Remimazolam attenuates traumatic brain injury-induced acute lung injury by suppressing pulmonary epithelial pyroptosis

Abstract

Background and objectives

TBI-induced acute lung injury (TBI-ALI), with an incidence rate of 22-25%, represents a critical determinant of secondary mortality. Remimazolam is a novel sedative that has shown potential for anti-inflammatory effects. However, whether remimazolam ameliorates TBI-ALI remains unclear.

Methods

We established a controlled cortical impact (CCI) mouse model of TBI and combined ATF3 knockdown with remimazolam administration to assess lung injury. Subsequently, we employed WB and mRNA-seq techniques to investigate the potential molecular mechanisms of remimazolam's effect on ALI. Finally, we conducted in vivo and in vitro experiments to validate our findings on these mechanisms.

Results

Remimazolam significantly mitigated TBI-ALI. Western blot and mRNA sequencing (mRNA-seq) analyses demonstrated that remimazolam inhibited post-TBI upregulation of activating transcription factor 3 (ATF3) and activation of the NOD-like receptor signaling pathway. In vitro experiments revealed that remimazolam reduced pyroptosis activation in mouse alveolar epithelial cells (MLE-12) by suppressing ATF3 expression, concurrently attenuating degradation of junctional proteins (ZO-1/E-cadherin). In vivo studies confirmed that remimazolam inhibited pulmonary epithelial pyroptosis and preserved blood-air barrier (BAB) integrity post-TBI, ultimately alleviating ALI progression.

Conclusion

Remimazolam mitigates TBI-ALI by suppressing post-traumatic ATF3 upregulation, thereby reducing NLRP3 inflammasome activation. This attenuates alveolar epithelial pyroptosis, preserves junctional protein integrity and BAB function, and ultimately ameliorates pulmonary pathology. These findings position remimazolam as a key therapeutic agent for neurotrauma-induced secondary organ dysfunction.

Introduction

Traumatic brain injury (TBI) is recognized as a global public health crisis, annually contributing to neurological deficits in over 60 million patients worldwide and incurring direct medical expenditures and socioeconomic burdens approximating $400 billion [1]. Its pathological progression extends beyond primary central nervous system damage, triggering multi-organ dysfunction through bidirectional brain-lung axis interactions. Notably, TBI-induced acute lung injury (TBI-ALI), with an incidence rate of 22–25%, represents a critical determinant of secondary mortality [2, 3]. Elucidating the regulatory mechanisms underlying TBI-ALI holds significant clinical implications for improving patient outcomes.

In recent years, pyroptosis-a Gasdermin family protein-mediated programmed cell death modality-has garnered significant scientific attention. This process is centrally regulated by the NOD-like receptor protein 3 (NLRP3) inflammasome, which recruits the ASC adaptor protein to assemble pro-caspase−1 into oligomeric complexes, ultimately driving Gasdermin D (GSDMD) cleavage, plasma membrane pore formation, and maturation/release of interleukin−1β (IL−1β) and interleukin−18 (IL−18) [4, 5]. Notably, the regulatory roles of pyroptosis-related signaling pathways in post-TBI peripheral organ injury remain incompletely elucidated.

Activating transcription factor 3 (ATF3), a critical regulatory node in stress responses, participates in inflammatory homeostasis maintenance through epigenetic modifications and transcriptional regulation [6]. Mechanistic studies demonstrate that under hypoxic stress, ATF3 directly binds to the NLRP3 promoter region to potentiate its transcriptional activation, a process hypothesized to serve as a critical regulatory node for ATF3-mediated pyroptosis modulation [7–12]. However, systematic investigations into whether ATF3 modulates TBI-ALI pathogenesis via pyroptotic pathways remain unreported in current literature.

Remimazolam, a novel ultra-short-acting benzodiazepine targeting γ-aminobutyric acid type A (GABA_A) receptors, has emerged as a preferred agent for critical care sedation due to its favorable pharmacokinetic profile characterized by rapid onset and organ-independent metabolism [13]. Beyond its sedative properties, emerging preclinical evidence highlights its potent anti-inflammatory effects, including suppression of NLRP3 inflammasome activation and cytokine storm mitigation [14, 15]. However, its modulatory effects on pyroptotic signaling pathways and therapeutic utility in TBI-ALI remain unexplored, warranting mechanistic investigation.

