Circular RNA circVAPA modulates macrophage pyroptosis in sepsis‐induced acute lung injury through targeting miR‐212‐3p/Sirt1/Nrf2/NLRP3 axis

Abstract

Sepsis‐induced acute lung injury (ALI) is an inflammatory condition involving the pyroptosis of macrophages. This study investigated the role of circular RNA hsa_circ_0006990 (circVAPA) in regulating macrophage pyroptosis in ALI and the underlying mechanisms. The expression pattern of circVAPA was examined in the mouse model of ALI and in the LPS‐treated RAW264.7 macrophage cell line. Lung tissue damage was evaluated by haematoxylin and eosin staining, immunohistochemistry and a myeloperoxidase activity assay. The molecular mechanisms were investigated by luciferase reporter assay, western blot, RT‐qPCR and ELISA. circVAPA was down‐regulated in the lung tissues of ALI mice and LPS‐induced RAW264.7 cells. circVAPA over‐expression alleviated lung tissue injury and dampened LPS‐induced pyroptosis and Th17‐associated inflammatory responses. miR‐212‐3p was identified as a target of circVAPA, and miR‐212‐3p negatively regulated the expression of Sirt1. Sirt1 knockdown largely abolished the effect of circVAPA over‐expression on pyroptosis. CircVAPA/miR‐212‐3p/Sirt1 axis also regulates Nrf2 and NLRP3 expression upon LPS challenge. By targeting miR‐212‐3p, circVAPA over‐expression negatively regulates the expression of Sirt1 and pyroptosis‐related factors (Nrf2 and NLRP3), which alleviates the inflammatory damages in sepsis‐induced ALI.

1. INTRODUCTION

The term sepsis is used to describe a complication which can be seen following trauma or severe infection. It can lead to multiple organ dysfunction and septic shock, such as acute lung injury (ALI). ¹ ALI induced by sepsis is due to serious inflammatory damage in lung tissues. In recent years, the number of patients with severe sepsis has increased by about 1.5% every year worldwide and the mortality rate of resulting ALI is about 40%, which thus poses a serious health threat to septic patients. ² After the onset of sepsis‐induced ALI, excessive inflammation is associated with lung fibrosis and impaired lung function. Sepsis‐induced ALI is characterized by diffuse macrophage infiltration into lung tissues, va scular permeability disturbance and pulmonary oedema. ³ , ⁴ The recruitment of macrophages can also trigger undesirable activation of other immune cells such as Th17 cells. ⁵ , ⁶ However, the exact pathogenic mechanisms underlying macrophage activation in ALI are still unclear. Since there is no effective treatment to prevent the progression of ALI, understanding the mechanisms underlying macrophage activation could provide insights into the formulation of intervention strategies.

Pyroptosis is a form of inflammatory cell death associated with inflammatory damages in tissues. ⁷ The process of  pyroptosis is dependent on inflammasome‐dependent activation of caspase‐1 and Gasdermin D (GSDMD), which eventually leads to cell rupture and release of pro‐inflammatory cytokines including interleukin‐1β (IL‐1β) and interleukin‐18 (IL‐18). ⁸ , ⁹ In this pathway, the NLR family pyrin domain containing 3 (NLRP3) inflammasome recruits apoptosis‐related spot‐like proteins to activate caspase‐1 which cleaves GSDMD and pro‐forms of IL‐1β and IL‐18. ¹⁰ , ¹¹ Although the NLRP3‐dependent inflammasome plays a crucial role in anti‐microbial infection defence, excessive activation of inflammasome in pyroptosis culminates in uncontrolled inflammatory tissue damage. ¹² It is well‐known that NLRP3‐dependent inflammasome activation can induce macrophage pyroptosis in ALI. ¹³ Although this pathway has been implicated in the progression of ALI, the mechanisms governing pyroptosis activation in macrophages in ALI remain to be elucidated. ¹⁴ , ¹⁵ , ¹⁶

Recently, Nrf2/HO‐1 pathway activation has been reported to inhibit inflammasome activity. ¹⁷ , ¹⁸ Nrf2 (NFE2L2 NFE2 like bZIP transcription factor 2) is a nuclear transcription factor orchestrating antioxidant defence upon oxidative stress, and it mediates the transcription of various antioxidant genes and enzymes to neutralize reactive oxygen species to mitigate oxidative damages. ¹⁹ , ²⁰ Therefore, Nrf2 is recognized as the master regulator of cytoprotective genes with anti‐inflammatory and antioxidant functions. ²¹ In addition, sirtuin 1 (Sirt1) has been shown to play a pivotal role in preventing lung injury caused by inflammation, ²² and its activation could facilitate Nrf2‐mediated antioxidant signalling. ²³ Therefore, the Sirt1/Nrf2 signalling axis may be involved in the progression of sepsis‐induced ALI. Understanding the molecular interplay of Sirt1, Nrf2 and NLRP3 could shed light on the regulation of pyroptosis and inflammatory responses during ALI.

Circular RNA (circRNA) is a class of non‐coding RNA with circular structure. The dysregulation of circRNAs has been widely reported in different pathophysiological conditions. There is also evidence that circRNAs may play regulatory roles in sepsis‐induced ALI. ²⁴ , ²⁵ As a member of circRNA, circVAPA (hsa_circ_0006990) has been found to be up‐ or down‐regulated in various tumours and diseases, ²⁶ , ²⁷ but its potential engagement in ALI has not been reported. In this study, we found the down‐regulation of circVAPA in the LPS‐induced ALI mouse model and LPS‐treated macrophage cell line. By targeting miR‐212‐3p, circVAPA modulated the expression of downstream factors including Sirt1, Nrf2, NLRP3 and impinged on macrophage pyroptosis. Over‐expression of circVAPA could alleviate inflammatory damages in the lung tissues of the ALI model, and attenuate the expression of Th17 markers such as IL‐17A and IL23. These data suggest that targeting circVAPA could serve as a potential strategy to ameliorate lung tissue damages in sepsis‐induced ALI.

2. MATERIALS AND METHODS

2.1. Sepsis‐induced ALI model in mice

Wild‐type C57BL/6 mice were raised under standard humidity and temperature conditions with 12 h dark/light cycle. Mice were randomly divided into 4 groups (N = 6 in each group). The ALI model was induced by LPS (L2880, Sigma Aldrich, WI, USA). In the sham group mice were injected with placebo (PBS); in the LPS group mice were injected with LPS (10 mg/kg, i.p); in the LPS + vector group mice were injected with LPS (10 mg/kg, i.p) and empty expression vector; and in the LPS + circVAPA group mice were injected with LPS (10 mg/kg, i.p) and circVAPA expression vector. After anaesthesia 72 hours‐post‐treatment the mice were sacrificed. The left lung was collected for histological and molecular analysis, and the right lung was perfused with sterile saline for three times before bronchoalveolar lavage fluid (BALF) collection and macrophage isolation. A portion of the left lung tissues was weighed and homogenized in a lysis buffer to obtain a supernatant. Another portion of the left lung was rinsed with saline and fixed in formalin for histological evaluation.

The animal experiments were approved by the Animal Care and Use Committee at the First Affiliated Hospital of Fujian Medical University.

2.2. Haematoxylin and eosin (HE) staining

H&E staining was performed using HE Stain Kit (C1015S, Beyotime, Beijing, China). The left lung tissue was fixed in formalin and then dehydrated with ethanol. The tissues were embedded in paraffin and cut into 5 μm sections. The sections were deparaffinized and rehydrated, and stained with haematoxylin aqueous solution for 5 min. After rinsing with distilled water (dH2O), Bluing Reagent was applied to completely cover the tissue sections for 2 min. After further washing with dH2O, the section was dehydrated in absolute alcohol, followed by staining with Eosin Y Solution for 2–3 min. The section was rinsed using absolute ethanol for three times and then mounted on to a slide. The images were recorded under a microscope (CX23, Olympus, Japan). A blind lung injury scoring system was used to document the lung injury. Briefly alveolar congestion, alveolar wall thickening and oedema, and fibrosis were scored on the 0 ~ 3 scale for each item (0: None, 1: Mild, 2: Moderate, 3: Severe), with a maximum score of 9 for each sample. Ten randomly selected fields from each sample with approximately the same number of alveoli were assessed by two pulmonary pathologists in a blinded fashion.

2.3. Immunohistochemistry (IHC)

The levels of RORγt, IL‐17α and IL‐23 in the left lung were detected by IHC. The collected lung tissues were prepared as 5 μm sections in paraffin as described previously. After deparaffinization and rehydration, antigen retrieval was performed by heating the section in citrate unmasking solution (SignalStain® Citrate Unmasking Solution (10X) (#14746), Cell Signaling Technologies, CA, USA) at 95°C for 10 min. Sections were cooled on bench top for 30 min, and then washed in dH₂O three times for 5 min each. The sections were further incubated in 3% hydrogen peroxide for 10 min., and then blocked for 1 h at room temperature in TBST buffer with 5% normal Goat Serum. The following primary antibodies were used for staining: anti‐RORγt (ab113434), anti‐IL‐17α (ab79056) and anti‐IL‐23 (ab190356) (1:200, Abcam, Cambridge, UK) for 18 h at 4°C. The rabbit IgG was used as negative control (sham) for the staining. The section was soaked with three drops of SignalStain® Boost Detection Reagent (HRP, Rabbit #8114, Cell Signaling Technologies) for 30 min at room temperature, followed by the addition of 300 μL SignalStain® substrate (#8059, Cell Signaling Technologies) for 5 min. After rinsing with dH₂O twice, the section was counter‐stained by haematoxylin for 1 min, and the images were recorded using a light microscope (CX23, Olympus, Japan).

2.4. Measurement of pulmonary oedema

Pulmonary oedema was evaluated by dry/wet weight method. A small piece of left lung tissue was taken from each group for rapid determination of wet weight (W), and then placed in a high temperature oven at 80°C for 48 h for dehydration before weighing (D). The dry/wet ratio of lung tissue was calculated as D/W × 100%.

2.5. Myeloperoxidase (MPO) Activity

MPO kit (KCW20262, Mlbio, Shanghai, China) was used to detect the activity of MPO in lung tissue as described previously. ²⁰ According to the instructions, the tissue was weighed and mixed with the homogenizing medium for 30 min incubation at 37°C. 5% of tissue homogenate was collected and centrifuged at 10,000× g for 10 min. A quantity of 100 μL supernatant was mixed with 50 μL reaction reagent at 37°C for 30 min. The activity of MPO was recorded by a spectrophotometer using absorbance at 460 nm, and the value was normalized to the tissue weight.

2.6. ELISA assay

Lung tissue homogenate, lung lavage fluid or the lysate of RAW264.7 cells were used for cytokine determination by ELISA. The commercial ELISA kits for IL‐1β (PI301), IL‐18 (PT513) and IL‐6 (PI326) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). A quantity of 100 μL sample was used to measure concentrations of the cytokines. The samples were added to the capture‐antibody‐coated plate for 1 h incubation at 37°C. After a wash step to remove unbound material, 100‐μL biotin‐labelled detection antibody was added for 30 min incubation, which was followed by the staining with 100 μL streptavidin–HRP. A volume of 50 μL chemiluminescent detection reagents were used for signal development, and the optical density of samples and standards was measured at 450 nm using a microplate reader (Infinite 200 PRO; Tecan, CA, USA). The concentration of each cytokine was measured based on the linear regression of the standards.

2.7. Cell culture and transfection

The mouse macrophage line RAW264.7 was cultured in DMEM supplemented with 10% foetal bovine serum (26,140,087, Thermo Fisher Scientific, CA, USA) and 1% penicillin/streptomycin (SV30010, Hyclone, CA, USA) at 37°C with 5% CO₂. Lipofectamine™2000 (11,668,019, Invitrogen, Shanghai, China) transfection reagents were used to transfect circVAPA plasmid or vector, Sirt1 siRNA, miR‐212‐3p mimic or the corresponding controls in to RAW264.7 cells. CircVAPA expression plasmid, Sirt1 siRNA and the corresponding controls were synthesized by Genepharma (Shanghai, China). miR‐212‐3p mimic and miR‐NC were purchased from RiboBio Biotech (Guangzhou, China). Briefly, cells were seeded in 6‐well plates at a density of 5 × 10⁵ cells/well. Twenty‐four h later, 100 nm of siRNA/miRNA mimic or 6 μg of plasmid was added into 100 μL Opti‐MEM® I Reduced‐Serum Medium (31,985,062, Invitrogen, CA, USA), and then 6 μL Lipofectamine 2000 reagent was added for 10 min incubation at room temperature. The mixture was added to cell culture for 48 transfection before further experimental analysis.

