4.25% represent 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, respectively. *P o0.05 vs. control.


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Fig. 1. (continued)

upregulated in the 2.5% Dianeal, 4.25% Dianeal, Rotenone, and Antimycin A groups compared with that of the control group (Fig 2A). Furthermore, we pretreated HMrSV5 cells with NLRP3 siRNA and found that NLRP3 knockdown significantly blocked the upregulation of IL-1β induced by mitochondrial ROS (Fig. 2B).

3.3. RSV induced mitophagy/autophagy in HMrSV5 cells RSV induced LC3-II accumulation, a hallmark of autophagy. Rapamycin was used as a positive control for autophagy induction (Fig. 3A). Knockdown of ATG5, which is a protein



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Fig. 2. Mitochondrial ROS upregulate IL-1β expression through NLRP3 inflammasome activation. A. HMrSV5 cells were treated with 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, Rotenone (10 μM), Antimycin A (50 μg/ml), or TTFA (10 μM) for 12 h. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated. B. HMrSV5 cells stably expressing siRNA against NLRP3 were treated with 4.25% Dianeal, Rotenone (10 μM), Antimycin A (50 μg/ml) and MSU (NLRP3 inflammasome activator, 150 μg/ ml) for 12 h. Supernatants (SN) and cell extracts (Input) were analysed by western blotting as indicated. D1.5%, D2.5%, D4.25% represent 1.5% Dianeal, 2.5% Dianeal, 4.25% Dianeal, respectively. *P o 0.05.

essential for autophagosome formation [23], led to decreased RSV-induced LC3-II accumulation (Fig. 3B). In addition, electron microscopy images of HMrSV5 cells treated for 48 h with RSV (50 μmol/L) displayed typical hallmarks of autophagy, including accumulation of numerous vesicles with distinct double membranes (Fig. 3C). Additionally, mitochondria (Mitotracker) and

GFP-LC3 colocalization was confirmed using confocal microscopy (Fig. 3D). 3.4. RSV induced mitophagy/autophagy via AMPK activation RSV treatment increased AMPK phosphorylation in HMrSV5


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cells (Fig. 4A). In addition, to identify the upstream signal of AMPK activation by RSV, the effect of RSV on LKB1 phosphorylation over time was assessed. RSV treatment resulted in a notable increase in LKB1 phosphorylation (Fig. 4A). In contrast, RSV treatment decreased mTOR phosphorylation in HMrSV5 cells (Fig. 4A). To address the role of AMPK, LKB1, and mTOR on RSV-induced autophagy, we knocked down both the α1 and α2 AMPK subunits, LKB1, and mTOR using RNA interference. The combination of both siRNAs led to a nearly complete abrogation of AMPKα and LKB1 and a remarkably decreased LC3-II accumulation (Fig. 4B, a and b), indicating that AMPK and its upstream kinase LKB1 mediated the effect of RSV on autophagy. In contrast, knockdown of mTOR led to LC3-II accumulation (4B, c).

3.5. The effect of mitophagy/autophagy regulation on mitochondrial ROS and IL-1β expression through NLRP3 inflammasome activation To further explore the role of mitophagy/autophagy on NLRP3 inflammasome activation, we next induced mitophagy/ autophagy by RSV. Additionally, we inhibited mitophagy/autophagy specifically through 3-MA (autophagy inhibitor) treatment or downregulation of ATG5 and Beclin1 (a Bcl-2- and PI3KIII-interacting protein), which is another protein essential for autophagosome formation [23]. Increased mitochondrial ROS concentrations and IL-1β upregulation were confirmed following inhibition but not induction of mitophagy/autophagy (Fig. 5A a, b, and c). In addition, ATG5 and Beclin1 downregulation sensitised cells to IL-1β release induced by MSU or

nigericin, which is an NLRP3 inflammasome activator. RSV treatment attenuated this effect (Fig. 5B).

4. Discussion The introduction of PD more than three decades ago has provoked much interest in mesothelial cell biology. In the peritoneal cavity, HPMCs represent the largest population of resident cells, whose primary function is to provide a non-adhesive and protective layer against foreign particle invasion and injury to the peritoneum consequent to chemical or surgical insult [24]. Our previous study demonstrated that conventional glucose-based PD solutions downregulated the expression of Toll-like receptors 2 and 4 (TLR2/TLR4) by HPMCs and triggered hyporesponsiveness to pathogen-associated molecular patterns (PAMPs). These data indicated that long-term application of conventional glucose-containing PD solutions may increase the risk of microbe invasion and peritoneal infection [22]. In this study, we demonstrated that treatment with conventional glucose-based PD solutions in HMrSV5 cells resulted in increased ROS production, which was accompanied by total mitochondrial upregulation. These findings were similar to previous reports [25–28], although some studies used pure glucose as a stimulus. This study is closely linked with the PD clinical practice. We also found that by blocking key enzymes of the mitochondria respiratory chain of ROS production, ROS-generating mitochondria were observed when complex I and III inhibitors Rotenone and antimycin A, respectively, but not complex II inhibitor TTFA, were added to HMrSV5 cells. These results were in agreement