This study aims to elucidate the regulatory mechanisms of remimazolam on pyroptosis and inflammatory responses in traumatic brain injury-associated acute lung injury. Building upon preliminary sequencing data, we hypothesize that remimazolam attenuates pulmonary epithelial pyroptosis and inflammation via ATF3 downregulation, thereby suppressing NLRP3 inflammasome activation.

Methods

Animal experiments

All animal experiments conducted in this study were approved by the Laboratory Animal Ethics Committee of Shandong First Medical University (Approval No. W202406120616). C57BL/6 wild-type mice (12 weeks old, weighing 16–20 g) were obtained from Vital River Laboratories (Beijing, China) and maintained in a stable environment with controlled temperature and humidity (22 ± 2 °C, 50 ± 10%) and a 12-hour light/dark cycle, with free access to water and food.

The animal model was established based on previous studies [16]. Mice in the TBI group were anesthetized with isoflurane in a gas mixture containing 70% N2O and 30% O2. A bone flap (3.5 mm) was removed at the intersection of the right lower frontal fontanel and the coronal suture, and a controlled cortical impact (CCI) device was used to inflict brain trauma with specific parameters: a tip diameter of 3.5 mm, an impact velocity of 6 m/s, and an impact depth of 2 mm. After completion of the procedure, the scalp was sutured, and the mice were allowed to recover on a warm heating pad. The mice in the sham surgery group underwent craniotomy without CCI. Postoperatively, mice in the remimazolam group received six intraperitoneal administrations of remimazolam (5 mg/kg/h), while mice in the non-remimazolam group were administered an equivalent volume of phosphate-buffered saline (PBS) via the same route [17].

For in vivo siRNA-ATF3 transfection, mice received tail vein injections containing si-ATF3 (2.5 µg/g body weight), 10% glucose solution, DEPC-treated water, and Entranster™ in vivo transfection reagent (Engreen, Cat# 18668−11−1, Beijing, China) in a total volume of 200 µL, according to the manufacturer’s protocol. Animals were fasted for 24 h prior to the procedure with ad libitum access to water. Surgical interventions were performed 72 h post-transfection [18].

Mice were euthanized by cervical dislocation at 12–24 h postoperatively, with subsequent collection of lung tissues, plasma, and bronchoalveolar lavage fluid (BALF) for downstream experimental analyses. Before harvesting mouse lung tissue, a small incision was made in the left atrial appendage of the mouse heart. Using a 20 ml syringe filled with saline, the right ventricle was cannulated to perfuse the lungs until they turned completely white.

Hematoxylin and Eosin (H&E) staining and lung injury scoring

To assess morphological alterations in pulmonary tissues, lung specimens were fixed in 4% neutral-buffered formalin, paraffin-embedded, and sectioned at 5 μm thickness. These sections were subsequently stained with hematoxylin and eosin and examined under a light microscope (Nikon, Tokyo, Japan). To quantify lung injury severity in mice, a semi-quantitative scoring system ranging from 0 (no injury) to 4 (severe injury) was applied for histopathological classification [19]. For the ALI score, three random non-overlapping fields of view per lung section were scored, and the average was calculated. All histological assessments were performed in a blinded manner to ensure objectivity.

Wet/Dry (W/D) weight ratio

To evaluate the severity of pulmonary edema, the W/D weight ratio was employed. The right lung lobes of mice were harvested, gently blotted with sterile gauze to remove surface moisture, and the wet weight was immediately measured. Following this, the tissues were desiccated in a 60 °C oven for 48 h and re-weighed to determine the dry weight. The W/D ratio was subsequently calculated by dividing the wet weight by the dry weight.

Immunofluorescent (IF) staining

Lung tissue sections were permeabilized with immunostaining permeabilization buffer for 5 min, then blocked with 5% BSA for 30 min at room temperature and incubated with primary antibody diluted in 5% BSA overnight at 4 °C. After washing away the primary antibody, the specimens were incubated with red fluorescent Alexa Fluor 594 rabbit anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cell nuclei were stained with DAPI for 5 min. Changes in ZO-1 and E-cadherin were observed using confocal microscopy.

