Quercetin, a natural flavonoid, protects against hepatic ischemia–reperfusion injury via inhibiting Caspase-8/ASC dependent macrophage pyroptosis

Graphical abstract

Highlights

• •Quercetin, a natural flavonoid, protected mice from hepatic ischemia–reperfusion injury.
• •Quercetin inhibited the cleavage of GSDMD in macrophages during hepatic ischemia–reperfusion injury.
• •Quercetin inhibited NLRP3 and AIM2 inflammasome-induced macrophage pyroptosis.
• •Quercetin inhibited ASC assembly to suppress activation of NLRP3 and AIM2 inflammasome via blocking the binding between Caspase-8 and ASC.
• •The anti-hepatic ischemia–reperfusion effect of quercetin could not be duplicated in myeloid cells-specific GSDMD-knockout mice.

Abstract

Introduction

Hepatic ischemia–reperfusion injury (IRI) is an inevitable adverse event following liver surgery, leading to liver damage and potential organ failure. Despite advancements, effective interventions for hepatic IRI remain elusive, posing a significant clinical challenge. The innate immune response significantly contributes to the pathogenesis of hepatic IRI by promoting an inflammatory cytotoxic cycle. We have reported that blocking GSDMD-induced pyroptosis in innate immunity cells protected hepatic IRI from inflammatory injury. However, the search for effective pyroptosis inhibitors continues.

Objectives

This study aims to evaluate whether quercetin, a natural flavonoid, can inhibit GSDMD-induced pyroptosis and mitigate hepatic IRI.

Methods

We established the hepatic IRI murine model and cellular pyroptosis model to evaluate the efficacy of quercetin.

Results

Quercetin effectively alleviated hepatic IRI-induced tissue necrosis and inflammation. We found that during hepatic IRI, the cleavage of GSDMD occurred in hepatic macrophages, but not in other non-parenchymal cells. Quercetin inhibited the cleavage of GSDMD in macrophages. Moreover, we found that quercetin blocked the ASC assembly to inhibit the formation of NLRP3 inflammasomes and AIM2 inflammasomes, suppressing macrophage pyroptosis. Co-immunoprecipitation experiments confirmed that quercetin inhibited the interaction between ASC and Caspase-8, which is the mechanism of ASC complex and inflammasome formation. Overexpression of Caspase-8 abolished the anti-pyroptosis effect of quercetin in NLRP3 and AIM2 inflammasome signaling. Furthermore, we found that the hepatoprotective activity of quercetin was reduced in myelocytic GSDMD-deficient mice.

Conclusion

Our findings suggest that quercetin has beneficial effects on hepatic IRI. Quercetin could attenuate hepatic IRI and target inhibition of macrophage pyroptosis via blocking Caspase-8/ASC interaction. We recommend that quercetin might serve as a targeted approach for the prevention and personalized treatment of hepatic IRI in perioperative patients.

Introduction

Hepatic ischemia reperfusion injury (IRI) is an inevitable complication following liver surgery and transplantation [1]. Hepatic IRI can lead to liver injury and, in severe cases, may result in hepatic failure and multiple organ dysfunction syndrome [1], [2], significantly reducing postoperative survival rates [3]. In addition, hepatic IRI is a principal risk factor of fibrosis and post-surgical cancer recurrence [2], [4]. Despite extensive research, effective pharmacological treatments for hepatic IRI are scarce. Although liver transplantation is the only feasible option for IRI-induced hepatic failure, only a small proportion of patients benefit from liver transplantation due to the limited source of donors [4]. Consequently, hepatic IRI remains a formidable challenge for clinicians [1], [5].

Vascular control is inevitably required during most liver surgical options, which leads to interruption of blood flow and local ischemia and hypoxia [6]. When restored blood flow, it will exacerbate damage to hypoxic hepatocytes through a cascade of signaling events [7]. The initial phase of IRI is featured by oxidative stress-mediated hepatocyte death [8]. Due to that hepatocytes contain a high abundance of mitochondria, excessive reactive oxygen/nitrogen species are produced from damaged mitochondria to amplificated hepatocytes injury [4]. The subsequent phase involves immune cell activation triggered by damaged hepatocytes, leading to cytokine release and a secondary inflammatory response [1]. Activated innate immune cells play a crucial role in this process, contributing to further hepatocyte damage [4], [9]. Importantly, the local sterile inflammatory response driven by innate immunity has been accepted to be the central of hepatic IRI [4], [7]. Targeting the reperfusion-mediated inflammatory response is a crucial therapeutic target [7].

Pyroptosis, a highly pro-inflammatory form of cell death, has been increasingly understood in recent years. Pyroptosis is described as a regulated cell death executed by gasdermin (GSDM) family [10]. GSDMD is the earliest reported member of GSDM family which causes macrophage pyroptosis during pathogen infection [11]. GSDMD is an evolutionarily conserved gene expressed in a variety of cells [12]. We previously found that myeloid-specific GSDMD deficiency, but not in hepatocytes, alleviated inflammatory injury in hepatic IRI mice [13], suggesting that targeting pyroptosis could be a viable intervention strategy.