Fig. 3. RSV induces mitophagy/autophagy in HMrSV5 cells. A. HMrSV5 cells were incubated for 48 h with different RSV concentrations. Cell lysates were analysed by SDSPAGE. Proteins were blotted with anti-LC3 and GAPDH antibodies. Rapamycin (Rapa, 100 nmol/L) was used as a control for autophagy induction. *Po 0.05, #Po 0.05 vs. RSV (0 μMol/L). B. HMrSV5 cells stably expressing siRNA against ATG5 were treated with different RSV concentrations for 48 h. Cell lysates were analysed by SDS-PAGE. Proteins were blotted with anti-LC3 and anti-ATG5 antibodies. C. HMrSV5 cells were incubated for 48 h with 50 μMol/L of RSV. After inclusion, preparations were observed with an electron microscope mounted with a CCD camera. D. HMrSV5 cells expressing GFP-LC3 were stimulated with 50 μMol/L of RSV for 48 h. The colocalization of mitochondria and GFP-LC3 dots was analysed using confocal microscopy. *P o0.05, #Po 0.05.



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Fig. 3. (continued)

with previous findings in the human THP1 macrophage cell line [29]. Secretion of the key inflammatory cytokine IL-1β is a consequence of phagocyte activation and promotes a multitude of metabolic, physiologic, inflammatory, haematologic, and immunologic effects. Excessive or prolonged IL-1β generation can cause widespread tissue damage, which is associated with numerous acute and chronic inflammatory human diseases. IL-1β production is critically regulated by the NLPR3 inflammasome [30,31]. There is accumulating evidence that links the sensing of cellular stress signals to a direct pathophysiological role of NLRP3 activation in a wide range of autoinflammatory and autoimmune disorders [32]. Our in vitro data demonstrated that mitochondrial ROS production from high glucose dialysate exposure or mitochondria respiratory chain key enzyme blockade upregulated IL-1β expression, while NLRP3 downregulation blocked this effect. These results suggest that the NLRP3 inflammasome mediated mitochondrial ROS induced-IL-1β secretion in HMrSV5 cells. Further exploration is needed to examine whether a similar mechanism occurs in NLRP3-/- mice. Dostert [5] and Zhou et al. [29] demonstrated that ROS generated by a nicotinamide adenine dinucleotide phosphate oxidase upon particle phagocytosis or mitochondrial ROS, respectively, can trigger the NLRP3 inflammasome. Subsequent NLRP3 inflammasome activation leads to IL-1β secretion in the human THP1 macrophage cell line. Interestingly, the latter research group further determined that mitochondrial ROS were from the accumulation of damaged, ROS-generating mitochondria, which were caused by mitophagy/autophagy blockade. Therefore, we further investigated the effect of mitophagy/autophagy on NLRP3 inflammasome activation. Although some

RSV-induced autophagy research has been performed in other cell types [16–20], information about RSV-induced autophagy in HPMCs was absent. In the current study, we demonstrated that RSV induced mitophagy/autophagy in HMrSV5 cells, which was indicated by LC3-II formation, accumulation of numerous vesicles with distinct double membranes, and colocalization of mitochondria and GFP-LC3. To further confirm the effect of RSVinduced autophagy, siRNA knockdown of ATG5 was used to block autophagy in HMrSV5 cells. Down-regulation of ATG5 decreased RSV-induced LC3-II formation. Previous reports have indicated that RSV may activate AMPK, an intracellular sensor of energy status that is activated to reserve cellular energy content and serves as a key regulator of cell survival or death in response to pathological stress [33–35]. Our current study also revealed that RSV increased AMPK phosphorylation and phosphorylation of its upstream kinase LKB1 but decreased mTOR phosphorylation. Furthermore, following AMPKα and LKB1 downregulation, we observed a remarkably decreased LC3-II accumulation, which may indicate that AMPK and LKB1 mediate the effect of RSV on autophagy. Puissant et al. [16] demonstrated that RSV could induce autophagy in chronic myelogenous leukaemia cells. RSV also stimulated AMPK, thereby inhibiting the mTOR pathway. AMPK knockdown impaired RSV-induced autophagy. Shin and colleagues [36] reported that RSV induced AMPK and LKB1 activation and played a protective role in mitochondrial dysfunction in HepG2 cells, a human hepatocytederived cell line. Previous reports have revealed that RSV may first activate SIRT1, which is a member of the conserved family of NAD þ -dependent deacetylases and ADP-ribosyltransferases involved in numerous fundamental cellular processes [37]. This then leads to AMPK activation via deacetylation and activation



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Fig. 4. RSV induces mitophagy/autophagy via AMPK activation. A. Lysates from HMrSV5 cells treated for various times with 50 μMol/L of RSV were analysed by SDS-PAGE. Phosphorylation and non-phosphorylation expression levels of AMPKα, LKB1, and mTOR were determined using specific antibodies. aP o0.05 (p-AMPK/AMPK) and bPo 0.05 (p-LKB1/LKB1) represent RSV treatment different time points versus 0h respectively. cPo 0.05 (p-mTOR/mTOR) represents RSV treatment different time points versus 24 h. B. HMrSV5 cells were transfected with a combination of two siRNAs directed against AMPKα1 and AMPKα2 or an siRNA directed against LKB1 and m-TOR. Protein levels and phosphorylation status of AMPKα (a), LKB1 (b), m-TOR (c), and LC3 cleavage were visualised by immunoblotting.