Enzyme-Linked immunosorbent assay (ELISA)

Levels of IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) were quantified using a commercially available ELISA kit (Reed Biotech, Wuhan, China) following the manufacturer’s standardized protocols.

Cell culture and transfection

Mouse lung epithelial cells (MLE-12) were obtained from the FuHeng Cell Center (Shanghai, China). The cells were cultured in DMEM/F-12 supplemented with 1% streptomycin/penicillin solution and 10% fetal bovine serum (Lonsera, Shanghai, China). Cells were stimulated with lipopolysaccharide (LPS; 200 ng/mL, 4 h) followed by adenosine triphosphate (ATP; 2 mM, 45 min).

Plasmid transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s technical bulletin. si-ATF3 and oe-ATF3 were designed and synthesized by GenCefe Biotech Co., Ltd. The sequences of si-NC and si-ATF3 are as follows:

si-NC	sense	5’-UUCUCCGAACGUGUCACGUTT-3′
	anti-sense	5’-ACGUGACACGUUCGGAGAATT-3′
si-ATF3#1	sense	5’-GGCUCAGAAUGGACGGACATT-3′
	anti-sense	5’-UGUCCGUCCAUUCUGAGCCTT-3′
si-ATF3#2	sense	5’-CCAAGUGUCGAAACAAGAATT-3′
	anti-sense	5’-UUCUUGUUUCGACACUUGGTT-3′
si-ATF3#3	sense	5’-GACACCCUUUGUCAAGGAATT-3′
	anti-sense	5’-UUCCUUGACAAAGGGUGUCTT-3′

mRNA sequencing

Lung tissues were harvested from mice and stored in tissue RNA preservation solution (M6100, New Cell & Molecular Biotech). Total RNA was isolated from pulmonary tissues using TRIzol reagent (Invitrogen, USA). RNA integrity was assessed by Shanghai Bioprofile Technology Co., Ltd. utilizing a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Qualified RNA libraries were sequenced on the Illumina NovaSeq 6000 platform with a paired-end 150 bp (PE150) configuration. Differentially expressed genes (DEGs) were identified using stringent thresholds of adjusted p-value (padj) < 0.05 and absolute fold change (|FC|) ≥ 2.

Western blot

Proteins were extracted from lung tissue and cells with RIPA buffer containing protease inhibitors. Protein concentration was then determined by BCA protein assay kit (Solarbio, Cat: PC0020). Equal amounts of protein were subjected to SDS-PAGE followed by immunoblotting with specific antibodies. The primary antibodies used are as follows: anti-ATF3 (ab207434, Abcam), anti-NLRP3 (#15101, CST), anti-Gasdermin D (#10137, CST), anti-ZO-1 (21773-1-AP, Proteintech), anti-E-cadherin (20,874-1-AP, Proteintech), anti-GAPDH (60,004-1-Ig, Proteintech), anti-Caspase-1 (#3866, CST), anti-TMS1(R013826,Epizyme). The next day, bands were incubated with peroxidase-conjugated secondary antibodies and analyzed by an enhanced chemiluminescence system (Bio-Rad, CA, USA), and quantitative analysis was performed by ImageJ software (National Institutes of Health, NIH, USA).

Statistical analysis

All experiments were conducted as biological replicates, with sample size determined by statistical power requirements. Data are expressed as mean ± standard deviation (SD). All data passed the Shapiro-Wilk normality test (n < 50). Comparisons between groups were made using a t-test (two-sided). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests were used for comparison of data from multiple groups, respectively. P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results

TBI induces ALI by compromising the structural integrity of the blood-air barrier (BAB)

The BAB’s integrity is maintained through precise regulation of alveolar epithelial tight junctions (ZO-1/occludin complexes) and adherens junctions (E-cadherin/β-catenin complexes) [20, 21]. Disruption of the BAB represents a hallmark pathological feature of TBI-ALI [22–24]. Western blot analysis revealed decreased expression of ZO-1 and E-cadherin in murine lung tissues at 12 h post-TBI compared to sham-operated controls, with further reductions observed at 24 h post-injury (Fig. 1A). Consistent with these findings, immunofluorescence staining also showed reduced ZO-1 and E-cadherin expression (Fig. 1B-D).