Quercetin is a natural flavonoid widely distributed in various foods, such as apples, berries, cherries, and citrus fruits [14], has an estimated daily intake ranging from 37 to 600 mg [15]. Purified quercetin is also used as a dietary supplement [16]. Quercetin has been reported to have anti-oxidant, anti-cancer, anti-inflammatory, and anti-allergic activities [14], [17]. In preclinical models, quercetin has been shown to reduce liver inflammation in conditions such as chemical liver injury, nonalcoholic fatty liver disease, cholestatic liver injury, and alcoholic hepatitis [18], [19], [20], [21], [22]. Importantly, quercetin has been shown to alleviate parenchymal organ IRI, including in the heart, kidney, and brain [14], [23], and a specific study has highlighted its capacity to mitigate hepatic IRI in rodents [24]. Although quercetin's ability to reduce ROS and inhibit proinflammatory factor expression is documented [23], the underlying mechanisms by which it protects against IRI remain underexplored. Recent findings indicate that quercetin inhibits pyroptosis in immune cells [25], [26], suggesting a novel therapeutic angle. Therefore, we hypothesize that quercetin could serve as a potential pharmacotherapy targeting pyroptosis signaling pathways for the treatment of hepatic IRI. In this study, we established a hepatic IRI mouse model and macrophage pyroptosis model to evaluate the effect of quercetin and explored the molecular mechanism underlying its protective effects against hepatic IRI.

Material and method

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Experimental Animal Ethical Committee, the Shanghai University of Traditional Chinese Medicine, Shanghai, China (Approval no. PZSHUTCM211009020). This study did not involve human patients.

Animal

Wild-type male C57BL/6J mice were obtained from Shanghai JieSiJie Laboratory Animal Company (China). GSDMD ^flox/flox Lysm^Cre mice (C57BL/6 background) were generated and bred as previously described [13], and male GSDMD ^flox/flox Lysm^Cre were used in the experiments.

Hepatic IRI mouse model and treatment

The warm hepatic IRI mice were duplicated as previously described [27]. In summary, the mice were freed from the hepatic portal vein, blocked the blood supply to the left lobe and mid hepatic lobe for 90 min (ischemia), and then the blood vessels were opened (reperfusion) stitched. At 0.5 h after reperfusion, the mice were once intraperitoneal injected with quercetin (12, 25, or 50 mg/kg) (MB1938, Meilunbio, China), N-Acetylcysteine (NAC, 100 mg/kg) (MB1735, Meilunbio, China), or the same volume (0.2 mL) of PBS. The mice were sacrificed 6 h after reperfusion. The serum and live samples were collected.

Drug safety experiment

A short-term drug safety experiment was performed. Wild-type male C57BL/6J mice were randomly divided into: control group, QE 3 days group, and QE 7 days group. The mice were intraperitoneally administrated with QE (50 mg/kg, QD) or PBS for 3 days or 7 days. The body weight was measured every day. On the last day, mice were sacrificed and the serum, liver, heart, and kidney samples were collected.

Liver enzyme level assay

The serum alanine transaminase (ALT) and aspartate transferase (AST) were measured using detection kits (Nanjing Jiancheng Bioengineering Institute, China).

ELISA

Cytokines were measured using the ELISA Kits (IL-1β, 70-EK201B, MultiSciences, China; TNF-α, EMC102a, NeoBioscience, China).

H&E and TUNEL staining

The tissue samples were fixed in formalin. The liver sections were stained with hematoxylin and eosin (H&E) (C0105S, Beyotime, China) or the TUNEL detection kit (C1098, Beyotime, China).

Histochemistry and immunofluorescence

The liver sections were heated in the EGTA buffer for antigen repair. For histochemistry, the following antibodies were used: F4/80 (28463–1-AP, Proteintech, China), MPO (SKU:023, Biocare Medical, USA). For immunofluorescence, follows antibodies were used: N-GSDMD antibody (ab215203, Abcam, UK), F4/80 antibody (QA17A29, Biolegend, USA), ASC antibody (#67824, Cell Signaling, USA). A DAPI dye was used for the nuclear staining (C1005, Beyotime, China).

Real-time qPCR

Total RNA was extracted and reverse transcript to cDNA as previously described [13]. Quantitative PCR was processed using a PCR detection system (Quant Studio 3, Thermo Fisher, USA). Relative gene expression was normalized with respect to β-actin. Mitochondrial DNA damage was detected as previously described [28].

Cell culture and treatment

The bone marrow-derived macrophages (BMDMs) and mouse macrophage-like cell line RAW 264.7 cells were used. The BMDMs were generated as previously described [29]. To obtain RAW 264.7-ASC cells, RAW 264.7 were transfected with ASC-plasmid (Genechem, China) using FuGENE HD Transfection Reagent (E2311, Promega, USA) for 48 h. The macrophage pyroptosis was duplicated as previously described [30]. To induce NLRP3-mediated pyroptosis, BMDMs or RAW 264.7-ASC were treated with 200 ng/mL LPS (L2630, Sigma-Aldrich, USA) for 4 h followed by 20 μM Nigericin (S6653, Selleck, China) for 1.5 h. Meanwhile, cells were treated with quercetin (12–50 μM, MB1938, Meilunbio, China). After Nigericin treatment for 1.5 h, the cells were acquired for further testing. To induce AIM2-mediated pyroptosis, BMDMs or RAW 264.7-ASC were transfected with 2 μg Poly (dA:dT) (Invivogen, USA) using FuGENE HD for 16 h with/without quercetin. To induce AIM2-mediated pyroptosis, BMDMs were transfected with 1 μg LPS using FuGENE HD for 16 h with/without quercetin. To overexpress Caspase-8, RAW 264.7 was pre-transfected with Caspase-8 plasmid (Genechem, China) and ASC plasmid for 48 h before the drug challenge.

