Sirtuin 3 attenuates sepsis-induced acute lung injury by suppressing NF-κB/NLRP3/GSDMD-mediated pyroptotic signaling

Abstract

BACKGROUND Sepsis is a major global health threat and frequently leads to acute lung injury (ALI). Pyroptosis has been increasingly implicated in the inflammatory progression of sepsis-induced ALI. Sirtuin 3 (Sirt3), a mitochondrial deacetylase, can regulate inflammatory injury and cellular stress responses. However, its role in sepsis-induced ALI and pulmonary pyroptosis remains incompletely understood.

METHODS Sirt3 expression was measured in peripheral blood mononuclear cells (PBMCs) and neutrophils from septic patients (n=10) and healthy controls (n=5). Wild-type (WT) and Sirt3 knockout (Sirt3^−/−) mice were subjected to cecal ligation and puncture (CLP). Lung injury, inflammatory responses, transcriptomic changes, and nuclear factor-κB (NF-κB)/NLRP3/gasdermin D (GSDMD)-related signaling were evaluated. In vitro, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of Sirt3 knockdown and overexpression on lipopolysaccharides (LPS)/nigericin-induced pyroptotic injury.

RESULTS Sirt3 expression was reduced in circulating immune cells from septic patients. In mice, CLP decreased pulmonary Sirt3 expression, and Sirt3 deficiency aggravated lung histological injury, pulmonary edema, and systemic inflammatory responses. Transcriptomic analysis revealed enrichment of immune- and pyroptosis-related pathways in septic lungs from Sirt3^−/− mice. Consistently, Sirt3 deficiency increased NF-κB/NLRP3/GSDMD-associated signaling, as shown by increased p-NF-κB/NF-κB, NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, N-terminal gasdermin D (N-GSDMD), and GSDMD levels. In HUVECs, Sirt3 knockdown exacerbated LPS/nigericin-induced cell injury, whereas Sirt3 overexpression preserved cell viability and reduced lactate dehydrogenase (LDH) release.

CONCLUSION Sirt3 downregulation is associated with aggravated sepsis-induced ALI and enhanced NF-κB/NLRP3/GSDMD-related pyroptotic signaling. These findings suggest that Sirt3 may serve as a potential regulatory node in endothelial inflammatory injury during sepsis-induced ALI.

INTRODUCTION

Sepsis represents a major global health burden and is a leading cause of mortality among hospitalized patients.^[1,2] It is clinically characterized by life-threatening organ dysfunction arising from an imbalanced host response to infection.^[3,4] The lung is particularly vulnerable to sepsis, and septic patients frequently develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), which are associated with poor outcomes.^[5] Despite advances in mechanical ventilation and treatment protocols, the mortality rate of sepsis complicated by ARDS remains as high as 32.9%.^[6] Therefore, identifying the molecular mechanisms that drive inflammatory lung injury in sepsis remains clinically important.

Pyroptosis is an inflammatory form of lytic programmed cell death that contributes to immune activation and tissue injury. Inflammasome activation promotes caspase-mediated cleavage of gasdermin D (GSDMD), generating N-terminal GSDMD (N-GSDMD), which forms membrane pores and drives the release of proinflammatory mediators.^[7] Clinical and animal studies have shown increased caspase-1 activity, inflammasome activation, and GSDMD cleavage in sepsis-associated ARDS or septic lung injury,^[8,9] suggesting that pyroptosis in the lungs is involved in sepsis-induced ALI.

Sirtuin 3 (Sirt3) is a mitochondrial NAD⁺-dependent deacetylase that regulates mitochondrial metabolism, redox balance, and stress responses,^[10] and has been increasingly recognized as a cytoprotective factor against organ injury.^[11] In sepsis, Sirt3 has been reported to attenuate oxidative stress, inflammasome activation, and organ dysfunction.^[12-14] A study suggested that serum Sirt3 levels are associated with infection severity and poor prognosis in sepsis patients.^[15] However, its role in sepsis-induced ALI, particularly its relationship with pulmonary pyroptosis and endothelial injury, remains incompletely understood.

In this study, we examined Sirt3 expression in circulating immune cells from septic patients and investigated its role in sepsis-induced ALI using a cecal ligation and puncture (CLP) mouse model. Transcriptomic analysis was performed to identify Sirt3-associated inflammatory and pyroptosis-related pathways in lungs of septic mice. In addition, loss- and gain-of-function experiments in endothelial cells were performed to evaluate whether Sirt3 modulates lipopolysaccharide (LPS)/nigericin-induced pyroptotic injury. Together, this study aimed to clarify the potential protective role of Sirt3 in sepsis-induced ALI and its association with NF-κB/NLRP3/GSDMD-related pyroptotic signaling.

METHODS

Sample collection

Ten septic patients and five healthy controls were included. Sepsis was diagnosed according to the Sepsis-3 criteria.^[4] To evaluate the clinical relevance of Sirt3 in sepsis, Sirt3 mRNA expression was measured in circulating immune cells from septic patients and healthy controls. Peripheral blood was collected, and peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated within 2 h.

Animals and experimental design

We examined whether Sirt3 deficiency affected sepsis-induced ALI in vivo. Male C57BL/6N mice and Sirt3 knockout mice on a C57BL/6N background were used. Sirt3 knockout mice were generated by CRISPR/Cas9 genome editing (Cyagen Inc., China; KOCMP-64384-Sirt3-B6N-VA). All animal procedures were approved by the Laboratory Animal Ethics Committee of China-Japan Friendship Hospital (ZRDWLL240042).

Sepsis was induced by CLP. The mice underwent laparotomy followed by CLP, whereas the sham mice underwent laparotomy without ligation or puncture. The detailed procedures are provided in supplementary file 1.

After acclimatization, the wild-type (WT) and Sirt3^−/− mice were randomly assigned to each genotype for sham or CLP surgery, with six mice initially included per group. Blood and lung tissues were collected 24 h after surgery. Because the samples were allocated to different assays, the number of assay-specific biological replicates varied and is indicated in the corresponding figure legends.

Histological analysis

The right middle lobe lung tissues of mice were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histopathological evaluation. Lung injury severity was scored according to previously described criteria.^[16] Histological assessment was conducted in a single-blinded manner by an experienced investigator who was unaware of group allocation.

Lung wet/dry ratio

The wet weight of the freshly isolated left lung was recorded immediately after excision. The tissue was subsequently dried at 70 °C for 24 h until its weight stabilized. The dry weight was recorded to calculate the wet/dry weight ratio.

Enzyme-linked immunosorbent assay (ELISA)

Serum interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels were measured using commercial ELISA kits, as listed in supplementary Table 1.

Transcriptome analysis

To explore the molecular changes associated with Sirt3 deficiency during sepsis-induced lung injury, RNA-seq analysis was performed on lung tissues. Lung samples from four groups of mice were randomly collected for transcriptomic analysis: WT+sham (n=3), Sirt3^−/−+sham (n=3), WT+CLP (n=3), and Sirt3^−/−+CLP (n=4). Total RNA extraction, library preparation, sequencing, and data processing were performed. The primary comparison of interest was Sirt3^−/−+CLP versus WT+CLP, which was used to identify Sirt3-dependent transcriptional changes under septic conditions. For exploratory screening, candidate differentially expressed genes (DEGs) were defined as genes with |log2 fold change|>1 and nominal P<0.05. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the clusterProfiler package^[17] in R. Detailed RNA-seq procedures and bioinformatic preprocessing are provided in supplementary file 2.

Western blotting

We further examined whether Sirt3 deficiency was associated with activation of the NF-κB/NLRP3/GSDMD pyroptosis-related pathway. Total protein was extracted from lung tissues or human umbilical vein endothelial cells (HUVECs), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking, the membranes were incubated with primary antibodies against Sirt3, p-NF-κB, NF-κB, NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), pro-caspase-1, cleaved caspase-1, gasdermin D (GSDMD), N-GSDMD, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The protein bands were visualized and quantified by densitometry. Detailed information on the antibodies and reagents is listed in supplementary Table 1.

Cell culture and Sirt3 modulation

To determine whether Sirt3 influences endothelial pyroptotic injury, loss- and gain-of-function experiments were performed in HUVECs. HUVECs were obtained from procell and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin under standard conditions (37 °C, 5% CO₂). To establish an in vitro pyroptosis model, cells were primed with LPS (2 μg/mL) for 3 h and then stimulated with nigericin (5 μmol/L) for 1 h. Information on the cells and key reagents used for the cell experiments is provided in supplementary Table 1.

For Sirt3 knockdown, HUVECs were transfected with negative control siRNA (si-NC) or Sirt3-specific siRNA (si-Sirt3) using Lipofectamine 3000. For Sirt3 overexpression, HUVECs were transduced with negative control lentivirus (LV-NC) or Sirt3-overexpressing lentivirus (LV-Sirt3). Knockdown and overexpression efficiency were verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The detailed transfection and transduction procedures are provided in supplementary files 3 and 4.

Cell viability and cytotoxicity assays

Cell viability and cytotoxicity were assessed using cell counting kit 8 (CCK-8) and lactate dehydrogenase (LDH) assays, respectively, as listed in supplementary Table 1.

qRT-PCR

Total RNA was extracted using TRIzol reagent and reverse-transcribed into complementary DNA (cDNA). qRT-PCR was performed using SYBR-based detection. Relative mRNA expression was normalized to that of GAPDH and calculated using the 2^−ΔΔCt method. The primer sequences are listed in supplementary Table 2.

Statistical analysis

The data were presented as the mean±standard error of the mean (SEM). Comparisons between two groups were performed using Student’s t test, and multiple-group comparisons were analyzed by one-way analysis of variance (ANOVA) followed by appropriate post hoc tests. Statistical analyses were performed using SPSS 25.0, with a P-value < 0.05 considered significant.

RESULTS

Sirt3 expression was reduced in sepsis, and Sirt3 deficiency aggravated CLP-induced lung injury

The general clinical data of sepsis patients, including age, sex, vital signs, blood gas analysis results, inflammatory markers, and quick Sequential Organ Failure Assessment (qSOFA) score, are listed in supplementary Table 3. Compared with healthy controls, septic patients had significantly lower Sirt3 mRNA expression in both neutrophils and PBMCs (Figure 1A, B).

Figure 1. Clinical relevance and in vivo phenotype. A, B: Sirt3 mRNA expression in neutrophils and PBMCs from septic patients (n=10) and HCs (n=5); C: Sirt3 protein expression in lung tissues; D: representative H&E staining of lung tissues from different experimental groups; Scale bars: 1000 μm (low magnification) and 50 μm (high magnification); E: quantification of lung injury scores based on histopathological assessment; F: lung wet/dry weight ratio (n=4); G-I: serum levels of IL-1β, TNF-α, and IL-6 (n=5). The data are presented as the mean±SEM. ^*P<0.05, ^**P<0.01, ^***P<0.001. Sirt3: sirtuin 3; PBMCs: peripheral blood mononuclear cells; HCs: healthy controls; WT: wild-type; CLP: cecal ligation and puncture; IL-1β: interleukin-1β; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; SEM: standard error of the mean; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

In WT mice, CLP reduced Sirt3 protein expression in lung tissues, whereas Sirt3 expression was markedly diminished in Sirt3^−/− mice (Figure 1C). Histological analysis showed that CLP induced obvious lung injury, including alveolar structural disruption, inflammatory cell infiltration, and alveolar edema. These pathological changes were more pronounced in the Sirt3^−/− mice after CLP (Figure 1D). The lung injury scores and lung wet/dry weight ratios were significantly higher in the Sirt3^−/−+CLP group than in the WT+CLP group (Figure 1E, F). In addition, serum IL-1β, TNF-α, and IL-6 levels were further increased in Sirt3^−/− mice following CLP (Figure 1G, H, I). These findings suggest that Sirt3 deficiency aggravates CLP-induced lung injury and systemic inflammatory responses.

Sirt3 deficiency enhanced inflammatory and pyroptosis-associated signaling in lungs of septic mice

A comparison between Sirt3^−/−+CLP and WT+CLP screened 327 candidate DEGs (supplementary Table 4). GO enrichment analysis of these candidate DEGs revealed enrichment of immune- and inflammation-related terms, including innate immune response, pyroptotic inflammatory response, and canonical inflammasome complex (Figure 2A). Additional KEGG enrichment analysis showed enrichment of inflammatory signaling pathways, including the NOD-like receptor, NF-κB, Toll-like receptor, and TNF signaling pathways (supplementary Figure 1).

Figure 2. Transcriptomic analysis and NF-κB/NLRP3/GSDMD pyroptosis signaling. A: GO enrichment analysis was performed using candidate DEGs from the Sirt3^-/-+CLP versus WT+CLP mice comparison. B: representative Western blots showing the protein expression of p-NF-κB, NF-κB, NLRP3, ASC, cleaved caspase-1, pro-caspase-1, N-GSDMD, GSDMD, and GAPDH in lung tissues; C-I: quantitative analysis of p-NF-κB/NF-κB, NLRP3/GAPDH, ASC/GAPDH, cleaved caspase-1/GAPDH, pro-caspase-1/GAPDH, N-GSDMD/GAPDH, and GSDMD/GAPDH expression. All the data are presented as the mean±SEM. n=3-4 mice per group for Western blotting quantification, as indicated in individual panels. ^*P<0.05, ^**P<0.01, ^***P<0.001. N-GSDMD: N-terminal fragment of GSDMD; Sirt3: sirtuin 3; WT: wild-type; CLP: cecal ligation and puncture; BP: biological process; CC: cellular component; MF: molecular function.

Western blotting analysis showed that the p-NF-κB/NF-κB ratio was increased in the lungs of septic mice but further increased in those of the Sirt3^−/−+CLP group (Figure 2B, C). In parallel, NLRP3 and ASC protein levels were elevated in Sirt3^−/− mice after CLP (Figure 2B, D, E). Caspase-1 activation was markedly increased in Sirt3-deficient lungs, as evidenced by elevated levels of cleaved caspase-1 (Figure 2B, F), whereas pro-caspase-1 levels remained largely unchanged (Figure 2B, G). In parallel, we observed a significant increase in the N-GSDMD, the functional pore-forming moiety that executes pyroptotic cell death (Figure 2B, H), along with elevated full-length GSDMD (Figure 2B, I). These results indicate that the loss of Sirt3 was associated with increased NF-κB/NLRP3/GSDMD-related pyroptotic signaling in septic lung tissue.

Sirt3 modulated LPS/nigericin-induced endothelial pyroptotic injury in vitro

Sirt3 knockdown was confirmed by Western blotting and RT-qPCR in si-Sirt3-transfected cells (Figure 3A, B). Conversely, compared with negative control lentivirus (LV-NC), lentiviral transduction with LV-Sirt3 significantly increased Sirt3 expression (Figure 3C, D ).

Figure 3. Sirt3 regulates endothelial pyroptosis in vitro. A-B: the knockdown efficiency of Sirt3 was verified by Western blotting and qRT-PCR; C-D: the overexpression efficiency of Sirt3 was confirmed by Western blotting and qRT-PCR; E-F: cell viability was assessed by a CCK-8 assay; G-H: LDH release in the culture supernatants; I-J: representative brightfield images of HUVECs under different treatments. The white arrows indicate cells with a pyroptotic morphology. Scale bar = 25 μm. All the data are presented as the mean±SEM. n=3 independent experiments for Western blotting and qRT-PCR; n=5 replicate wells per group for the CCK-8 and LDH assays. ^*P<0.05; ^**P<0.01; ^***P<0.001. LPS: lipopolysaccharide; Ni: nigericin; LDH: lactate dehydrogenase; qRT-PCR: quantitative real-time polymerase chain reaction; HUVECs: human umbilical vein endothelial cells; CCK-8: cell counting kit 8; Sirt3: sirtuin 3; SEM: standard error of the mean; CON: control.

Following LPS priming and nigericin stimulation, HUVECs showed reduced cell viability, increased LDH release, and morphological changes consistent with pyroptotic cell death. Sirt3 knockdown further decreased cell viability and increased LDH release under LPS/nigericin stimulation (Figure 3 E, G). In contrast, Sirt3 overexpression preserved cell viability and reduced LDH release (Figure 3 F, H). Bright-field microscopy showed that LPS/nigericin-induced cell swelling and membrane bubble-like structures were aggravated by Sirt3 knockdown but alleviated by Sirt3 overexpression (Figure 3 I, J). These findings suggest that Sirt3 attenuates endothelial pyroptotic injury under septic-like inflammatory stimulation. Consistent with these findings, the results of the qRT-PCR analysis revealed that Sirt3 knockdown increased, whereas Sirt3 overexpression decreased, the expression of selected inflammatory and pyroptosis-related genes in response to LPS/nigericin stimulation (supplementary Figures 2, 3).

DISCUSSION

This study investigated the potential role of Sirt3 in sepsis-induced ALI by integrating clinical observations, a CLP mouse model, transcriptomic analysis, and endothelial cell experiments. The Sirt3 mRNA expression was reduced in neutrophils and PBMCs from septic patients. In mice, CLP decreased pulmonary Sirt3 expression, and Sirt3 deficiency aggravated lung histological injury, pulmonary edema, and systemic inflammatory responses. Transcriptomic analysis further suggested that Sirt3 deficiency was associated with enrichment of immune- and pyroptosis-related pathways under septic conditions. Consistent with these findings, Western blotting revealed increased NF-κB/NLRP3/GSDMD-related signaling in the lungs of septic Sirt3^−/− mice. In HUVECs, Sirt3 knockdown exacerbated, whereas Sirt3 overexpression attenuated, LPS/nigericin-induced endothelial injury. Together, these findings suggest that Sirt3 may act as a protective factor against sepsis-induced lung injury, partly through limiting pyroptosis-associated inflammatory signaling.

Neutrophils are the key effectors of innate immune responses during the initial phase of sepsis. Their dysregulated activation and excessive accumulation in the lungs can exacerbate endothelial activation, barrier dysfunction, and leukocyte recruitment, all of which drive the progression of sepsis-induced ALI and ARDS by amplifying local inflammation and vascular permeability.^[18]PBMCs also undergo profound functional changes during sepsis, and previous studies have linked PBMC pyroptosis and immune cell transcriptomic remodeling to disease severity and mortality.^[19,20] In this context, our finding that Sirt3 expression was reduced in both neutrophils and PBMCs provides clinical evidence that Sirt3 dysregulation is not limited to injured lung tissue but may also reflect systemic immune disturbance during sepsis. Although the clinical cohort was limited in size, these observations support the relevance of Sirt3 in the host immune response and provide a clinical basis for further mechanistic investigation.

Sirt3 has been reported to protect against several forms of sepsis-associated organ injury, including acute kidney injury,^[12] myocardial damage,^[21] gastrointestinal injury,^[22] and disruption of the endothelial barrier.^[14] Consistent with these studies, we observed reduced Sirt3 protein expression in lung tissues and more severe lung injury in Sirt3^-/- mice after CLP. Our transcriptomic profiling revealed that the loss of Sirt3 fundamentally reshaped the pulmonary response to sepsis. The enrichment of pathways related mainly to innate immune responses, NF-κB signaling, pyroptosis, and the canonical inflammasome complex suggests that the loss of Sirt3 promotes a transcriptional environment permissive for inflammasome activation. These findings are consistent with studies showing that Sirt3 can suppress NLRP3 activation and mitigate inflammatory injury in sepsis models.^[23]