Fig. 1.

TBI induces ALI by compromising the structural integrity of the blood-air barrier (BAB). A Western blotting was used to detect the expression levels of ZO-1 and E-cadherin in mouse lung tissues (n = 3). B and D Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: ZO-1, Scale bar: 100 μm. C and D Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: E-cadherin, Scale bar: 100 μm. E Lung injury score was evaluated via H&E staining of the lung tissue of the mice (n = 6). F W/D weight ratio of mouse lung tissue (n = 6). G Levels of IL-1β, IL-6, and TNF-α in mouse BALF (n = 6) Statistical significance was determined by one-way ANOVA as appropriate

To systematically evaluate the pathological consequences of BAB disruption, we performed quantitative histopathological analysis of lung tissues using a modified Smith scoring system. H&E staining demonstrated hallmark features of ALI in TBI groups, including alveolar septal thickening with neutrophilic infiltration, intra-alveolar erythrocyte extravasation, and progressive alveolar collapse. The W/D weight ratio exhibited significant elevation at 24 h post-TBI compared to both sham-operated controls and the 12-hour post-TBI group, confirming progressive pulmonary edema formation (Fig. 1E-F). Furthermore, ELISA revealed markedly increased concentrations of IL-1β, IL-6, and TNF-α in BALF at 24 h post-TBI (Fig. 1G).

The above results suggest that TBI may promote ALI by disrupting alveolar junctional proteins.

Remimazolam attenuates TBI-ALI by preserving junctional protein integrity

To investigate the protective effects of remimazolam on TBI-ALI, we administered remimazolam or an equivalent volume of PBS to mice in the TBI and sham groups after CCI surgery, following previously described methods. WB analysis revealed significant downregulation of ZO-1 and E-cadherin protein expression in TBI group lungs, whereas remimazolam treatment restored their expression levels to near-baseline (Fig. 2A). The results of fluorescence staining were consistent with the above results (Fig. 2B-E).

Fig. 2.

Remimazolam Attenuates TBI-ALI by Preserving Junctional Protein Integrity. A Western blotting was used to detect the expression levels of ZO-1 and E-cadherin in mouse lung tissues (n = 3). B and D Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: ZO-1, Scale bar: 100 μm. C and E Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: E-cadherin, Scale bar: 100 μm. F Lung injury score was evaluated via H&E staining of the lung tissue of the mice (n = 6). G W/D weight ratio of mouse lung tissue (n = 6). H Levels of IL-1β, IL-6, and TNF-α in mouse BALF (n = 6) Statistical significance was determined by one-way ANOVA as appropriate

Histopathological evaluation via H&E staining demonstrated marked histopathological improvement in remimazolam-treated groups compared to TBI + PBS controls, characterized by reduced neutrophilic infiltration, diminished pulmonary hemorrhage areas, and attenuated alveolar collapse (Fig. 2F). Gravimetric analysis showed remimazolam significantly lowered the W/D weight ratio, confirming mitigation of pulmonary edema (Fig. 2G). ELISA further revealed remimazolam’s suppressive effects on BALF levels of IL-1β, IL-6, and TNF-α (Fig. 2H).

The data collectively indicate that remimazolam may mitigate TBI-ALI progression by stabilizing ZO-1 and E-cadherin.

Remimazolam mitigates TBI-ALI via the NOD-like receptor signaling pathway

To further investigate the molecular mechanisms underlying remimazolam’s protective effects in TBI-ALI, whole-transcriptome sequencing was performed on murine lung tissues. KEGG pathway enrichment analysis demonstrated that differentially expressed genes (DEGs) in the TBI group were predominantly enriched in immune and inflammatory processes, whereas this enrichment was significantly attenuated in the TBI mice treated with remimazolam (TBI + R group) (Fig. 3A, B). Focusing specifically on immune-related pathways, we observed a pronounced enrichment of the NOD-like receptor signaling pathway after TBI, which was substantially reduced following remimazolam treatment (Fig. 3C, D). Consistently, Gene Set Enrichment Analysis (GSEA) further confirmed the suppression of NOD-like receptor–related signaling in the TBI + R group (Fig. 3E).