Cell viability assay

The cell viability was detected using CellTiter-Lumi Steady Plus kit (C0069S, Beyotime, China). The same volume of CellTiter-Lumi buffer was added to each well of the culture plate. The fluorescence intensity was detected.

Propidium iodide (PI)-staining and lactate dehydrogenase (LDH) detection

The cells were incubated with PI solution (10 μg/mL, dissolved in PBS) (ST511, Beyotime, China). The images were recorded using a fluorescence microscope. The supernatant of medium was collected and the LDH level was detected (C0016, Beyotime, China).

Flow cytometry

Cells were stained with an Annexin V-FITC + PI kit (C1062, Beyotime, China). Stained cells were detected by the Flow cytometry system (FACS Canto II, BD Biosciences, USA). Annexin V-/PI + and Annexin V+/PI + cells were defined as the death cells. In parallel, the single-cell suspension from IRI mice was separated using the Gentle MACS system (Miltenyi Biotec, Germany). The single-cell suspension was fixed with formalin and 0.1 % Triton X-100 for 15 min and washed by PBS for flow cytometry analysis. The following antibodies were used: APC-CD45 (17–0451-82, Invitrogen, USA), PE-F4/80 antibody (QA17A29, Biolegend, USA), BV510-CD11b (562950, BD Biosciences) and Alexa Fluor 488-conjugated (ab236553, Abcam, UK) N-GSDMD antibody (ab215203, Abcam, UK). The data were analyzed by FlowJo (BD Biosciences, USA).

Catalase activity test and malondialdehyde detection

The liver samples were homogenized by the Lysis buffer. Catalase activity kit (S0051, Beyotime, China) and malondialdehyde (MDA) detection kit (S0131, Beyotime, China) were used for evaluating oxidative stress levels.

Mitochondrial membrane potential detection

The fresh liver tissues were acquired from IRI mice. Mitochondria were isolated using a tissue mitochondria isolation kit (C3606, Beyotime, China). The isolated mitochondria were stained using a JC-1 assay kit (C2003, Beyotime, China). The fluorescence intensity of monomer and aggregate were detected at 530 nm and 590 nm respectively.

ASC oligomer cross-linking

The ASC cross-linking was proceeded as previously described [31]. Cells were lysed with Triton Buffer (0.5 % Triton X-100). The pellet was re-suspended in Triton Buffer, and cross-linked for 30 min with 2 mM disuccinimidyl suberate (DSS) (MB0842, Meilunbio, China). The pellets were collected after centrifugation and dissolved in the loading buffer for western blot.

Co-immunoprecipitation (IP) and western blot

For Co-IP, proteins were dissociated from cells using IP Lysis buffer (87787, Thermo Fisher, USA). The lysate was added with 10 % Protein A agarose (P2175S, Beyotime, China) and ASC anti-body (sc-514414, Santa Cruz, USA) at 4 °C overnight. The mixture was centrifuged to obtain the supernatant for further procedure of western blot. The protein was dissociated from cells or liver tissues using RIPA Lysis Buffer (89900, Thermo Fisher, USA). Purified proteins were mixed with a loading buffer and separated on gels by SDS-PAGE. The antibodies are shown as follows: β-actin (A3854) was purchased from Sigma-Aldrich (USA); ASC (#67824S), NLRP3 (#15101S), Caspase-1 p20 (#4199S), and Caspase-8 (#4790S) were purchased from Cell Signaling (USA); GSDMD (ab209845), Caspase-1 (ab124812), and Caspase-11 (ab180673) were purchased from Abcam (UK); AIM2 (sc-293174) were purchased from Santa Cruz (USA). Three independent experiments were proceeded. The repeating blotting was shown in supplementary materials (Sup.4).

Statistical analysis

Statistical analysis was performed using GraphPad Prism. All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) was used for the comparison among groups. Once a statistically significant change was found (p < 0.05), a post hoc comparison was performed using Fisher's LSD test.

Quercetin protected mice from hepatic IRI

Hepatic IRI represents a significant challenge during the perioperative period [32], necessitating the exploration of potential pharmacological interventions. In our study, mice subjected to hepatic IRI were treated with either quercetin or PBS. Gradient doses of quercetin (12, 25, 50 mg/kg) were used according to previous reports [24]. In addition, NAC was used as a positive control drug [33]. At 0.5 h after reperfusion, the mice were once intraperitoneal injected with quercetin or NAC. We observed that quercetin alleviated liver injury and dose-dependently down-regulated the liver enzyme levels (ALT/AST) (Fig. 1A–B). Moreover, quercetin significantly reduced Suzuki tissue damage scores and necrotic area (Fig. 1C). Quercetin significantly reduced TUNEL-positive hepatocytes (Fig. 1D–E). We observed that 50 mg/kg quercetin showed a comparable protective effect compared with NAC. To test the tolerance of short-term, we administered 50 mg/kg quercetin to mice for 3 or 7 days. The result showed that mice exhibit a good tolerance to quercetin (Sup.1).