Both in vivo and in vitro data indicated that reduced Sirt3 expression enhanced pyroptotic injury. In Sirt3^−/− mice, GSDMD expression was elevated in CLP-injured lungs, whereas in HUVECs, Sirt3 knockdown amplified pyroptotic features, including increased cell death and characteristic large pyroptotic bubbles. These observations are consistent with those of a previous study showing that pyroptosis, mediated by NLRP3 inflammasome activation and GSDMD cleavage, contributes to sepsis-induced lung injury.^[24] Mechanistically, Sirt3 appears to restrain the activity of the NFκB/NLRP3/GSDMD pyroptosis axis, a key pathway driving inflammatory cell death in sepsis-induced ALI. Previous reports have shown that loss of Sirt3 enhances inflammasome activation, leading to increased caspase-1 cleavage and GSDMD-mediated pyroptosis.^[23,25] In endothelial cells, Sirt3 knockdown increases the transcription of NF-κB, NLRP3, ASC, caspase-1, and proinflammatory cytokines, whereas in vivo, Sirt3 deficiency promotes inflammasome protein accumulation in the lungs. These findings align with the well-established model whereby NF-κB provides a priming signal for NLRP3 transcription, which, upon activation, triggers caspase1 cleavage and GSDMD-dependent pyroptotic cell death.^[26] Our cellular and animal data, together with those of prior studies, suggest that Sirt3 functions as a critical brake on inflammasome-mediated pulmonary pyroptosis during sepsis.

Pulmonary endothelial injury is a central event in sepsis-induced ALI and is linked to inflammatory stimulation, barrier disruption, vascular hyperpermeability, pulmonary edema, and the progression of lung injury.^[27,28] Recent studies have shown that endothelial pyroptosis disrupts vascular integrity and aggravates organ injury in sepsis models.^[29-31] Consistent with these findings, Sirt3 depletion in vascular tissues promotes mitochondrial superoxide accumulation and NF-κB activation, linking impaired Sirt3 signaling to inflammatory vascular dysfunction.^[32] In the present study, gain- and loss-of-function experiments in HUVECs provided functional evidence supporting the protective role of Sirt3 in endothelial inflammatory regulation. These data suggest that endothelial Sirt3 may regulate inflammasome-driven pyroptosis and preserves vascular barrier function during sepsis, thereby mitigating the development of septic lung injury. Although pulmonary endothelial cells are more lung specific, HUVECs are an established human endothelial model for studying conserved inflammatory and injury responses under septic-like stimulation; therefore, our in vitro data support a general endothelial protective role of Sirt3 and complement the in vivo findings.

Several limitations should be acknowledged. First, the clinical cohort size was relatively small, and Sirt3 expression in clinical samples was assessed only at the mRNA level; therefore, larger cohorts with protein-level validation are needed to confirm its clinical relevance. Second, although Sirt3 deficiency was associated with increased NF-κB/NLRP3/GSDMD-related pyroptotic signaling, the direct acetylation substrates of Sirt3 in this pathway were not identified. Third, in vivo Sirt3 rescue experiments were not performed, limiting the strength of causal inference in the animal model. Future studies using prospective clinical cohorts, Sirt3 rescue strategies, and acetylation-focused mechanistic approaches will be important to validate and extend these findings.

CONCLUSION

Sirt3 downregulation was associated with aggravated sepsis-induced lung injury and enhanced NF-κB/NLRP3/GSDMD-related pyroptotic signaling. Targeting Sirt3 may represent a potential strategy for limiting endothelial inflammatory injury in sepsis-induced ALI, although further mechanistic and clinical validation is needed.

Funding: This study was supported by grants from the National Natural Science Foundation of China (82272196) and the National High Level Hospital Clinical Research Funding (2022-NHLHCRF-YS-03).

Ethical approval: For the clinical component of the study, the study protocol was reviewed and approved by the Ethics Committee of China-Japan Friendship Hospital (approval number: 2024-KY-417-1), and all experimental procedures were conducted in accordance with the principles of the Declaration of Helsinki. For animal experiments, all procedures were approved by the Laboratory Animal Ethics Committee of China-Japan Friendship Hospital (approval number: ZRDWLL240042).

Conflicts of interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

Contributors: GQZ and WJZ designed the study. WJZ, NG, and RL conducted the animal experiments. WJZ oversaw all in vitro experiments. WJZ, SY, and HTL performed the western blot analyses. WJZ analyzed the data and drafted the manuscript. GQZ, STY, and WJZ critically revised the manuscript. All authors read and approved the final version of the manuscript.