Fig. 3.

Remimazolam Mitigates TBI-ALI by Modulating ATF3 and NOD-like Receptor Pathways. A Results of up-regulated biological processes via KEGG enrichment analysis. B Results of down-regulated biological processes via KEGG enrichment analysis. C Results of up-regulated immune system pathways via KEGG enrichment analysis. D Results of down-regulated immune-system pathways via KEGG enrichment analysis. E Enrichment plot of NOD-like receptor pathways via GSEA analysis. F Western blotting was used to detect the expression level of NLRP3 in MLE-12 cells (n = 3). G Western blotting was used to detect the expression levels of ZO-1 and E-cad in MLE-12 cells (n = 3). H Levels of IL-1β, IL-6, and TNF-α in the supernatant of serum-free culture medium of MLE-12 cells (n = 6) Statistical significance was determined by one-way ANOVA or two-sided Student’s t-test as appropriate

To further validate the importance of NLRP3, we conducted in vitro experiments using CY-09 (MCE, Cat#HY-103666), a specific NLRP3 inhibitor. WB results indicated that CY-09 significantly inhibited NLRP3 expression (Fig. 3F) and attenuated the pyroptosis-induced decrease in ZO-1 and E-cadherin expression (Fig. 3G). Additionally, ELISA results demonstrated that inhibiting NLRP3 markedly reduced the release of pyroptosis-associated inflammatory factors (Fig. 3H).

Collectively, these findings indicate that remimazolam mitigates the progression of TBI-ALI, at least in part, by suppressing NLRP3 inflammasome activation.

ATF3 activates pulmonary epithelial pyroptosis via NLRP3 inflammasome signaling

Analysis of mRNA sequencing results showed that in lung tissue from TBI group, 986 genes were up-regulated and 455 were down-regulated compared to the Sham group. In the TBI + R group, 1022 genes were up-regulated and 1066 were down-regulated versus the TBI group. Volcano plot analysis revealed significant upregulation of ATF3 expression in the TBI group, which was markedly downregulated in the TBI + R group compared to TBI group (Fig. 4A).

Fig. 4.

ATF3 Activates Pulmonary Epithelial Pyroptosis via NLRP3 Inflammasome Signaling. A Volcano plot of DEGs between the lung tissues of the Sham, TBI and TBI + R groups. B Western blotting was used to detect the expression levels of ATF3 in MLE-12 cells (n = 3). C Western blotting was used to detect the expression levels of NLRP3, ATF3 and GSDMD in MLE-12 cells (n = 3). D Western blotting was used to detect the expression levels of ZO-1 and E-cadherin in MLE-12 cells (n = 3). E Levels of IL-1β, IL-6, and TNF-α in the supernatant of serum-free culture medium of MLE-12 cells (n = 6). F and G Western blotting was used to detect the expression levels of ZO-1, E-cadherin, NLRP3 and ATF3 in MLE-12 cells (n = 3) Statistical significance was determined by one-way ANOVA as appropriate

To dissect ATF3’s modulation of pyroptosis, an in vitro model was established using LPS and ATP stimulated MLE-12 alveolar epithelial cells. siRNA-mediated ATF3 knockdown was achieved via Lipofectamine 3000 transfection (Fig. 4B). WB analysis demonstrated LPS/ATP-induced activation of the NLRP3 inflammasome, evidenced by upregulated NLRP3 and cleaved GSDMD-N expression. ATF3 silencing significantly attenuated these effects, confirming its pro-pyroptotic role (Fig. 4C). Notably, LPS/ATP stimulation reduced ZO-1 and E-cadherin expression, while ATF3 knockdown restored their protein levels (Fig. 4D). ELISA results showed that si-ATF3 effectively suppressed the release of NLRP3-induced pro-inflammatory cytokines (Fig. 4E).

To further investigate the relationship between remimazolam, ATF3, NLRP3, and junctional proteins, we overexpressed ATF3 by transfecting the oe-ATF3 plasmid into the MLE-12 pyroptosis model. Western blot results showed that the overexpression of ATF3 attenuated remimazolam’s protective effects on the tight junction proteins ZO-1 and E-cadherin and attenuated its inhibitory effects on NLRP3 and ATF3 (Fig. 4F, G). These findings confirm that ATF3 is a key factor in remimazolam-mediated pyroptosis inhibition.

TBI induces ALI via the ATF3/NLRP3/GSDMD pathway

WB analysis confirmed elevated ATF3 protein levels in murine lung tissues at 24 h post-TBI. ATF3 and NLRP3 protein levels decreased after remimazolam treatment, consistent with transcriptomic sequencing data and in vivo experiments (Fig. 5A-C).

Fig. 5.

TBI Induces ALI via the ATF3/NLRP3/GSDMD Pathway. A-C Western blotting was used to detect the expression levels of NLRP3 and ATF3 in mouse lung tissues (n = 3). D and E Western blotting was used to detect the expression levels of ATF3 in mouse lung tissues (n = 3). F-H Western blotting was used to detect the expression levels of ZO-1, E-cadherin, NLRP3, GSDMD-N, ASC, Caspase-1 and ATF3 in mouse lung tissues (n = 3). I and K Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: ZO-1, Scale bar: 100 μm. J and L Immunofluorescence staining of mouse lung tissues. Blue: DAPI; Red: E-cadherin, Scale bar: 100 μm. M and N Western blotting was used to detect the expression levels of NLRP3, GSDMD-N, ASC, Caspase-1 and ATF3 in mouse lung tissues (n = 3). O Lung injury score was evaluated via H&E staining of the lung tissue of the mice (n = 6). P W/D weight ratio of mouse lung tissue (n = 6). Q Levels of IL-1β, IL-6, and TNF-α in mouse BALF (n = 6) Statistical significance was determined by one-way ANOVA or two-sided Student’s t-test as appropriate

ATF3 knockdown in mice was achieved using Entranster™ in vivo transfection reagent (Fig. 5D-E).

WB showed markedly increased protein levels of NLRP3, GSDMD-N, ASC, and Caspase-1 in lung tissues of TBI mice vs. the Sham group, accompanied by decreased levels of ZO-1 and E-cadherin due to pyroptosis. siRNA-mediated ATF3 silencing reduced NLRP3, GSDMD-N, ASC and Caspase-1 protein levels while mitigating TBI-induced downregulation of ZO-1 and E-cadherin (Fig. 5F-H). Meanwhile, immunofluorescence staining revealed that the expression levels of ZO-1 and E-cadherin were significantly restored following ATF3 knockdown (Fig. 5I-L).

To more directly observe remimazolam’s inhibitory effect on post-TBI pyroptosis, protein levels of NLRP3, GSDMD-N, ASC, and Caspase-1 in mouse lung tissue were measured. WB results showed that remimazolam significantly suppressed the assembly and activation of the NLRP3 inflammasome in mouse lung tissue after TBI (Fig. 5M-N).

H&E staining demonstrated that ATF3 knockdown significantly attenuated TBI-induced lung histopathology, including reduced alveolar hemorrhage and inflammatory infiltration (Fig. 5O). Gravimetric analysis showed a marked reduction in the W/D ratio in the ATF3-silenced group compared to TBI controls, indicating improved vascular integrity (Fig. 5P). Concurrently, ELISA revealed decreased concentrations of IL-1β, IL-6, and TNF-α in BALF (Fig. 5Q).

In summary, remimazolam alleviates TBI-ALI by inhibiting NLRP3-induced pyroptosis of alveolar epithelial cells through downregulating ATF3.

Discussion

This study is the first to delineate the protective mechanism whereby remimazolam mitigates TBI-ALI by targeting the ATF3/NLRP3/GSDMD regulatory axis. We demonstrate that TBI induces ATF3 activation via the brain-lung axis, driving NLRP3 inflammasome assembly and GSDMD-mediated pyroptosis, which disrupts alveolar epithelial junctional proteins (ZO-1/E-cadherin) and ultimately precipitates inflammatory cascades and acute lung injury. Remimazolam attenuates pyroptotic signaling and preserves junctional integrity through ATF3 suppression, thereby ameliorating TBI-ALI progression. These findings establish a novel theoretical framework for multi-organ protection post-TBI and propose targeted therapeutic strategies against neurotrauma-induced pulmonary complications.