Fig. 1.

Quercetin protected mice from hepatic IRI. Hepatic IRI mice were treated with quercetin (QE), N-Acetylcysteine (NAC), or PBS. The protective effect of QE on liver injury. (A): The H&E staining of liver. (B): The liver enzyme levels. (C): The necrotic area and Suzuki score. (D-E): The TUNEL staining and quantification. Data are represented as mean ± SD. **p < 0.01, *p < 0.05. ns: not significant.

Quercetin alleviated oxidative stress and inhibited liver inflammation

The pathophysiological mechanism of hepatic ischemia–reperfusion injury (IRI) includes initial hepatocellular oxidative stress and subsequent innate immune response-induced inflammatory cytotoxic cycle [4], [8]. We first detected oxidative stress levels in the liver. The results showed that quercetin inhibited the level of hepatic MDA and loss of catalase in IRI mice (Fig. 2A). Moreover, we detected mitochondrial homeostasis and damaged mitochondrial DNA levels in liver tissues. We purified mitochondria from the liver of IRI mice, and the JC-1 test showed that quercetin protected mitochondrial homeostasis and quercetin suppressed the mitochondrial DNA damage (Fig. 2B). Given that inflammatory response is the crucial pathological mechanism during hepatic IRI [5], we examined the inflammatory cell infiltration and inflammatory factor levels in mice. Immunohistochemistry staining showed that macrophages (F4/80-positive) and neutrophils (MPO-positive) were increased after hepatic IRI (Fig. 2C). Quercetin decreased the infiltration of macrophages and neutrophils. Moreover, quercetin inhibited inflammatory factors, including TNF-α and IL-1β (Fig. 2D). These results indicated that quercetin could alleviate oxidative stress and inhibit innate immune activation.

Fig. 2.

Quercetin inhibited oxidative damage and inflammation. Hepatic IRI mice were treated with quercetin (QE) or PBS. The protective effect of QE on oxidative damage and inflammatory response. (A): The relative caralase ativity and the hepatic malondialdehyde (MDA) level. (B): The damged mitochondrial DNA level and the mitochondrial membrane potential detection (JC-1 test). (C): The inflammatory cell infiltration of liver. The F4/80 (macrophage) and MPO (neutrophil) staining of liver pathological section. (D): The serum level of IL-1β and TNF-α; The mRNA level of hepatic IL-1β and TNF-α. Data are represented as mean ± SD. **p < 0.01.

Quercetin inhibited the pyroptosis signaling in hepatic IRI mice

To elucidate the mechanism of quercetin's protective effects against IRI, we initially investigated whether quercetin directly targets hepatocytes by employing a hypoxic reoxygenation model in primary mouse hepatocytes—a recognized method to simulate oxidative stress and mitochondrial dysfunction characteristic of IRI [34]. However, quercetin did not alleviate hypoxic reoxygenation-induced hepatocyte damage (Sup.2A). Hence, we turned our attention to the innate immune response to understand the underlying principle of quercetin. Previously, we have reported that GSDMD knockout (KO) in myeloid cells ameliorated hepatic IRI [1] and recent studies have suggested that quercetin has an anti-pyroptosis effect [21]. Given that quercetin inhibited IL-1β, a representative marker of pyroptosis [12], we hypothesized that quercetin could regulate the pyroptosis signaling during hepatic IRI. As expected, quercetin inhibited the cleavage of GSDMD in hepatic IRI mice livers (Fig. 3A). GSDMD-induced pyroptosis signaling is defined into the inflammasome signaling (Caspase-1-dependent) and noncanonical signaling (Caspase-11-dependent) [35]. In the present study, we found that quercetin inhibited the Caspase-1 signaling (Fig. 3A). NLRP3 inflammasome signaling is caused by TLR4 and damage-associated molecular patterns (DAMPs) [36]. AIM2 inflammasome signaling can be activated by double-stranded DNA, such as mitochondrial (mt) DNA [37]. DAMPs and mtDNA are typical damage signal molecules during IRI [38]. We observed that AIM2 and NLRP3 were up-regulated after hepatic IRI, while quercetin effectively inhibited the activation of AIM2 and NLRP3. In conclusion, quercetin inhibited cleavage of GSDMD after hepatic IRI and suppressed the upstream signaling of GSDMD, including NLRP3, AIM2, and Capsase-1 signaling (Fig. 3A). Our past study showed that Caspase-11 signaling did not activate during hepatic IRI [13]. Consistent with our previously published data we have not detected the cleaved Caspase-11 expression in the liver of IRI mice (Sup.2C). Taken together, we suggested that quercetin could inhibit the NLRP3 and AIM2-induced pyroptosis signaling during Hepatic IRI.

Fig. 3.

