Abstract
Objective
NLRP3 inflammasome-mediated pyroptosis of macrophage acts essential roles in the progression of sepsis-induced acute lung injury (ALI). Tangeretin (TAN), enriched in citrus fruit peel, presents anti-oxidative and anti-inflammatory effects. Here, we aimed to explore the potentially protective effect of TAN on sepsis-induced ALI, and the underlying mechanism of TAN in regulating NLRP3 inflammasome.
Material and methods
The effect of TAN on sepsis-induced ALI and NLRP3 inflammasome-mediated pyroptosis of macrophage were examined in vivo and in vitro using a LPS-treated mice model and LPS-induced murine macrophages, respectively. The mechanism of TAN regulating the activation of NLRP3 inflammasome in sepsis-induced ALI was investigated with HE staining, Masson staining, immunofluorescent staining, ELISA, molecular docking, transmission electron microscope detection, qRT-PCR, and western blot.
Results
TAN could evidently attenuate sepsis-induced ALI in mice, evidenced by reducing pulmonary edema, pulmonary congestion and lung interstitial fibrosis, and inhibiting macrophage infiltration in the lung tissue. Besides, TAN significantly suppressed inflammatory cytokine IL-1β and IL-18 expression in the serum or bronchoalveolar lavage fluid (BALF) samples of mice with LPS-induced ALI, and inhibited NLRP3 inflammasome-mediated pyroptosis of macrophages. Furthermore, we found TAN inhibited ROS production, preserved mitochondrial morphology, and alleviated excessive mitochondrial fission in LPS-induced ALI in mice. Through bioinformatic analysis and molecular docking, Polo-like kinase 1 (PLK1) was identified as a potential target of TAN for treating sepsis-induced ALI. Moreover, TAN significantly inhibited the reduction of PLK1 expression, AMP-activated protein kinase (AMPK) phosphorylation, and Dynamin related protein 1 (Drp1) phosphorylation (S637) in LPS-induced ALI in mice. In addition, Volasertib, a specific inhibitor of PLK1, abolished the protective effects of TAN against NLRP3 inflammasome-mediated pyroptosis of macrophage and lung injury in the cell and mice septic models.
Conclusion
TAN attenuates sepsis-induced ALI by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis, and TAN is a potentially therapeutic candidate against ALI through inhibiting pyroptosis.
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Introduction
Sepsis-induced ALI is a life-threatening organ dysfunction, which is the major cause of admission and death in hospital intensive care units [1]. Numerous studies report that sepsis-induced ALI is closely associated with diffuse inflammatory cell infiltration, followed with uncontrolled inflammatory responses, alveolar-capillary barrier damage, and pulmonary physiological dysfunction [2, 3]. Although great achievements have been made in lung-protective mechanical ventilation, prone positioning, and extracorporeal membrane oxygenation, the mortality rate of sepsis-induced ALI is still high [4, 5]. Therefore, developing effective pharmacotherapies for sepsis-induced ALI is urgently needed.
Macrophages are the major leukocyte population of innate immune cells infiltrating into the lungs in sepsis and ALI, and play essential roles in producing pro-inflammatory factors [6, 7]. Suppression of macrophages-mediated excessive inflammatory response could evidently improve pulmonary function and survival status in sepsis [8,9,10]. Recently, more and more studies report that pyroptosis, a pro-inflammatory type of regulated cell death, acts an important role in the pathogenesis of sepsis and ALI [11,12,13]. Generally, pyroptosis is usually triggered by assemble inflammasomes which promote the formation of active caspase-1 via the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and then lead to cleave gasdermin D (GSDMD) and release of interleukin-1β (IL-1β) and IL-18 [14,15,16]. Among the pyroptosis-related inflammasomes, nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome-mediated pyroptosis is the most widely studied, and is closely involved in sepsis and ALI [17, 18]. Fully clarifying the regulatory mechanism of NLRP3 inflammasome-mediated pyroptosis is essential for developing the effective pharmacotherapies for sepsis-induced ALI.