2.8. RNA extraction and RT‐qPCR

Total RNA was extracted from tissues or cells using TRIzol reagent (15,596,026, Thermo Fisher Scientific, CA, USA). After quantification, 1 μg of total RNA was used for reverse‐transcription into cDNA using First Strand cDNA Synthesis Kit (R21101, Vazyme, Beijing, China) kit. AceQ® qPCR SYBR® Green Master Mix (Q111‐02, Vazyme, Beijing, China) was used for qPCR detection on the 7500 Real Time PCR System (Applied Biosystems, CA, USA). GAPDH was used as the internal reference for target gene normalization by 2^−ΔΔCt method. All primers were synthesized by Anhui Universal Biosynthesis (Anhui, China).

2.9. Western blot assay

Cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail (P0013C, Beyotime, Beijing, China). Total protein was quantitated by a BCA Protein assay kit (P0009, Beyotime, Beijing, China). SDS‐PAGE electrophoresis was performed to separate protein bands and the separated protein samples were transferred to a PVDF membrane (P2005, Beyotime, Beijing, China). Subsequently, the membrane was blocked with 5% skimmed milk for 2 h. The membrane was labelled with anti‐Sirt1 (ab12193), anti‐Nrf2 (ab62352), anti‐NLRP3 (ab4207), anti‐ASC (ab236996), anti‐GSDMD‐N (ab215203), anti‐pro‐caspase 1 (ab286125), anti‐caspase 1 p20/p22 (ab207802) and anti‐actin (ab209857) (at 1:1000 dilutions, Abcam, Cambridge, UK) antibodies for 12 h at 4°C, followed by further incubation with the secondary antibodies (ab131368 and ab96899 at 1:300 dilutions) for 2 h. The membrane was washed four times with TBST buffer and the protein bands were visualized using an enhanced chemiluminescence kit (sc‐2048, Santa Cruz, TX, USA) and photographed on a gel imager system (Bio‐Rad, CA, United States). The densitometry analysis was performed with Image J software (Bethesda, MD, USA).

2.10. Dual luciferase reporter assay

The wild‐type‐binding sites between circVAPA/miR‐212‐3p and miR‐212‐3p/Sirt1 mRNA 3'UTR were predicted by Starbase (https://starbase.sysu.edu.cn/starbase2/) or Tarrget Sacn online tools (http://www.targetscan.org/TargetScan). The corresponding wild‐type (WT)‐binding sites or mutated‐binding sites (MUT) at circVAPA and the 3'UTR of Sirt1 mRNA were cloned into the pmirGLO luciferase reporter vector (E1330, Promega, WI, USA). The luciferase reporter containing WT or Mut‐binding sites (circVAPA‐WT, circVAPA‐MUT, Sirt1‐WT or Sirt1‐MUT) was co‐transfected into RAW264.7 cells together with miR‐NC or miR‐212‐3p mimic using Lipofectamine 2000. Then, 48‐h post‐transfection, the relative luciferase activities were measured using Dual‐Luciferase Reporter Assay Kit (E1910, Promega, WI, USA) on a luminescence microplate reader (Infinite 200 PRO; Tecan, CA, USA). The relative firefly luciferase activity in the reporter plasmid was normalized to that of Renilla luciferase.

2.11. Data analysis

The data analysis involved in the study was conducted using GraphPad Prism 6.0. (GraphPad software, NY, USA). The statistical difference between two groups was compared using unpaired student's t tests. Comparisons among multiple groups were analysed using one‐way analysis of variance (ANOVA) with Tukey's post hoc test for pairwise comparison. All experimental data are expressed as mean ± SD. p < .05 indicates a statistical significance.

3. RESULTS

3.1. Over‐expression of circVAPA attenuates sepsis‐induced ALI

To investigate the role of circVAPA in sepsis‐induced ALI, we constructed a mouse model of ALI induced by LPS, with the PBS injection as the control. H&E staining confirmed that the lung tissue of mice in the LPS group was severely damaged, with disrupted lumen, alveolar congestion, alveolar wall thickening and oedema, and increased fibrosis; while the level of lung tissue injury in the LPS plus circVAPA over‐expression group was significantly alleviated, with an H&E staining showing more organized lumen structure (Figure 1A). We detected the level of circVAPA in the lung tissues in all experimental groups, and found that circVAPA level was reduced in the LPS group when compared to the sham group, and the administration of circVAPA expression vector indeed increased circVAPA level in the lung tissue (Figure 1B). Further, the pulmonary oedema determined by the dry/wet method showed that the wet–dry ratio of lung tissue in the LPS group was significantly increased when compared to that of the sham group, which significantly decreased after circVAPA over‐expression (Figure 1C).