Quercetin inhibited macrophage pyroptosis in hepatic IRI mice. Hepatic IRI mice were treated with quercetin (QE) or PBS. The effect of QE on pyroptosis signaling in the liver. (A): The GSDMD signaling in IRI liver. (B): The activation level of GSDMD in nonparenchymal cell (NPC) and hepatocyte (HC) in hepatic IRI; GSDMD activation level in F4/80⁺ macrophages and F4/80^- cells. (C): The ratio of N-GSDMD positive macrophage using flow cytometry. (D): The colocalization of N-GSDMD and F4/80⁺ macrophage in liver. Data are represented as mean ± SD. **p < 0.01.

Quercetin inhibited the cleavage of GSDMD in macrophages during hepatic IRI

GSDMD cleavage, a crucial step in pyroptosis, can occur in multiple cell types [10]. We previously reported that the cleavage of GSDMD during hepatic IRI occurred in innate immune cell cells, instead of hepatocytes [11]. Consistent with previous results, we determined the cleaved GSDMD only in nonparenchymal cells (NPCs), but not in hepatocytes (Fig. 3B). Macrophage is the largest number of immune cell types in hepatic NPCs [39], which is the first cell type found to undergo GSDMD-induced pyroptosis [40]. It is accepted that macrophage pyroptosis plays a key proinflammatory role in inflammatory response [41]. Therefore, we hypothesized that hepatic macrophages occurred pyroptosis during hepatic IRI. We separated macrophages (F4/80 positive cells) from the liver after hepatic IRI by magnetic beads separation. Cleaved GSDMD was detected in the separated macrophages, and quercetin reduced the expression of cleaved GSDMD (Fig. 3B). Interestingly, cleaved GSDMD was not detected in other NPCs (F4/80 negative cells) (Fig. 3B). In addition, we analyzed the N-GSDMD + macrophage using flow cytometry. The result showed that quercetin significantly reduced the ratio of N-GSDMD positive macrophages (Fig. 3C). Further, we labeled macrophages (F4/80-antibody, red fluorescence) and cleaved GSDMD (N-GSDMD antibody, yellow fluorescence) on liver sections from hepatic IRI mice. The results showed almost all cleaved-GSDMD co-localized with macrophages. We found that vast N-GSDMD positive macrophage is located in non-necrotic liver tissue and a spot of them in the necrotic area in the IRI group. Quercetin reduced the ratio and quantity of N-GSDMD-positive macrophages (Fig. 3D). These results proved that quercetin displays a remarkable anti-pyroptosis effect in vivo.

Quercetin inhibited NLRP3 inflammasome-induced macrophage pyroptosis in vitro

To further elucidate the anti-pyroptosis effect of quercetin, we established in vitro macrophage pyroptosis models. We treated BMDMs with LPS plus nigericin to induce NLRP3 inflammasome-induced pyroptosis [30], [42]. The activation of NLRP3 inflammasome requires two signals, including TLR4 signaling and cellular damage signaling. LPS plus nigericin are the widely recognized treatment for inducing NLRP3 inflammasome pyroptosis in macrophages. LPS bonded to TLR4, providing the first signal, and nigericin, a potassium ion vector, transferred potassium ions into the cytoplasm, providing the second signal. There is a special form of DNA damage in the early stage of pyroptosis before the event of phosphatidylserine eversion [35]. We stained the dead cells using propidium iodide (PI). PI could combine damaged DNA through GSDMD pore, being an early marker of pyroptosis [43]. Quercetin dose-dependently inhibited the rate of LPS + nigericin-induced death cells (PI-positive cells) (Fig. 4A–B). In parallel, we tested the cellular damage using Annexin V plus PI staining. We have detected vast PI+/ Annexin V- cells (over 60 %), a small quantity of PI+/ Annexin V + cells (approximately 6–10 %), and few PI-/ Annexin V + cells. We found that quercetin inhibited dose-dependently inhibited cellular damage (Fig. 4A–B). Moreover, quercetin also dose-dependently enhanced cell viability (Fig. 4C). To further study the effect of quercetin on macrophage pyroptosis, we examined key makers of pyroptosis. ASC is the essential component of inflammasome and mediates Caspase-1 pyroptosis signaling. The aggregation of ASC is a hallmark of inflammasome formation which is called ASC spot. We observed that ASC spot in LPS + NRI challenged BMDMs and quercetin reduced the ratio of ASC spot (Fig. 3D). The release of LDH and IL-1β is a well-recognized feature of pyroptosis [43]. We found that quercetin dose-dependently inhibited LDH and IL-1β levels in the medium (Fig. 4E). LPS + nigericin treatment leads to activation of Caspase-1 and GSDMD with leakage of NLRP3 inflammasome. Quercetin blocked Caspase-1/GSDMD signaling (Fig. 4F-G). Further, we detected the expression of ASC dimer/monomer in cell lysates. Results showed that quercetin inhibited the oligomerization of ASC (Fig. 4F–G).

Fig. 4.

Quercetin inhibited NLRP3 inflammasome-induced macrophage pyroptosis in vitro.The effect of quercetin (QE) on LPS + nigericin- mediated pyroptosis in bone marrow-derived macrophages (BMDMs). (A-B): The PI staining and the Annexin V-PI staining. (C): The cell vitality test. (D): The immunofluorescent staining of ASC spot. (E): The medium IL-1β leve and LDH level. (F-G): The NLRP3 signaling and ASC oligomerization detection. Data are represented as mean ± SD. **p < 0.01, *p < 0.05.

Quercetin inhibited AIM2 inflammasome-induced macrophage pyroptosis in vitro

We next duplicated AIM2/Caspase-1 induced macrophage pyroptosis, respectively. Unlike NLRP3 inflammasomes, AIM2 inflammasome activation only requires one signal, such as leaked DNA [37]. While macrophages phagocytosed damaged cells, the leaked DNA could activate AIM2 inflammasomes to cause pyroptosis. Poly (dA:dT) is a factitious repetitive synthetic double-stranded DNA sequence that could activate AIM2 inflammasome signaling. In line with the effect of quercetin on NLRP3 inflammasomes, quercetin dose-dependently inhibited the rate of dead cells and increased cell viability of the Poly (dA:dT)-transfected BMDMs (Fig. 5A–C). In addition, quercetin inhibited the ratio of ASC spot-positive cells and inhibited LDH and IL-1β (Fig. 5D–E). Mechanistically, quercetin blocked the Caspase-1/GSDMD signaling and leakage of AIM2 inflammasomes (Fig. 5F–G). As expected, quercetin inhibited the oligomerization of ASC (Fig. 5F–G). Although, Caspase-11 did not play a role during hepatic IRI, we detect the effect of quercetin on Caspase-11 induced macrophage pyroptosis. Caspase-11-induced cleavage of GSDMD and pyroptosis is independent of Capase-1 or ASC assembly [43]. Intracellular LPS could directly activate Capase-11, leading to cleavage GSDMD and pyroptosis. We transfected LPS to BMDMs and replicated the Caspase-11/GSDMD-induced macrophage pyroptosis. Interestingly, quercetin fails to regulate cell death once again (Sup.3A–D). Western blot showed that quercetin did not suppress the cleavage of Caspase-11 and GSDMD (Sup.3E–F). Overall, we suggested that quercetin could inhibit NLRP3/ AIM2-induced macrophage pyroptosis.

Fig. 5.

Quercetin inhibited AIM2 inflammasome-induced macrophage pyroptosis in vitro. The effect of quercetin (QE) on poly(dA:dT)-mediated pyroptosis in bone marrow-derived macrophages (BMDMs). (A-B): The PI staining and the Annexin V/ PI staining. (C): The cell vitality test. (D): The immunofluorescent staining of ASC spot. (E): The medium IL-1β leve and LDH level. (F-G): The AIM2 signaling and ASC oligomerization detection. Data are represented as mean ± SD. **p < 0.01, *p < 0.05.

Quercetin blocked the binding between Caspase-8 and ASC to inhibit ASC assembly

Our study showed quercetin inhibited NLRP3 and ASC inflammasome-induced macrophage pyroptosis as well as ASC oligomerization in macrophages treated with different stimuli. However, quercetin did not regulate ASC-independent Caspase-11 signaling-induced macrophage pyroptosis. These results implied an upstream mechanism for quercetin targeting ASC oligomerization. The expression of inflammasome elements (increased protein levels of NLRP3/AIM2 and ASC) and ASC oligomerization-induced inflammasome are the two steps of inflammasome formation [44]. Importantly, the upstream activation signals of NLRP3 and AIM2 are distinct. Quercetin did not regulate ASC expression. Therefore, we hypothesized that quercetin controlled the assembly of ASC. Previous research has shown that ASC assembly occurred on a molecule scaffold composed of Capase-8 and FADD [45], [46]. Our data displayed that quercetin did not alter the protein levels of ASC, Capase-8, and FADD (Fig. 6A–B and Sup.2D). Therefore, we further detected the protein–protein interaction between ASC and Capase-8/FADD. Co-IP showed that quercetin inhibited the binding between caspase-8 and ASC in both LPS + nigericin and Poly (dA:dT)-transfected BMDMs respectively (Fig. 6A–B). To confirm whether quercetin inhibited ASC assembly and pyroptosis via blocking caspase-8 and ASC, we aimed to overexpress Caspase-8 into the macrophage. Since primary BMDMs are difficult to molecular clone, we applied the macrophage-like cells RAW 264.7-ASC for the rescue experiments. We overexpressed Caspase-8 into RAW 264.7-ASC and challenged it with LPS + nigericin or Poly (dA:dT), respectively. Caspase-8 overexpression did not activate pyroptosis signaling in RAW 264.7-ASC; In addition, caspase-8 overexpression did not affect Caspase-1/GSDMD signaling. Importantly, we found that caspase-8 overexpression weakened the effect of quercetin (Fig. 6C–D). These results indicated that quercetin inhibited ASC assembly and Caspase-1/GSDMD-induced pyroptosis by blocking the binding between Caspase-8 and ASC.

Fig. 6.