of the AMPK kinase LKB1 [38–40]. Chen and colleagues [20] observed that RSV increased SIRT1 expression and AMPK activity in the human umbilical vein endothelial cell line HUVEC. RSV-induced autophagy in HUVECs was abolished in the presence of AMPK or SIRT1 inhibitors, as well as following AMPK and SIRT1 siRNA transfection. Zhang et al. [41] demonstrated

that RSV induced autophagy in hepatocytes. RSV increased SIRT1 and phosphor-AMPK levels in HepG2 cells. Incubation with AMPK or SIRT1 inhibitors or with AMPK or SIRT1 siRNA abolished RSV-mediated autophagy. Our results were consistent with these findings. However, we did not investigate the role of SIRT1 in RSV-mediated autophagy in current study.



However, the elevated mitochondrial ROS concentrations and IL-1β upregulation were confirmed following inhibition but not induction of mitophagy/autophagy. We also observed that downregulation of ATG5 or Beclin1 sensitised cells to MSU- or nigericin-induced IL-1β release, and RSV attenuated this effect.

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Taken together, we conclude that mitophagy/autophagy regulation plays an essential role in NLRP3 inflammasome activation and subsequent IL-1β secretion in HPMCs. In conclusion, our study demonstrated that in HPMCs, exposure to conventional high glucose-based PD solutions

Fig. 5. Effect of mitophagy/autophagy regulation on mitochondrial ROS production and IL-1β expression through NLRP3 inflammasome activation. A. HMrSV5 cells stimulated with 3-MA (10 mM) for 24 h, cells stably expressing siRNA against Beclin1 or ATG5, or cells treated with RSV (50 μMol/L) for 48 h were stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry. In addition, supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated (c). B. HMrSV5 cells stably expressing siRNA against Beclin1 or ATG5 or cells treated with RSV (50 μMol/L) for 48 h were treated with MSU (150 μg/ml) and nigericin (15 μM) for 12 h. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated. *P o 0.05.



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increased ROS production, which can trigger the NLRP3 inflammasome, whose subsequent activation leads to IL-1β secretion. Mitophagy/autophagy inhibition increased ROS production, NLRP3 inflammasome activity, and IL-1β expression. In addition, RSV can induce mitophagy/autophagy via AMPK

activation and may protect HPMCs from ROS-NLRP3-mediated inflammatory injury. These data may provide a basis for further development of a potential therapeutic strategy for protecting the peritoneum membrane during long-term PD. Future studies should confirm these findings in vivo.



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Fig. 5. (continued)

Acknowledgement This work was supported by the National Natural Science Foundation of China (81200555).

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Autophagy in pulmonary macrophages mediates lung inflammatory injury via NLRP3 inflammasome activation during mechanical ventilation.

NLRP3 inflammasome activation by mitochondrial ROS in bronchial epithelial cells is required for allergic inflammation.

Genipin inhibits NLRP3 and NLRC4 inflammasome activation via autophagy suppression.

WITHDRAWN: Schisandrin B suppresses NLRP3 inflammasome activation to alleviate myocardial ischemia reperfusion injury via maintaining mitochondrial autophagy.

Isoflurane attenuates lipopolysaccharide-induced acute lung injury by inhibiting ROS-mediated NLRP3 inflammasome activation.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

Resveratrol inhibits NLRP3 inflammasome activation by preserving mitochondrial integrity and augmenting autophagy.

Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice.

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri.

Reperfusion Injury.

Fluoxetine-enhanced autophagy ameliorates early brain injury via inhibition of NLRP3 inflammasome activation following subrachnoid hemorrhage in rats.

Lentinan exerts synergistic apoptotic effects with paclitaxel in A549 cells via activating ROS-TXNIP-NLRP3 inflammasome.

Acute Exercise-Induced Mitochondrial Stress Triggers an Inflammatory Response in the Myocardium via NLRP3 Inflammasome Activation with Mitophagy.

TXNIP Pathway in Mesangial Cells.

Andrographolide ameliorates OVA-induced lung injury in mice by suppressing ROS-mediated NF-κB signaling and NLRP3 inflammasome activation.

NLRP3 inflammasome activation is involved in Ang II-induced kidney damage via mitochondrial dysfunction.

NLRP3 inflammasome activation by Paracoccidioides brasiliensis.

ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection.

reperfusion Injury through inhibiting NLRP3 inflammasome activation.

Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome.

Mechanism of NLRP3 inflammasome activation.

Methylsulfonylmethane inhibits NLRP3 inflammasome activation.

or NF-κB-mediated NLRP3 inflammasome activation by promoting Nrf2 antioxidant pathway.

Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage.