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. 2024;15(4):289–296. doi: 10.5847/wjem.j.1920-8642.2024.059

Effect of tubastatin A on NLRP3 inflammasome activation in macrophages under hypoxia/reoxygenation conditions

Hao Li 1, Chang Liu 1, Ying Cui 1, Panpan Chang 2,3,4,✉, Wei Chong 1,✉
PMCID: PMC11265631  PMID: 39050221

Abstract

BACKGROUND:

There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock (HS). The aim of this study was to explore the potential of the histone deacetylase 6 (HDAC6)-specific inhibitor tubastatin A (TubA) to suppress nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation in macrophages under hypoxia/reoxygenation (H/R) conditions.

METHODS:

The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8 (CCK8) assay. Briefly, 2.5 μmol/L TubA was used with RAW264.7 cells under H/R condition. RAW264.7 cells were divided into three groups, namely the control, H/R, and TubA groups. The levels of reactive oxygen species (ROS) in the cells were detected using fluorescence microscopy. The protein expression of HDAC6, heat shock protein 90 (Hsp90), inducible nitric oxide synthase (iNOS), NLRP3, gasdermin-D (GSDMD), Caspase-1, GSDMD-N, and Caspase-1 p20 was detected by western blotting. The levels of interleukin-1β (IL-1β) and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA).

RESULTS:

HDAC6, Hsp90, and iNOS expression levels were significantly higher (P<0.01) in the H/R group than in the control group, but lower in the TubA group than in the H/R group (P<0.05). When comparing the H/R group to the control group, ROS levels were significantly higher (P<0.01), but significantly reduced in the TubA group (P<0.05). The H/R group had higher NLRP3, GSDMD, Caspase-1, GSDMD-N, and Caspase-1 p20 expression levels than the control group (P<0.05), however, the TubA group had significantly lower expression levels than the H/R group (P<0.05). IL-1β and IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group (P<0.01), but significantly lower in the TubA group compared to the H/R group (P<0.01).

CONCLUSION:

TubA inhibited the expression of HDAC6, Hsp90, and iNOS in macrophages subjected to H/R. This inhibition led to a decrease in the content of ROS in cells, which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1β and IL-18.

Keywords: Hemorrhagic shock, Hypoxia/reoxygenation, Macrophage, NLRP3, Tubastatin A

INTRODUCTION

Fluid resuscitation after hemorrhagic shock (HS) can result in ischemia/reperfusion injury,[1,2] which is characterized by an excessive inflammatory response triggered by cellular hypoxia and reoxygenation.[3] Macrophages, a crucial component of innate immunity,[4] are highly sensitive to hypoxia/reoxygenation (H/R) and release inflammatory factors, including interleukin-1β (IL-1β) and IL-6. The excessive secretion of these factors can lead to organ damage.[5-7]

The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important component of the inflammatory response and is mainly expressed in macrophages.[8-10] The NLRP3 inflammasome comprises three components: the NLRP3 protein (sensor), apoptosis-associated speck-like protein (ASC) (adapter), and Caspase-1 (effector).[11] NLRP3 recruits Caspase-1 through ASC to form active Caspase-1 p33 (CARD + p20)/p10.[12] Caspase-1 (p33/p10) then cleaves Pro-IL-1β and Pro-IL-18 into IL-1β and IL-18. Additionally, caspase-1 (p33/p10) cleaves the pyroptosis effector protein gasdermin-D (GSDMD) into GSDMD-N, leading to the formation of pores in the cell membrane, which ultimately causes cell pyroptosis.[13] Recent evidence suggests that NLRP3 inflammasome activation in RAW264.7 cells under H/R conditions leads to an excessive inflammatory response.[14-16] However, no effective drug for inhibiting the ischemia/reperfusion injury caused by fluid resuscitation after HS or a specific target for suppressing NLRP3 inflammasome activation in macrophages under H/R conditions is available at present.

Histone deacetylase 6 (HDAC6) controls the deacetylation of histone and non-histone proteins.[17] HDAC6 is involved in several physiological and pathological processes.[18,19] Tubastatin A (TubA) specifically inhibits HDAC6.[20] Chang et al[21] demonstrated that TubA effectively improved the survival rate of a rat model of HS. TubA inhibited HDAC6 in mouse bone marrow-derived macrophages (iBMDM) co-stimulated with lipopolysaccharide (LPS) and nigericin in a mouse sepsis model, which ultimately inhibited NLRP3 inflammasome activation,[22] suggesting that TubA has the potential to inhibit NLRP3 inflammasome activation in macrophages during H/R. However, its exact mechanism of action remains unknown.

The heat shock protein 90 (Hsp90), a molecular chaperone, is a non-histone substrate of HDAC6.[23] Its primary role is to preserve the activated state of a target protein by maintaining its conformation.[24] HDAC6 promotes Hsp90, which ensures the stability of the target protein.[25] TubA inhibits Hsp90 by inhibiting HDAC6 in macrophages.[26] Knocking out Hsp90 in macrophages decreases the expression of inducible nitric oxide synthase (iNOS) and the production of reactive oxygen species (ROS).[27] Increased ROS promote NLRP3 inflammasome activation in macrophages under H/R.[14] Therefore, we proposed the following hypothesis: TubA can inhibit HDAC6 in macrophages under H/R conditions and attenuate Hsp90 and iNOS, leading to a reduction in ROS production and weakening NLRP3 inflammasome activation. The main objective of this study was to investigate the effect of TubA on NLRP3 inflammasome activation in H/R-induced macrophages, to provide new therapeutic strategies for treating systemic inflammatory responses induced by fluid resuscitation after HS.

METHODS

Reagents

Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA, 11995); fetal bovine serum (FBS, Gibco, USA, 10099141); Tub A (Selleck, USA, 804912); cell-counting kit-8 (CCK8, Abbkine, China, C0041); ROS assay kit-highly sensitive DCFH-DA (Dojindo, Japan, R252); RIPA lysate (Beyotime, China, P0013B); PMSF (Beyotime, China, ST506); alkaline phosphatase inhibitor (Beyotime, China, ST019); micro BCA protein assay kit (Thermofisher, USA, 23235); membrane blocking solution (Solarbio, China, SW3010); TBST (Solarbio, China, T1087); anti-HDAC6 antibody (Cell Signal Technology, USA, #7612S); anti-NLRP3 antibody (Cell Signal Technology, USA, #15101); anti-caspase 1 p20 antibody (Cell Signal Technology, USA, 89332S); anti-GSDMD antibody (Abcam, UK, ab209845); anti-caspase-1 antibody (Abcam, UK, ab138483); anti-Hsp90 antibody (Abcam, UK, ab59459); anti-iNOS antibody (Abcam, UK, ab178945); goat anti-mouse HRP (Abcam, UK, ab6728); goat anti-rabbit HRP (Abcam, UK, ab6721); ECL chemiluminescence substrate (Tanon, China,180-501); IL-1β mouse ELISA kit (AssayGenie, Ireland, MOFI00058), and IL-18 mouse ELISA kit (Boster, China, EK0433) were used.

Cell culture and H/R treatment

The mouse leukemic monocyte/macrophage cell line, RAW264.7, was purchased from the American Type Culture Collection (USA, TIB-71). RAW264.7 cells were cultured under the following conditions: complete medium was used for culture at 37 °C in an atmosphere with 5% CO2. When the cells had grown to a 70% confluence, H/R treatment was performed. The cells were divided into three groups: (1) control group, in which the cells were cultured in a serum-free medium at 21% O2 for 6 h, followed by a complete medium at 21% O2 for 6 h; (2) H/R group, in which the cells were subjected to hypoxia in serum-free medium at 1% O2 for 6 h, followed by reoxygenation in complete medium at 21% O2 for 6 h; and (3) TubA group, in which the cells were subjected to hypoxia with 2.5 μmol/L TubA prepared in serum-free medium at 1% O2 for 6 h, followed by reoxygenation with 2.5 μmol/L TubA in a complete medium at 21% O2 for 6 h. After treatment, the cells and supernatants were collected for analysis.

Cell viability

A CCK8 assay was used to assess the viability of RAW264.7 cells under H/R conditions with varying concentrations of TubA (0, 2.5, 5, 10, and 20 μmol/L). A total of 4×105 cells/mL were seeded in 96-well plates with 100 μL of medium. After the cells were subjected to a normal culture conditions, H/R treatment was administered. At the end of the treatment, 10 μL of CCK8 reagent was added to each well and incubated for 30 min at 37 °C. The absorbance was measured at 450 nm using a microplate reader.

ROS assay

The ROS production in the cells was assayed using a fluorescence microscope. The measurements were performed following the manufacturer’s instructions. The fluorescence intensity was observed using a fluorescence microscope by selecting the fluorescein isothiocyanate (FITC) channel.[28]

Western blotting analysis

The total protein content of the cells was extracted and quantified using a micro BCA protein assay kit. Subsequently, the proteins were denatured and subjected to electrophoresis and membrane transfer. The membranes were then treated with a membrane-blocking solution for 1 h and incubated with primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature. Finally, a chemiluminescent imaging system was used for image acquisition.

Enzyme-linked immunosorbent assay (ELISA)

The levels of IL-1β and IL-18 in the cell supernatant were measured using the IL-1β mouse ELISA kit and IL-18 mouse ELISA kit, respectively. Measurements were conducted following the manufacturer’s instructions, and a microplate reader was used for detection.

Statistical analysis

Statistical analysis was performed using SPSS 26.0 software (IBM, USA). Differences between groups were evaluated using one-way analysis of variance (ANOVA), followed by Bonferroni post-hoc multiple comparison tests. A P-value less than 0.05 was considered statistically significant. GraphPad Prism 9 software was used for data visualization.

RESULTS

Effect of different concentrations of TubA on the viability of macrophages under H/R

To determine the most suitable concentration of TubA, we evaluated the viability of RAW264.7 cells treated with different concentrations of TubA (0, 2.5, 5, 10, and 20 μmol/L) under H/R using a CCK8 kit. As shown in Figure 1, significant changes in cell viability were detected. Specifically, compared to that in the 0 μmol/L group, the cell viability increased in the 2.5 μmol/L group (P<0.01). However, the cell viability gradually decreased in the 5 and 10 μmol/L groups compared to the 2.5 μmol/L treatment group (P<0.05).

Figure 1.

Figure 1

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Effect of different concentrations of TubA on the viability of macrophages under H/R. Compared with 0 μmol/L, **P<0.01; compared with 2.5 μmol/L, #P<0.05, ##P<0.01 (n=4). H/R: hypoxia/reoxygenation; TubA: tubastatin A.

Effect of TubA on the expression of HDAC6, Hsp90, and iNOS in macrophages under H/R

The expressions of HDAC6, Hsp90, and iNOS in RAW264.7 cells were detected using western blotting. As shown in Figure 2, the expressions of HDAC6, Hsp90, and iNOS were higher in the H/R group than those in the control group (P<0.01). In addition, the TubA group exhibited decreased expressions of HDAC6, Hsp90, and iNOS compared with the H/R group (P<0.05).

Figure 2.

Figure 2

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Effect of TubA on the expression of HDAC6, Hsp90, and iNOS in macrophages under H/R. A: western blotting results of the protein expression of HDAC6, Hsp90, and iNOS in RAW264.7 cells; B: the relative protein expression levels. Compared with the control group, **P<0.01; compared with the H/R group, ##P<0.01, #P<0.05 (n=3). The concentration of TubA was 2.5 μmol/L. H/R: hypoxia/reoxygenation; TubA: tubastatin A.

Effect of TubA on the levels of ROS in macrophages under H/R

The ROS levels in RAW264.7 cells were measured using fluorescence microscopy. As shown in Figure 3, the fluorescence intensity of ROS was significantly higher in the H/R group than that in the control group (P<0.01). Additionally, compared with the H/R group, the TubA group exhibited a significant decrease in the fluorescence intensity of ROS (P<0.05).

Figure 3.

Figure 3

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Effect of TubA on the ROS in macrophages under H/R. A: ROS levels in RAW264.7 cells measured under fluorescence microscopy; B: semi-quantitative analysis of ROS levels in each group. Compared with the control group, **P<0.01; compared with the H/R group, #P<0.05 (n=3). The concentration of TubA was 2.5 μmol/L. H/R: hypoxia/reoxygenation; TubA: tubastatin A.

Effect of TubA on NLRP3 inflammasome activation in macrophages under H/R

Western blotting was performed to assess the expression of key proteins involved in the NLRP3 inflammasome pathway. As depicted in Figure 4, compared with those in the control group, the protein levels of NLRP3, GSDMD, caspase-1, GSDMD-N, and caspase-1 p20 in the H/R group were significantly higher (P<0.05). Additionally, compared with those in the H/R group, the protein levels of NLRP3, GSDMD, caspase-1, GSDMD-N, and caspase-1 p20 in the TubA group were significantly lower (P<0.05).

Figure 4.

Figure 4

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Effect of TubA on NLRP3 inflammasome activation in macrophages under H/R. A: western blotting analysis of the protein expressions of NLRP3, GSDMD, caspase-1, GSDMD-N, and caspase-1 p20 in RAW264.7 cells. B: the relative expression levels of the NLRP3, GSDMD, caspase-1, GSDMD-N, and caspase-1 p20 proteins. Compared with the control group, **P<0.01, *P<0.05; compared with the H/R group, #P<0.05 (n=3). The concentration of TubA was 2.5 μmol/L. H/R: hypoxia/reoxygenation; TubA: tubastatin A.

Effect of TubA on IL-1β and IL-18 secretion by macrophages under H/R

ELISA was performed to measure the levels of IL-1β and IL-18 in the supernatants of RAW264.7 cells. As shown in Figure 5, the secretion of IL-1β and IL-18 in cell supernatants increased significantly compared to that in the control group (P<0.01). Additionally, the TubA group exhibited a significant decrease in the secretion of IL-1β and IL-18 in cell supernatants compared to the H/R group (P<0.01).

Figure 5.

Figure 5

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Effect of TubA on IL-1β and IL-18 secretion in macrophages under H/R. A: IL-1β secretion by RAW264.7 cells; B: IL-18 secretion by RAW264.7 cells. Compared with the control group, **P<0.01; compared with the H/R group, ##P<0.01 (n=3). The concentration of TubA was 2.5 μmol/L. H/R: hypoxia/reoxygenation; TubA: tubastatin A; IL-1β: interleukin-1β.

DISCUSSION

Fluid resuscitation after HS can lead to ischemia/reperfusion injury, which can trigger an excessive inflammatory response.[29] Macrophages play a crucial role in sensing and initiating the inflammatory response during H/R.[6,15,30] Our study demonstrated that the upregulation of HDAC6, Hsp90, and iNOS led to an increase in ROS levels in macrophages under H/R. ROS facilitate NLRP3 inflammasome activation, resulting in the secretion of IL-1β and IL-18.

As a multifunctional protein, HDAC6 plays a role in various physiological and pathological processes.[19,31] Under H/R, the expression of HDAC6 increases as an adaptive response to maintain normal cell function.[32] Similarly, Hsp90 expression increases under this stress.[33,34] Elevated expression of HDAC6 and Hsp90 prevents iNOS degradation, leading to increased iNOS expression.[35] Increased iNOS expression resulted in increased ROS production in RAW264.7 cells.[36] Under H/R, the elevated ROS levels in RAW264.7 cells cause expression of NLRP3, ASC, and Caspase-1 p20 to rise, which in turn trigger the activation of the NLRP3 inflammasome.[14] Previous findings also confirmed that the NLRP3 inflammasome is activated in H/R-stimulated RAW264.7 cells.[15,37] HDAC6 inhibition is therefore required to reduce excessive downstream inflammatory responses.