Quercetin blocked the interaction between Caspase-8 and ASC to inhibit ASC assembly and pyroptosis. (A-B): The effect of quercetin (QE) on FADD/Caspase-8/ASC complex of (A) NLRP3 inflammasome-induced and (B) AIM2 inflammasome-induced macrophages pyroptosis. (C-D): Caspase-8 overexpression (OE) abolished the inhibitory effect of quercetin on NLRP3 inflammasome-induced (C) and AIM2 inflammasome-induced (D) macrophage pyroptosis. Data are represented as mean ± SD. **p < 0.01, ns: not significant.

GSDMD knockdown blocked quercetin-induced hepatoprotective effect in IRI mice

Given that quercetin could reduce hepatic IRI injury and inhibit macrophage pyroptosis in vitro, we hypothesized the hepatoprotective effect of quercetin may depend on the regulation of GSDMD-induced macrophage pyroptosis. To test this speculation, we bred the transgenic mouse deficient in GSDMD in myeloid cells (GSDMD^flox/flox Lyzm^cre mice). The results showed no significant differences in hepatic injury between the quercetin group and the PBS group in GSDMD^flox/flox Lyzm^cre mice (Fig. 7A). IHC staining showed that quercetin no longer inhibited the infiltration of macrophages and neutrophils (Fig. 7B–C). In addition, quercetin treatment did not improve the cytokines levels in GSDMD-deficient mice (Fig. 7D). Overall, these results indicated that modulation of GSDMD-induced macrophage pyroptosis was the key mechanism for quercetin-induced hepatoprotective effects.

Fig. 7.

GSDMD knockdown blocked quercetin-induced protective effect in IRI mice The effect of quercetin (QE) on myelocytic GSDMD-deficient mice with hepatic IRI. (A): The liver injury level of hepatic IRI mice. The H&E staining of liver, Suzuki score, and necrotic area. The liver enzyme levels. (B-C): The inflammatory cell infiltration of liver. The F4/80 and MPO staining. (D)The serum level of IL-1β and TNF-α. Data are represented as mean ± SD. ns: not significant.

Discussion

In this study, we have determined that quercetin ameliorated liver injury and inflammation in hepatic IRI mice, concomitantly inhibiting macrophage pyroptosis. Initially, we observed that quercetin reduced the inflammatory response and oxidative stress in mice. Interestingly, our in vitro experiments revealed that quercetin does not directly protect hepatocytes. In parallel, our results indicate that quercetin could inhibit IL-1β, a representative marker of pyroptosis, during hepatic IRI. This finding shifted our focus towards the anti- pyroptosis effect of quercetin on immune cells, specifically macrophages. Our further study showed that the quercetin selectively inhibited NLRP3 and AIM2 inflammasomes-induced macrophage pyroptosis by blocking the binding between Caspase-8 and ASC. Furthermore, knockdown of GSDMD abolished the hepatoprotective effect of quercetin. Collectively, these findings position quercetin as a promising pharmacological agent or dietary supplement for mitigating hepatic IRI.

Pyroptosis is a non-apoptotic form of cell death [11]. Increasing evidence has implicated that GSDMD and pyroptosis involves in multiple liver diseases. For instance, GSDMD KO has been shown to reduce steatosis in steatohepatitis mice [47]. The activation of GSDMD has been detected in alcoholic liver disease, and GSDMD-KO alleviated hepatic inflammation and steatosis [48], [49]. We previously have reported that the GSDMD cleavage occurred in myeloid cells, but not in hepatocytes, and blocking GSDMD-induced pyroptosis signaling in innate immune cells reduced hepatic injury during IRI [13]. In the present study, we have uncovered that GSDMD cleavage and pyroptosis occurred in macrophages, but not in other nonparenchymal cells. Macrophage is the paramount innate immune cell and plays a role in immune surveillance and pathogen clearance [50]. The liver contains the largest proportion of macrophages among all solid organs in the body, Healthy livers contain abundant resident macrophages [51], [52]. During IRI, macrophages are considered to be the earliest activated immune cells [53], and are proposed to be the main producers of pro-inflammatory cytokines [54]. Our findings underscore the critical role of macrophage pyroptosis in the pathogenesis of hepatic IRI, suggesting that targeting this process may offer therapeutic benefits.

NLRP3 and AIM2 are the principal inflammasome signals leading to pyroptosis [55]. The inflammasomes are constituted by danger-signal sensor proteins (such as NLRP3 and AIM2), the adaptor protein ASC, and the downstream effector caspase-1 [56], [57]. TLR4 signaling and dsDNA can activate and promote the expression of NLRP3 and AIM2 respectively. ASC is then activated and self-associated [58]. Our results demonstrate that quercetin inhibited the ASC oligomerization, which was the reason why quercetin could regulate NLRP3 and AIM2 inflammasomes-induced pyroptosis, but not Caspase-11-induced pyroptosis. Importantly, Caspase-8 and FADD form the molecular scaffold which is essential for the ASC oligomerization [45], [46]. We found that quercetin blocked the protein binding between Caspase-8 and ASC, but not FADD and ASC. We concluded that quercetin might be a potent inhibitor of inflammasomes with great potential pharmacological action and application value.

Quercetin, a highly regarded natural small molecule, exhibits notable pharmacological activities across a range of diseases, particularly in IRI [17], [23]. Growing evidence showed that quercetin could alleviate multi-tissue IRI, including myocardial, gastrocnemius muscle, kidney, and brain [32], [59], [60], [61]. Specifically, previous studies have reported that quercetin reduced apoptotic hepatocytes in a hepatic IRI model [62] and quercetin liposomal nanoformulation could increase bioavailability to treat hepatic IRI [61]. Although quercetin is known to inhibit inflammatory factors and macrophage activation [14], the underlying mechanisms have remained largely unexplored. In this study, we found for the first time that quercetin against hepatic IRI was attributed to the regulation of pyroptosis in macrophages. Mechanically, quercetin targeted Caspase-8/ASC to inhibit inflammasome formation. This finding enhances our understanding of the molecular pathways through which quercetin mediates its hepatoprotective effects.

Recent advances have suggested that the formation and release of GSDMD pore do not always result in macrophage death [63], and the repair mechanism of GSDMD pore has been found [64]. The survival of these macrophages is crucial for liver regeneration, as they play significant roles in tissue repair and regeneration processes [65], [66]. We propose that quercetin treatment may rescue macrophages from death, which might contribute to liver regeneration and repair. We will investigate the potential role of rescuing macrophages and quercetin during post-liver injury for further research.

Hepatic ischemia reperfusion is an unavoidable complication following liver surgery [1]. Despite advances in medical science, many diseases, including hepatic ischemia–reperfusion, still lack effective pharmacological treatments. In these contexts, complementary and alternative therapies are accepted by the public [67]. These therapies often include personalized nutrition and dietary supplements, which can play a significant role in disease management [67], [68]. Quercetin is a well-known dietary supplement sourced from foods [69]. Intake of quercetin through personalized nutrition and dietary supplements may provide positive effects in several health conditions, including ischemia–reperfusion.

The liver is a mitochondria-rich organ and mitochondrial homeostasis is a critical 'gatekeeper' of the liver in response to external stimuli [70]. Hepatic ischemia–reperfusion injury (IRI) is well-known a mitochondrial dysfunction-induced disease [8]. Mitochondrial damage not only triggers ischemic necrosis of hepatocytes, but also induces innate immune activation, such as macrophage pyroptosis [37], leading to inflammatory injury. Given this, maintaining mitochondrial homeostasis is increasingly recognized as both a biomarker and a predictive factor in preventing ischemia–reperfusion injuries [70]. Recent advances in patient stratification and personalized medicine underscore the need for tailored therapeutic approaches [68], [71]. Given that quercetin contributes to alleviated mitochondrial homeostasis-induced inflammatory injury, we suggest that quercetin could serve as a potential targeted treatment for stratified patients, offering a preventive strategy specifically designed based on individual risk factors and mitochondrial health.

Conclusion

We found that the small-molecule quercetin effectively alleviated hepatic IRI by inhibiting GSDMD-induced macrophage pyroptosis. To our knowledge, we demonstrated for the first time that quercetin inhibited NLRP3 and AIM2 inflammasome formation by blocking the binding of ASC and Caspase-8. We suggested that quercetin might be a promising therapeutic agent for hepatic IRI, specifically targeting macrophage pyroptosis. We recommend that quercetin possesses positive effects on health and could be a targeted intervention for perioperative patients.

CRediT authorship contribution statement

Lin Jiacheng: Investigation, Methodology, Visualization, Writing – original draft. Li Fuyang: Investigation, Validation. Jiao Junzhe: Investigation, Validation, Formal analysis. Qian Yihan: Writing – review & editing. Xu Min: Formal analysis, Visualization. Wang Fang: Supervision, Data curation, Project administration. Sun Xuehua: Conceptualization, Funding acquisition. Zhou Tao: Resources, Funding acquisition. Wu Hailong: Conceptualization, Methodology, Formal analysis. Kong Xiaoni: Conceptualization, Methodology, Data curation, Supervision, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Fig. 1.

The drug safety experiment. The mice were treated with quercetin (QE, 50 mg/kg, ip.) three days or seven days. (A): The liver enzyme level. (B): The body weight cruve. (C): The H&E staining of liver, heart, and kidney. Data are represented as mean ± SD. ns: not significant.

Supplementary Fig. 2.

The supplementary data of in vitro experiment. (A): The effect of quercetin (QE) on hypoxia/reoxygenation (H/R) treated primary hepatocytes. (B): The effect of quercetin (QE) on cell viability in bone marrow-derived macrophages (BMDMs). (C): The detection of Caspase-11 activation in liver of IRI mice. (D): The statistical chart of FADD, Caspase-8, and ASC. Data are represented as mean ± SD. ns: not significant.

Supplementary Fig. 3.

The effect of quercetin (QE) on Caspase-11-induced macrophage pyroptosis in vitro. The effect of quercetin (QE) on LPS-mediated pyroptosis in bone marrow-derived macrophages (BMDMs). (A-B): The PI staining and the Annexin V/ PI staining. (C): The cell vitality test. (D): The medium IL-1β leve and LDH level. (E-F): The Caspase-11 signaling detection. Data are represented as mean ± SD. **p < 0.01, *p < 0.05. ns: not significant.