FIGURE 1.

Over‐expression of circVAPA attenuates sepsis‐induced ALI. (A) The pathological changes in mouse lung tissues of each experimental group was detected by H&E staining. The lung injury scores were summarized from the tissue sections of six mice in each group. (B) The expression levels of circVAPA were detected by qRT‐PCR in each group (n = 6 in each group). (C) The pulmonary oedema was assessed in each group by wet/dry tissue measurement method (n = 6 in each group). (D) The myeloperoxidase activity (MPO, a peroxidase enzyme abundantly expressed in neutrophil granulocytes) was detected in lung the tissues of each group (n = 6 in each group). (E) The levels of IL‐1β, IL‐6 and TNF‐α were detected by qRT‐PCR in each group (n = 6 in each group, each sample was assayed in triplicates). (F) FISH staining of circVAPA in the lung tissues of Sham and LPS model group. Images are the representative of three different samples. *p < .05; **p < .01; ***p < .001.

The examination of MPO (a peroxidase enzyme abundantly expressed in neutrophil granulocytes) activity in lung tissues showed that ativity was enhanced in the LPS group and that circVAPA over‐expression dampened its activity (Figure 1D). This indicates that circVAPA over‐expression could attenuate the recruitment and activity of neutrophils in the lung tissues upon LPS injection. In order to further assess the inflammatory conditions in the lung tissues, we analysed the relative expression of inflammatory factors by qRT‐PCR. The results showed that the mRNA levels of IL‐1β, IL‐6 and TNF‐α were significantly elevated in the LPS group, and that these up‐regulations were suppressed by the over‐expression of circVAPA (Figure 1E). To confirm the implication of circVAPA in LPS‐induced ALI, we performed fluorescence in situ hybridization (FISH) of circVAPA and the results confirmed the down‐regulation of circVAPA in LPS model group (Figure 1F). Together, these results indicate that circVAPA expression is repressed in the LPS‐induced ALI and the over‐expression of circVAPA could alleviate the inflammatory damages in ALI.

3.2. circVAPA attenuates macrophage pyroptosis in sepsis‐induced ALI through Sirt1/Nrf2/NLRP3 pathway

Next bronchoalveolar lavage fluid (BALF) was collected from the lung tissue and the macrophages were isolated. We performed Western blot to examine pyroptosis‐related proteins and the relative expression levels of Sirt1 and Nrf2 in macrophages of each experimental group. It was found that pyroptosis‐related proteins including NLRP3, ASC, GSDMD‐N (activated from) and caspase‐1 (p20 activated form) became up‐regulated in LPS‐induced group in comparison to the controls, while the over‐expression of circVAPA partially reversed these changes (Figure 2A). In contrast, Sirt1 and Nrf2 showed down‐regulation upon LPS treatment, and the over‐expression of circVAPA rescued their expression in LPS + circVAPA group (Figure 2A). In addition, ELISA measurement revealed that the levels of pro‐inflammatory cytokines including IL‐1β and IL‐18 in BALF were significantly increased in the LPS group, and circVAPA over‐expression partially suppressed the production of IL‐1β and IL‐18 (Figure 2B). These findings suggest that circVAPA may suppress the macrophage pyroptosis through the Sirt1/Nrf2/NLRP3 pathway.

FIGURE 2.

circVAPA relieves macrophage pyroptosis through the Sirt1/Nrf2/NLRP3 pathway. (A) The levels of Sirt1, Nrf2, NLRP3, ASC, GSDMD‐N and caspase 1 p20 were detected by Western blot in macrophages isolated from bronchoalveolar lavage fluid (BALF) of lung tissues in each group. Data are the summary of six samples from each group. (B) The expressions of IL‐1β and IL‐18 were detected in BALF of each group by ELISA. Data are the summary of six samples from each group. *p < .05; **p < .01; ***p < .001.

3.3. circVAPA modulates IL‐23/Th17 axis in sepsis‐induced ALI

IL‐23/Th17 axis balance plays a crucial role in the progression of sepsis‐induced ALI and the associated inflammation. ⁷ To this end, we investigated the infiltration of Th17 and the expression of IL‐23 by IHC in the lung tissues of each experimental group. The results showed that the staining signals of RORγt (Th17 master transcription factor), IL‐17α and IL‐23 in the lung tissues of the LPS group were significantly increased in comparison to the controls, while circVAPA over‐expression heavily reduced the staining signals of the above molecules (Figure 3A–C). Therefore, circVAPA over‐expression not only attenuates the inflammatory activation of macrophages but also reduces the recruitment of Th17 in sepsis‐induced ALI.

FIGURE 3.

circVAPA acts on IL‐23/Th17 axis balance in sepsis‐induced ALI. (A–C) The expression of RORγt, IL‐17α and IL‐23 were detected in lung tissues of each group by IHC. Red arrows indicate the positively stained cells. Data are the summary of six samples from each group. *p < .05; **p < .01; ***p < .001.

3.4. circVAPA modulates macrophage pyroptosis by targeting Sirt1/Nrf2/NLRP3 axis in vitro

To further verify the effect of circVAPA on Sirt1/Nrf2/NLRP3‐mediated macrophage pyroptosis, RAW264.7 cells were treated with LPS as an in vitro cell model. qRT‐PCR analysis showed the decreased level of circVAPA upon LPS treatment, and the transfection of circVAPA vector could increase circVAPA level in LPS‐induced RAW264.7 cells (Figure 4A). Consistent with the results of animal model, LPS treatment dampened the expression of Sirt1 and Nrf2 in RAW264.7 cells, while the expressions of pyroptosis‐related proteins (NLRP3, ASC, GSDMD‐N and caspase 1 p20) showed up‐regulation upon LPS induction. Moreover, the over‐expression of circVAPA could at least partially reverse the changes induced by LPS (Figure 4B). LPS challenge also promoted the production of IL‐1β and IL‐18 in the supernatant of RAW264.7 cell culture, and this effect was mitigated by circVAPA over‐expression (Figure 4C). Thus, these results further corroborate the in vivo observation that circVAPA regulates the macrophage pyroptosis through the Sirt1/Nrf2/NLRP3 axis.

FIGURE 4.

circVAPA inhibits Sirt1/Nrf2/NLRP3‐mediated macrophage pyroptosis. (A) The expressions of circVAPA was detected in RAW264.7 cells induced by LPS and transfected with an empty vector or circVAPA expression vector. (B) The expressions of Sirt1, Nrf2, NLRP3, ASC, GSDMD‐N and caspase 1 p20 were detected by Western blot in different groups. (C) The levels of IL‐1β and IL‐18 were detected in different groups by ELISA. All the data are the summary of three different experiments. *p < .05; **p < .01; ***p < .001.

3.5. circVAPA regulates Sirt1 by targeting miR‐212‐3p

Based on previous knowledge of circRNA, we hypothesized that circVAPA may also function as a molecular sponge for downstream miRNA to regulate Sirt1 and pyroptosis. To explore this we used the Starbase online tool which predicted potential interaction sites between circVAPA and miR‐212‐3p (Figure 5A). For further confirmation, we conducted dual luciferase reporter assay using the reporter containing wild‐type (circVAPA‐WT) or mutated‐binding sites (circVAPA‐MUT) in the presence of miR‐212‐3p mimic or miR‐NC control. miR‐212‐3p over‐expression inhibited the luciferase activity of circVAPA‐WT reporter, while this effect was abolished after mutation of the predicted‐binding sites (Figure 5A), indicating their interaction via the wild‐type‐binding sequences. Similarly, miR‐212‐3p might interact with the 3'UTR of Sirt1 mRNA as predicted by TargetScan database (Figure 5B). The luciferase reporter assay using Sirt1‐WT or Sirt1‐MUT reporter also supports the notion that miR‐212‐3p binds to the predicted wild‐type sequences within the 3'UTR of Sirt1 mRNA (Figure 5B). These results collectively imply that circVAPA/miR‐212‐3p axis regulates Sirt1 in macrophages. To further verify this point, RAW264.7 cells were transfected with control vector, circVAPA over‐expression vector, and circVAPA over‐expression vector together with miR‐NC or miR‐212‐3p mimic. circVAPA over‐expression significantly increased the level of sirt1, while the co‐transfection of miR‐212‐3p mimic abrogated the effect of circVAPA over‐expression on Sirt1 (Figure 5C). Therefore, circVAPA could regulate Sirt1 expression by targeting miR‐212‐3p in RAW264.7 cells.

FIGURE 5.

circVAPA regulates Sirt1 by targeting miR‐212‐3p. (A) The potential binding sites of circVAPA and miR‐212‐3p were predicted by Starbase database and their interaction was validated in RAW264.7 cells by dual‐luciferase reporter assay. (B) The potential binding sequences of miR‐212‐3p and 3'‐UTR of Sirt1 mRNA was predicted by TargetScan database, and their interaction was validated in RAW264.7 cells by dual‐luciferase reporter assay. (C) The effects of circVAPA over‐expression and miR‐212‐3p mimic on the protein levels of Sirt1 in LPS‐induced RAW264.7 cells was detected by Western blot. All the data are the summary of three different experiments.*p < .05; **p < .01; ***p < .001.

3.6. Sirt1 knockdown partially reverses the effect of circVAPA over‐expression on macrophage pyroptosis

To further investigate whether Sirt1 plays a key role in modulating the effect of circVAPA on macrophage pyroptosis, we applied synthesized Sirt1 siRNA which could efficiently knock down Sirt1 protein level in RAW264.7 cells (Figure 6A). In LPS‐induced RAW264.7 cells, the effects of circVAPA over‐expression on Sirt1, Nrf2 and pyroptosis‐related markers (NLRP3, ASC, GSDMD‐N and caspase 1 p20) were partially attenuated after the co‐transfection of Sirt1 siRNA (Figure 6B). In addition, the suppressive function of circVAPA over‐expression on LPS‐induced IL‐1β and IL‐18 production was also impaired upon Sirt1 siRNA co‐transfection (Figure 6C). The results suggest that Sirt1 at least partially mediates the effect of circVAPA on the regulation of LPS‐induced macrophage pyroptosis.

FIGURE 6.

Knockdown of sirt1 partially reverses the effect of circVAPA over‐expression on macrophage pyroptosis. (A) Knockdown efficacy of si‐Sirt1 was validated in RAW264.7 cells by Western blot. (B) The expressions of Sirt1, Nrf2, NLRP3, ASC, GSDMD‐N and caspase 1 p20 were detected by Western blot in RAW264.7 cells induced by LPS and transfected with empty vector or circVAPA expression vector, or transfected with circVAPA expression vector and Sirt1 siRNA. (C) The levels of IL‐1β and IL‐18 were detected in different groups by ELISA. All the data are the summary of three different experiments. *p < .05; **p < .01; ***p < .001.

4. DISCUSSION

Sepsis is a systemic inflammatory syndrome due to microbial infection, which can eventually cause damage in multiple organs. ²⁸ Previous studies have shown that the lung is one of the key organs susceptible to sepsis‐induced injuries. ²⁹ ALI is characterized by the recruitment of immune cells, elevated inflammatory reaction, fibrosis and degenerated lung functions. ³⁰ Nevertheless, the mechanisms underlying the inflammatory damages in ALI remain to be fully studied. In the in vivo mouse model of ALI and an in vitro model of RAW264.7 cell line induced by LPS, we reported the down‐regulation of circVAPA and found that circVAPA expression could alleviate LPS‐induced lung tissue damage. circVAPA over‐expression could ameliorate pulmonary oedema, attenuate the accumulation of inflammatory cells and hinder the release of inflammatory factors in the lung tissues. This is accompanied by diminished macrophage pyroptosis as demonstrated LPS‐induced RAW264.7 cells.

Excessive inflammation is one of the key features in the progression of sepsis‐induced ALI. The recruitment of immune cells such as macrophages and Th17 cells results in the over‐activation of immune responses in lung tissues, thereby promoting the onset and progression of inflammatory damage. ³¹ To investigate the effect of circVAPA on inflammation, we detected the expression of inflammatory factors in lung tissues of ALI model, and found that the circVAPA over‐expression could attenuate the up‐regulation of inflammatory factors such as IL‐1β, IL‐6 and TNF‐α in LPS‐induced ALI. In addition, since the glycoprotein MPO present in the intracellular granules is a marker of neutrophil recruitment, MPO activity can indicate neutrophil activity in the lung tissue. ³² As expected, the activity of MPO in ALI model mice was increased, while the over‐expression of circVAPA reduced MPO activity in LPS‐induced lung tissues. In addition, the activation IL‐23/Th17 axis is closely related to inflammatory damages including sepsis‐induced ALI. ³³ In our model, we found that RORγt, IL‐17α and IL‐23 levels in the lung tissues were significantly increased in LPS‐induced ALI mouse model, indicating the activation of IL‐23/Th17 axis; while the over‐expression of circVAPA significantly mitigated IL‐23/Th17 axis activation. Therefore, circVAPA can act as an immunosuppressive factor to dampen inflammatory responses in sepsis‐induced ALI.

The NLRP3‐dependent inflammasome is a key pathway to activate pyroptosis and promote inflammation in macrophages and other innate immune cells under pathological conditions. ³⁴ NLRP3‐mediated inflammasomes can activate caspase‐1 to cleave the pro‐form of IL‐1β for the maturation. In addition, caspase‐1 also cleaves gasdermin (GSDMD) and the N‐terminal of GSDMD could perforate the membrane for cytokine release. ⁷ , ³⁴ Accordingly, our data showed that LPS induction caused the increase of NLRP3, activated caspase‐1 p20 and GSDMD‐N in macrophages, which was associated with the elevation of IL‐1β production in the cell culture. However, the over‐expression of circVAPA significantly repressed pyroptosis activation and inhibited pro‐inflammatory cytokine production. Previous studies have implicated circRNAs in sepsis‐related inflammation. For example, circRNA 0001105 could protect the intestinal barrier against sepsis‐induced damage by attenuating inflammation and oxidative stress. ³⁵ circEXOC5 could significantly alleviate LPS‐induced release of inflammatory factors and improve ALI in mice. ³⁶ Our study added novel evidence regarding the role of circVAPA in sepsis‐induced ALI, which is in agreement with the notion that circRNAs can modulate sepsis‐induced inflammatory responses. ³⁷

Sirt1 is an NAD⁺‐dependent histone deacetylase, with versatile roles in cell signalling and inflammatory response. ³⁸ A previous study has shown that Sirt1 can mediate NF‐κB and p53 pathways in severe acute pancreatitis. ³⁹ Another study reported that the down‐regulation of Sirt1 attenuated the effect of miR‐30d‐5p depletion on hypoxia‐induced apoptosis. ⁴⁰ Our study revealed that miR‐212‐3p negatively regulated Sirt1 in RAW264.7 macrophages, and miR‐212‐3p was identified as a target of circVAPA. Sirt1 knockdown could largely abolish the effect of circVAPA over‐expression on macrophage pyroptosis. Therefore, CircVAPA/miR‐212‐3p/Sirt1 axis plays a key role in modulating LPS‐induced pyroptosis in macrophages.

Although the results for circVAPA and the relevant signalling axis molecules on the AKI animal model are encouraging, several questions require further studies to clarify. First, the precise mechanism governing circVAPA down‐regulation upon LPS treatment remains unknown. Next, how Sirt1 affects NLRP3‐dependent inflammasome activation also warrants further investigation. Since this study focused on the circVAPA/miR‐212‐3p/Sirt1 axis, we mainly studied the Sirt1/Nrf2 pathway. It is unclear whether circVAPA could also target other signalling processes to regulate NLRP3‐dependent inflammasome.

5. CONCLUSION

In conclusion, we reported that circVAPA is down‐regulated in sepsis‐induced ALI, and forced over‐expression of circVAPA can alleviate LPS‐induced ALI. The mechanism of the protective effects by circVAPA may lie in the suppression of NLRP3‐dependent inflammasome activation through miR‐212‐3p/sirt1 signalling axis in macrophages, which in turn dampens the accumulation of neutrophils/Th17 cells and inflammatory damages in the lung tissues. Future work is warranted to dissect the mechanisms governing circVAPA down‐regulation in sepsis‐induced ALI.

AUTHOR CONTRIBUTIONS

Conception and design: Yiming Lim, Yanjing Huang, Jinquan Lin. Data analysis and interpretation: Yanjing Huang, Jinquan Lin, Zhiwei Wu. Manuscript writing: All authors. Final approval of manuscript: All authors.

FUNDING INFORMATION

Natural Science Foundation of Fujian Province (No. 2020J01959).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Huang Y, Lin J, Wu Z, Li Y. Circular RNA circVAPA modulates macrophage pyroptosis in sepsis‐induced acute lung injury through targeting miR‐212‐3p/Sirt1/Nrf2/NLRP3 axis. Int J Exp Path. 2024;105:21‐32. doi: 10.1111/iep.12497