The HDAC6-specific inhibitor, TubA, can be used to treat HS[21,38,39] and inhibit NLRP3 inflammasome activation.[22] Therefore, TubA was used to inhibit the activation of HDAC6 and the NLRP3 inflammasome. Furthermore, TubA is an antineoplastic medication.[40] It may exhibit some toxicity to RAW264.7 cells, derived from a mouse leukemia macrophage line. In the absence of any intervention, 2.5, 5, and 10 μmol/L TubA had inhibitory effects on RAW264.7 cells, at low, medium, and high doses, respectively.[26] Therefore, we investigated the effects of 2.5, 5, 10, and 20 μmol/L TubA on the viability of RAW264.7 cells under H/R. Our findings indicated that 2.5 μmol/L TubA improved the viability of RAW264.7 cells under H/R. Our findings contrast with those of a previous study showing that 10 μM TubA inhibited NLRP3 inflammasome activation in iBMDM stimulated with LPS and nigericin.[21] This difference may be attributed to variations in the source of the macrophages and the specific intervention protocol used.

The primary function of TubA is the suppression of HDAC6 activity.[41] A previous study has demonstrated that TubA decreases the transcript levels of HDAC6.[42] Therefore, TubA inhibits the transcription of HDAC6, leading to a reduction in its expression. The stability of Hsp90 and HDAC6 was maintained through their interaction.[43] HDAC6 degradation leads to a reduction in Hsp90 expression. ROS and iNOS expression were suppressed by the decrease in Hsp90 expression.[27] The reduction in ROS inhibits NLRP3 inflammasome activation and results in decreased secretion of IL-1β and IL-18.

As depicted in Figure 6, TubA effectively inhibited the expressions of HDAC6, Hsp90, and iNOS in macrophages under H/R. This inhibition led to a decrease in the production of ROS, which subsequently hindered NLRP3 inflammasome activation and the release of IL-1β and IL-18. In a HS rat model, NLRP3 inflammasome activation resulted in myocardial ischemia/reperfusion injury[44] and pyroptosis of hippocampal neurons.[45] However, the therapeutic effects of TubA remain elusive. Therefore, in the subsequent phase of our study, we will investigate the effect of TubA on NLRP3 inflammasome activation in an animal model of fluid resuscitation following HS. This research provides a comprehensive theoretical foundation for the treatment of ischemia/reperfusion injury caused by fluid resuscitation after HS.

Figure 6.

Figure 6

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Potential mechanisms of tubastatin A (TubA) on NLRP3 inflammasome activation in macrophages under hypoxia/reoxygenation (H/R). The expression of HDAC6, Hsp90, and iNOS is increased in macrophages under H/R, resulting in elevated levels of ROS and NLRP3 inflammasome activation. Cleavage of GSDMD leads to the formation of GSDMD-N, which is then inserted into the cell membrane. This leads to an increase in the secretion of IL-1β and IL-18. TubA inhibits the expression of HDAC6, Hsp90, and iNOS in macrophages under H/R, thereby reducing ROS levels and inhibiting NLRP3 inflammasome activation. Additionally, TubA decreases the expression of GSDMD and GSDMD-N, along with the secretion of IL-1β and IL-18.

CONCLUSION

The specific HDAC6 inhibitor, TubA, was found to effectively inhibit the expressions of HDAC6, Hsp90, and iNOS in macrophages under H/R. Consequently, it reduced the levels of ROS and inhibited the activation of the NLRP3 inflammasome, further leading to a decrease in the secretion of IL-1β and IL-18. These findings can provide new therapeutic strategies for treating systemic inflammatory responses induced by fluid resuscitation after HS.

Footnotes

Funding: This work was supported by National Natural Science Foundation of China (82102315).

Ethical approval: This work involved the use of RAW264.7 cell lines and therefore no ethical approval was required. The authors declare no experimentation on human or animals were designed.

Conflicts of interest: The authors declare no conflicts of interest.

Author contributions: WC and PC contributed to the conception of the study. HL performed the experiment, performed the data analyses and wrote the manuscript. CL and YC helped perform the analysis with constructive discussions. All authors reviewed the manuscript.

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Articles from World Journal of Emergency Medicine are provided here courtesy of The Second Affiliated Hospital of Zhejiang University School of Medicine

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