Mitochondria, which are dynamic organelles, not only play important roles in energy production but also are closely involved in apoptosis and inflammasome activation [19]. The balance of mitochondrial dynamics is essential for mitochondrial function, and the disturbance of mitochondrial fusion and division induces overloaded mitochondrial reactive oxygen species (ROS) production, injured mitochondrial proteins and DNA, resulting in the activation of NLRP3 inflammasome [20,21,22]. Mitochondrial fission is regulated by Dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1), and excessive fission leads to mitochondrial fragmentation and dysfunction [23, 24]. Recent studies report that mitochondrial dysfunction acts essential roles in the pathogenesis of sepsis and ALI [25], and dexmedetomidine, hydrogen, and Kahweol have been identified to alleviate sepsis-induced ALI via regulating mitochondrial dynamics [26,27,28]. Thus, mitochondrial dynamics represents a powerful therapeutic target for sepsis-induced ALI therapy.
Nowadays, plant-derived natural products have attracted more and more attention for sepsis and ALI, owing to their valuable biological activities and low side effects. Tangeretin (TAN) is a natural polymethoxyflavone compound abundant in citrus fruit peel, and has multiple biological functions such as anti-viral, anti-cancer, anti-oxidation, and anti-inflammation [29,30,31]. TAN inhibits inflammatory response in osteoarthritis through regulating the MAPK/NF-κB signaling pathways [31]. TAN alleviates hepatic steatosis and oxidative stress in high fat diet-induced nonalcoholic fatty liver disease via regulating Nrf2 pathway [32]. Recently, TAN has been found to protect sepsis-induced myocardial dysfunction via regulating the PTEN/AKT/mTOR axis [33]. However, whether TAN protects sepsis-induced ALI and its underlying mechanism is completely unknown.
In the present study, we analyzed the effects of TAN on sepsis-induced ALI using a LPS-induced sepsis model. Furthermore, we explored the protective mechanism of TAN attenuating sepsis-induced ALI in vivo an in vitro. To the best of our knowledge, this is the first study indicating the protective effect of TAN on sepsis-induced ALI through inhibiting macrophage pyroptosis via the PLK1-AMPK-Drp1-NLRP3 axis.
Material and methods
Mice model of sepsis-induced ALI
The study protocol is approved by the Institutional Animal Care and Use Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (No. 2023017). Male C57BL/6 mice (6–8 weeks old) were purchased from the GemPharmatech Co., Ltd (Nanjing, China), and were housed in a room kept at 24 ± 2 °C temperature and approximately 40% humidity in a 12 h dark/12 h light cycle, and had free access to standard food and water. Sepsis-induced ALI was established using 10 mg/kg LPS (Sigma-Aldrich, USA) via intraperitoneal injection. TAN (50 mg/kg, B20646, ShanghaiyuanyeBio, China) or an equal amount of vehicle was intraperitoneally injected into mice 30 min before LPS administration. The dose of TAN was based on previous study [34].To explore the protective role of TAN for sepsis-induced ALI, 24 mice were randomly divided into Sham groups (n = 6), TAN (n = 6), LPS (n = 6), and LPS + TAN (n = 6). To investigate whether PLK1 is involved in the TAN’s protective effect, 24 mice were divided into four groups, including Sham group (n = 6), LPS group (n = 6), LPS + TAN (50 mg/kg) group (n = 6), and LPS + TAN + Volasertib (S2235, Selleck, USA) group (n = 6). Volasertib (20 mg/kg), a specific inhibitor of PLK1, was intraperitoneally injected into the mice 30 min before TAN administration. After 12 h of LPS stimulation, mice were euthanized to collect the lung bronchoalveolar lavage fluid (BALF), and lung tissue samples.
Hematoxylin–eosin (HE) staining and Masson staining
HE staining and Masson staining were performed as previously described [35, 36].
Calculation of weight loss rate
Weight loss is calculated as: weight loss rate = | (the weight of mice before execution-the weight of mice before intervention)/the weight of mice before intervention | × 100%.
Calculation of lung wet–dry weight ratio
After execution, mice fresh lungs were excised and then weighed to obtain the wet (W) weight and the dry (D) weight before and after desiccation at 80 °C for 24 h, respectively. Lung W/D weight ratio is calculated as: Lung W/D weight ratio = the W weigh/the D weight.
Calculation of lung injury scores
Lung injury scores were independently assessed and were scored by the Mikawa method [37]. Briefly, a semi-quantitative analysis of lung injury in mice was performed, according to the following criteria: (1) alveolar hemorrhage; (2) alveolar edema; (3) infiltration or aggregation of neutrophils in the alveolar or vascular; (4) alveolar wall thickening and/or hyaline membrane. Briefly, the histological changes were scored on a scale of 0–4 (0, normal; 1, mild; 2, moderate; 3, severe; 4, intense). A cumulative histology score for all parameters was calculated.
Enzyme-linked immunosorbent assay (ELISA)
The expression levels of inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the mice serum or the BALF were examined using the Mouse TNF-α ELISA Kit (EK282, Multi-Sciences, Hangzhou, China), and the Mouse IL-1β High Sensitivity ELISA Kit (EK201BHS, Multi-Sciences, Hangzhou, China), respectively, according to the manufacturer’s protocol.
Cell culture
Primary peritoneal macrophages were isolated from the C57BL/6 mice which were injected with 1 ml of 3% Brewer thioglycollate medium into the peritoneal cavity for 4 days. Then, 10 ml of PBS was administrated to the peritoneal cavity of the mouse, and peritoneal fluid was collected into a 50 ml tube and centrifuged to obtain the peritoneal exudate cells. Finally, the supernatant was removed, and cell pellet was resuspended in DMEM and kept in a CO2 incubator at 37 °C for 24 h for subsequent cell experiments. Primary peritoneal macrophages were stimulated with DMSO or TAN (2.5 µM) for 1 h, and then treated with 500 ng/mL LPS for 5.5 h before 2 mM ATP (BS215, Biosharp, China) was supplied for an additional 30 min. To investigate whether ROS is involved in the TAN’s protective effect in vitro, peritoneal macrophages were treated with the ROS inducer Rotenone (250 nM, S2348, Selleck, USA) 60 min before TAN treatment. To investigate whether PLK1 is involved in the TAN’s protective effect in vitro, peritoneal macrophages were treated with Volasertib (10 nM, S2235, Selleck, USA) 60 min before TAN treatment.
Immunofluorescence
After deparaffination, rehydration, and antigen retrieval, lung tissue sections at thickness of 5 μm were blocked with 5% BSA, stained with the primary antibodies, and then stained with the secondary antibodies, as previously described [38]. The primary antibodies, including F4/80 antibody (sc-52664, Santa Cruz, USA), ASC antibody (ab309497, Abcam, USA), GSDMD (20770-1-AP, Proteintech), PLK1 antibody (10305-1-AP, Proteintech), Phospho-DRP1 (Ser637) antibody (AF5791, Beyotime), and NLRP3 (68,102-1-Ig, Proteintech) were used.
Screening targets for TAN
The 2D structure of TAN was obtained from MolView (http://molview.org/), and then imported into the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) to obtain the potential target genes. ALI-related genes, and organelle biogenesis and maintenance-related genes were obtained from the GeneCards (https://auth.lifemapsc.com/), respectively. Then, the intersected genes were obtained by Venn diagram analysis (http://www.ehbio.com/test/venn/#/). The TAN and potential protein targets were subjected to molecular docking using AutoDock 4.0 software. A binding energy of − 5.0 kcal/mol was defined as having potential binding affinity.
Western blotting
Lung tissues were collected, lysed, quantified with BCA protein assay kit (P0010S, Beyotime), separated with 12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and then subjected to immunoblot analysis, as our previously described [38]. The antibodies against NLRP3 (68102-1-Ig, Proteintech), GSDMD (20770-1-AP, Proteintech), PLK1 antibody (10305-1-AP, Proteintech), AMPK antibody (10305-1-AP, 10929-2-AP), Phospho-AMPK (Thr172) antibody (AF3423, Affinity), Phospho-DRP1 (Ser637) antibody (AF5791, Beyotime), DRP1 antibody (12957-1-AP, Proteintech), Caspase1 antibody (81482-1-RR, Proteintech), and β-actin (81115-1-RR, Proteintech) were used.
Quantitative real-time PCR (qRT-PCR)
Total RNA of the lung tissues or the cells was extracted, inversely transcribed into cDNA, and then performed for qRT-PCR, as our previously described [38]. qRT-PCR primers were as follows: NLRP3, 5′-ATTACCCGCCCGAGAAAGG-3′ and 5′-TCGCAGCAAAGATCCACACAG-3′; IL-1β, 5′-GCAACTGTTCCTGAACTCAACT-3′ and 5′-ATCTTTTGGGGTCCGTCAACT-3′; IL-18, 5′-CCTACTTCAGCATCCTCTACTGG-3′ and 5′-AGGGTTTCTTGAGAAGGGGAC-3′, and GAPDH, 5′-TCAACAGCAACTCCCACTCTTCCA-3′ and 5′-CCCTGTTGCTGTAGCCGTATTCA-3′.
ROS detection
ROS levels in the frozen lung sections was detected using the DHE staining kit (BB-470515, Bestbio, China), and mitochondrial ROS levels in the primary peritoneal macrophages were detected using the MitoSOX Red staining kit (40778ES50, YEASEN, China), according to the manufacturer’s protocol, respectively. Then, the images were obtained under a fluorescence microscope (LSM880, ZEISS, Germany).
Transmission electron microscope (TEM) detection
TEM was performed as our previously described [39]. Briefly, small pieces of lung samples were fixed in 2.5% glutaraldehyde at least 2 h, washed with PBS for 15 min for total three times, then dehydrated in acetone of different concentrations and saturated with resin overnight. Lung sections (70 nm) were cut and stained with both uranyl acetate and lead citrate. Images were finally acquired by H-7650 (Hitachi, Tokyo, Japan).
JC1 staining
The mitochondrial membrane potential of primary peritoneal macrophages was detected using the JC-1 staining kit (C2006, Beyotime, China), according to the manufacturer's protocol. The cells were observed and photographed (LSM880, ZEISS, Germany).
Statistical analysis
Data were analyzed using the GraphPad Prism 5.0 software (GraphPad Software Inc.) Differences between groups were analyzed by the Student’s t test or one-way analysis of variance (ANOVA)/Bonferroni multiple-comparison post hoc test. p values (represented by asterisks), where *p < 0.05.
Results
TAN attenuates LPS-induced ALI in mice
We firstly explored the potential protective role of TAN on LPS-induced lung injury, and found the weight loss of the mice in the LPS group was significantly higher than that in the Sham group or the TAN group (Fig. 1A). While, TAN treatment notably attenuated LPS-induced weight loss in the LPS + TAN group (Fig. 1A). Besides, we evaluated pulmonary edema via detecting lung W/D ratio, and found that TAN treatment efficiently suppressed LPS-induced the increase of lung W/D ratio (Fig. 1B). Similarly, HE staining analysis showed that TAN treatment notably protected lung histomorphology in the mice with LPS-induced ALI, as evidenced by reducing pulmonary congestion, thickening of the alveolar wall and leukocyte accumulation (Fig. 1C). The scores for lung injury corresponded to these results (Supplementary Fig. 1A). Moreover, Masson staining analysis showed that TAN treatment also evidently alleviated lung interstitial fibrosis induced by LPS (Fig. 1D; Supplementary Fig. 1B). Collectively, these results indicate TAN could evidently attenuate sepsis-induced ALI in mice.
TAN attenuates inflammatory cytokine expression and inflammatory cell infiltration in mice with LPS-induced lung injury
Owing to inflammation acts essential roles in the pathogenesis of sepsis-induced ALI [2, 3], we further examined the expression levels of inflammatory cytokines, and found that TNF-α and IL-1β levels in the serum or BALF samples of LPS group were significantly increased, compared to those in the Sham group or the TAN group (Fig. 2A, B). TAN treatment notably reversed LPS-induced inflammatory cytokines in the LPS + TAN group (Fig. 2A, B). Furthermore, immunofluorescence analysis of the macrophage marker F4/80 in the mice lung tissues, and showed that TAN treatment could significantly inhibit LPS-induced macrophage infiltration in lung tissue (Fig. 2C).
TAN inhibits NLRP3 inflammasome-mediated-macrophage pyroptosis in LPS-induced ALI in mice
Given that NLRP3 inflammasome-mediated pyroptosis is an inflammatory cell death, and acts an important role in the pathogenesis of sepsis-induced ALI [17, 18], we further investigated whether the protective effect of TAN against sepsis-induced ALI is related to regulating NLRP3 inflammasome. As shown in Fig. 3A–C, the mRNA expression levels of NLRP3 and its downstream genes IL-1β and IL-18 were significantly increased in the lung tissues of LPS group, compared to those in the Sham group or the TAN group. However, TAN treatment evidently inhibited these gene expressions in the LPS + TAN group (Fig. 3A–C). Furthermore, Western blotting analysis also showed that TAN treatment evidently inhibited the protein expressions of NLRP3, the cleaved GSDMD which is a surrogate marker of pyroptosis, and the cleaved Caspase-1 in the LPS + TAN group, compared to those in the LPS group (Fig. 3D). Moreover, immunofluorescence analysis showed that TAN treatment significantly inhibited macrophage ASC and GSDMD expressions in the lung tissues of LPS + TAN group, compared to the LPS group (Fig. 3E, F), which further confirms that TAN inhibits NLRP3 inflammasome-mediated-macrophage pyroptosis in LPS-induced ALI in mice.
TAN inhibits ROS production and protects mitochondrial dynamics in LPS-induced ALI in mice
Considering that ROS is a crucial stimuli for the activation of NLRP3 inflammasome [40], and dysfunction of mitochondria could produce huge amount of ROS [41], we further explored whether the protective effect of TAN against NLRP3 inflammasome activation in sepsis-induced ALI is related to regulating ROS production. As shown in Fig. 4A, the ROS levels detected by DHE staining in the lung tissues of the LPS group were significantly increased, compared to those in the Sham group or the TAN group. TAN treatment evidently inhibited ROS production-induced by LPS in the LPS + TAN group (Fig. 4A). Furthermore, we detected the mitochondrial morphology in the lung tissues by TEM, and found that mitochondria were swollen, and round-shaped with smaller size and disrupted mitochondrial cristae in the LPS group, compared to those in the Sham group or the TAN group (Fig. 4B). Whereas, mitochondria were less swollen with elongated appearance and well-organized cristae in the LPS + TAN group, indicating that TAN preserves mitochondrial morphology and alleviates excessive mitochondrial fission in LPS-induced ALI in mice.
Moreover, we explored the protective effect of TAN on mitochondria in the peritoneal macrophages stimulated with LPS and ATP to induce pyroptosis. As shown in the supplementary Fig. 2, 2.5 µM TAN did not evidently influence the cell viability. Subsequently, we found that 2.5 µM TAN treatment significantly suppressed LPS + ATP-induced the depression of mitochondrial membrane potential detected using JC-1 staining and the accumulation of mitochondrial ROS detected using MitoSOX staining (Fig. 4C, D). However, the ROS activator Rotenone treatment notably reversed the protective effects of TAN on the decline of mitochondrial membrane potential dysfunction and mitochondrial ROS accumulation (Fig. 4C, D).
PLK1 is a potential target of TAN for treating sepsis-indued ALI
To clarify the molecular mechanism of TAN protecting mitochondrial morphology in sepsis-induced ALI, we obtained the 2D structure of TAN from MolView (http://molview.org/) (Fig. 5A), and then identified 100 potential target genes of TAN using SwissTargetPrediction database (Supplementary Table 1). Furthermore, 1643 ALI-related genes, and 296 organelle biogenesis and maintenance-related genes were obtained from the GeneCards, respectively (Supplementary Table 1). Through intersecting analysis, cyclin-dependent kinase 1 (CDK1) and PLK1 genes were identified (Fig. 5B). Moreover, the docking energy between TAN and CDK1 or PLK1 was − 7.72 kcal/mol and − 7.06 kcal/mol, respectively, which were both less than − 5.0 kcal/mol, indicating that TAN presents strong binding to CDK1 and PLK1. The binding mode of TAN and CDK1 or PLK1 was visualized by PyMoL (Fig. 5C, D).
Recent studies report that AMPK activation inhibits mitochondrial fission by promoting Drp1 phosphorylation (Ser-637) to attenuate vascular calcification in chronic kidney disease [42], myocardial ischemia–reperfusion injury [43], and allergic rhinitis [44]. PLK1 is identified to promote cholesterol efflux and alleviate atherosclerosis by regulating AMPK [45]. Thus, we speculate that TAN may protect mitochondrial morphology in sepsis-induced ALI through PLK1-AMPK-Drp1 axis. In the following experiments, we focused on PLK1.
TAN promotes PLK1-AMPK-Drp1 signaling in LPS-induced ALI in mice
Subsequently, we explored the effect of TAN on PLK1 expression in sepsis-induced ALI, and found that TAN treatment evidently increased PLK1 expression in the lung tissues, compared to that in the Sham group. Besides, PLK1 expression was significantly decreased in the LPS group, compared to those in the Sham group. Whereas, TAN treatment not only notably suppressed the LPS-induced reduction of PLK1 expression, but also increased AMPK phosphorylation and Drp1 phosphorylation (S637) in the LPS + TAN group, compared to the LPS group (Fig. 6A). Furthermore, immunofluorescence analysis also confirmed that TAN treatment significantly increased PLK1 expression and Drp1 phosphorylation (S637) in the lung tissues of the LPS + TAN group, compared to the LPS group (Fig. 6B, C). Taken together, these results indicate that TAN promotes PLK1-AMPK-Drp1 signaling in LPS-induced ALI in mice.
TAN suppresses NLRP3 inflammasome activation via regulating PLK1-Drp1 signaling in peritoneal macrophages stimulated with LPS and ATP
To explore whether the protective effect of TAN on sepsis-induced ALI was related to PLK1, LPS + ATP-stimulated peritoneal macrophages were pre-treated with the PLK1 inhibitor Volasertib or not. As shown in Fig. 7A, TAN treatment evidently inhibited LPS-induced reduction of PLK1 expression and Drp1 phosphorylation (S637), which was consistent with the result of in vivo (Fig. 6A). Besides, TAN treatment significantly suppressed LPS + ATP-induced NLRP3 expression, GSDMD and Caspase-1 activation (Fig. 6A). However, Volasertib treatment completely blocked the effects of TAN on PLK1 expression, Drp1 phosphorylation (S637), NLRP3 expression, and GSDMD and Caspase-1 activation in LPS + ATP-stimulated peritoneal macrophages (Fig. 7A, B). In addition, Volasertib treatment also blocked the protective effects of TAN on the decline of mitochondrial membrane potential dysfunction and promotion of IL-1β and IL-18 mRNA expressions in LPS + ATP-stimulated peritoneal macrophages (Fig. 7C, D). Collectively, these results indicate that TAN suppresses NLRP3 inflammasome activation via regulating PLK1-Drp1 signaling in LPS + ATP-stimulated peritoneal macrophages.
TAN attenuates LPS-induced ALI in mice via regulating PLK1/AMPK/DRP1/NLRP3 signaling axis
We further investigated the protective mechanism of TAN on sepsis-induced ALI using LPS-stimulated mice pre-treated with the PLK1 inhibitor Volasertib or not. As shown in Fig. 8A, B, and supplementary Fig. 3, Volasertib treatment significantly abolished the protective effects of TAN on the lung pathologic injury and lung interstitial fibrosis in LPS-induced ALI in mice. Furthermore, Volasertib treatment blocked the effects of TAN on PLK1 expression, AMPK phosphorylation, Drp1 phosphorylation (S637), NLRP3 expression, GSDMD and Caspase-1 activation in LPS-induced lung injured tissues (Fig. 8C). Besides, immunofluorescence analysis also confirmed that Volasertib treatment blocked the protective effect of TAN on NLRP3 inflammasome activation in LPS-induced lung injured tissues (Fig. 8D). Moreover, TUNEL analysis showed that Volasertib treatment not only abolished the protective effect of TAN on cellular apoptosis in LPS-induced lung injured tissues, but also aggravated cellular apoptosis (Fig. 8E). Overall, these results suggest that TAN attenuates LPS-induced ALI in mice via regulating PLK1/AMPK/DRP1/ NLRP3 signaling axis.
Discussion
The present study firstly reveals an important role of TAN in alleviating sepsis-induced ALI. TAN administration efficiently inhibited pulmonary edema, reduced reducing pulmonary congestion, thickening of the alveolar wall and leukocyte accumulation, and suppressed inflammatory cytokine expression and inflammatory cell infiltration in sepsis-induced ALI in mice. Mechanistically, TAN suppresses excessive mitochondrial fission, ROS production, and NLRP3 inflammasome-mediated pyroptosis through activating PLK1/AMPK/DRP1 signaling pathway in sepsis-induced ALI (Fig. 9). Our study indicates TAN is a potentially therapeutic candidate against sepsis-induced ALI, and uncovers a novel potential target of TAN, and deepens our understanding on the protective mechanism of TAN on macrophage pyroptosis.
As is known that macrophage pyroptosis plays an essential role in the pathogenesis of sepsis-induced ALI via triggering excessive inflammatory responses [17, 18]. Inhibition of macrophage pyroptosis could efficiently alleviate sepsis-induced ALI [46]. TAN has been reported to suppress osteoarthritis, and renal failure through inhibiting inflammation via regulating NF-κB signaling [31, 47]. In this study, we found that TAN treatment significantly inhibited macrophage pyroptosis via decreasing NLRP3 expression, and GSDMD and Caspase-1 activation in the sepsis-induced ALI. These in vivo and in vitro data strongly support the beneficial role of TAN on macrophage pyroptosis and sepsis-induced ALI.
ROS is known as a prominent stimulator to trigger the activation of NLRP3 inflammasome [48, 49], and dysfunction of mitochondrial fusion and fission produces amounts of ROS [50]. It is well accepted that Drp1 is a main promotor of mitochondrial fission [51], and phosphorylation at Ser-637 inhibits Drp1 activity while phosphorylation at Ser-616 activates Drp1 activity [52]. Previous study reported quercetin protected ethanol-induced hepatocyte pyroptosis through inhibiting ROS-NLRP3 signaling axis via promoting mitochondrial homeostasis [53]. Nodakenin attenuates knee osteoarthritis by regulating mitochondrial Drp1/ROS/NLRP3 axis [54]. Similar to these studies, our study found that TAN treatment could notably restored mitochondrial morphology, and inhibited excessive mitochondrial fission and ROS accumulation in the lung tissue with sepsis-induced ALI. Nowadays, Ser-637 of Drp1 has been reported to be phosphorylated by a kinase anchoring protein 1 (AKAP1) [55], and AMPK [42]. In this study, our study for the first time identified PLK1 is a potential target of TAN via bioinformatic analysis and molecular docking, and TAN alleviated sepsis-induced ALI through inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 (S637) signaling axis. Inhibiting PLK1 via Volasertib evidently blocked the promotive effects of TAN on the AMPK/DRP1(S637) signaling axis, and aggravated NLRP3 inflammasome activation in the lung tissue of septic mice. Besides, phosphorylation at Ser-637, Drp1 could also be phosphorylated at Ser-616 by the cyclin-dependent kinase (CDK) family [56], extracellular-signal-regulated kinase1/2 (ERK1/2) [57], and PKCδ [58]. Previous studies found that TAN inhibited ERK1/2 phosphorylation in a dose-dependent manner in human T47D mammary cancer cells [59], and in human glomerular mesangial cells [60]. Whether the protective role of TAN on macrophage pyroptosis and sepsis-induced ALI is also related to regulate ERK1/2/Drp1(S616) needs to be clarified in the following studies.
In addition to PLK1, our study also identified CDK1 was another potential target of TAN for treating sepsis-induced ALI. CDK1 is reported to promote cell cycle progression through the G2/M phase transition and activate homologous recombination DNA repair pathway [61]. CDK1 is highly expressed in several tumors, such as lung cancer, gastric cancer, and cervical cancer, and functions to promote the stemness of cancer cells, cell proliferation, and cell migration [62,63,64]. Given that CDK1 could phosphorylate Drp1 at Ser-616 [58], and phosphorylation at Ser-637 inhibits Drp1 activity [52], we speculate that the protective effect of TAN on macrophage pyroptosis and sepsis-induced ALI is also related to regulate CDK1/Drp1(S616) signaling axis, which needs to be investigated in the following studies.
In conclusion, our study indicates TAN attenuates sepsis-induced ALI by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis, and TAN is a potentially therapeutic candidate against ALI through inhibiting pyroptosis.
Data availability statement
All data generated or analyzed during this study are included in this published article and its supplementary information files.
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This study was supported by the National Natural Science Foundation of China (82004317), Guangzhou Science and Technology Plan Project (202102010322), and Guangdong Bureau of Traditional Chinese Medicine Project (20241115).
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Conceptualization, YL, YZ, ZS, BD, JZ, and ZZ; data curation, YL, YZ, GY, DZ, ZH, WG; methodology, YL, YZ, GY, DZ, ZH, WG; resources, BD, JZ, and ZZ; supervision, KA, ZS, BD, JZ, and ZZ; validation, YL, YZ, GY, DZ, ZH, WG; writing-original draft, YL, YZ, GY, DZ, ZH, WG, BD, JZ; writing-review and editing, YL, KA, ZS, BD, JZ, and ZZ. All authors have read and agreed to the published version of the manuscript.
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Liu, Y., Zhang, Y., You, G. et al. Tangeretin attenuates acute lung injury in septic mice by inhibiting ROS-mediated NLRP3 inflammasome activation via regulating PLK1/AMPK/DRP1 signaling axis. Inflamm. Res. 73, 47–63 (2024). https://doi.org/10.1007/s00011-023-01819-8
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DOI: https://doi.org/10.1007/s00011-023-01819-8