Current understanding of TBI-ALI pathogenesis involves multiple mechanisms, including coagulopathy, exosome-mediated epithelial ferroptosis [22, 25], and crucially, inflammasome-driven cytokine cascades [26]. The pioneering “neuro-respiratory-inflammasome axis” proposed by Kerr’s group provides a paradigm-shifting framework for understanding inflammasome centrality in TBI-ALI progression [27]. Our experimental data demonstrate significant activation of inflammasome-related effector molecules (IL-6, IL-1β, TNF-α) in post-TBI pulmonary tissues, with distinct temporal dynamics in ALI pathology. Notably, comparative temporal analysis revealed exacerbated lung injury at 24 h versus 12 h post-TBI, contrasting with Kerr’s reported ALI pathological peak at 4 h post-injury [28]. Furthermore, Saber et al. observed CD-1 mouse pulmonary injury spanning 1 h to 7 days post-lateral fluid percussion injury (LFPI) [29], suggesting strain-specific and modeling methodology-dependent variations in TBI-ALI trajectories.

Recent studies have progressively unveiled the multi-organ protective effects of remimazolam through diverse biological mechanisms. Current evidence indicates that this agent exerts tissue protection not only via modulation of inflammatory responses and apoptotic pathways, but also potentially through novel molecular targets. In hepatic injury models, Fang et al. demonstrated that remimazolam intervention significantly suppresses LPS-induced neutrophil and macrophage infiltration in rat livers, while reducing hepatic expression of pro-inflammatory cytokines (TNF-α, IL-6), thereby attenuating histopathological damage [15, 30]. Notably, Wen’s team made a groundbreaking discovery in neuroinflammation, showing that remimazolam reprograms the central nervous system inflammatory microenvironment by promoting microglial polarization toward the M2 phenotype [31]. Additionally, Li et al. first reported in acute lung injury models that remimazolam activates the PI3K/AKT signaling pathway to inhibit apoptosis in alveolar epithelial and vascular endothelial cells, offering new insights into its pulmonary protective mechanisms [17]. Our study innovatively reveals that remimazolam alleviates TBI-ALI by inhibiting pyroptosis, expanding its therapeutic repertoire in organ protection.

In the pathogenesis of ALI, the interplay between excessive inflammatory responses and programmed cell death constitutes a central mechanism. This study elucidates ATF3’s unique pro-inflammatory role in TBI-ALI. As a key member of the ATF/CREB family, ATF3 is widely recognized as a transcription hub rapidly induced during early stress responses [32–34]. However, prior research predominantly focused on its anti-inflammatory effects in pulmonary tissues [35, 36]. Aligning with emerging evidence, we demonstrate that ATF3 positively regulates alveolar epithelial pyroptosis via the NLRP3 inflammasome-dependent pathway [12].

Our study delineates remimazolam’s pivotal role in modulating the ATF3/NLRP3 pyroptosis axis in TBI-ALI. Integrated Western blot (WB) and transcriptomic analyses reveal that TBI upregulates ATF3 to aberrantly activate the NOD-like receptor signaling pathway, establishing an ATF3-NLRP3-GSDMD molecular cascade that culminates in pulmonary epithelial pyroptosis. Notably, the pyroptosis effector GSDMD-N disrupts BAB integrity by degrading ZO-1 and E-cadherin, providing novel insights into TBI-ALI pathogenesis at the junctional complex level. In terms of mechanism verification, the study adopts a dual model verification strategy. In vitro, LPS + ATP-induced MLE-12 pyroptosis models demonstrated that ATF3 knockdown suppresses NLRP3 inflammasome assembly, inhibits GSDMD pathway activation, and reverses ZO-1/E-cadherin downregulation. In vivo, CCI-induced TBI murine models confirmed that remimazolam attenuates TBI-ALI via ATF3 inhibition, with efficacy comparable to ATF3-specific siRNA intervention.

There are also some deficiencies in this study. While the remimazolam administration protocol adopted from prior studies effectively inhibited TBI-ALI progression, optimal plasma drug concentrations in mice remain undetermined. Although we validated remimazolam’s suppression of ATF3 and ATF3’s activation of NLRP3, the precise mechanisms underlying ATF3-NLRP3 transcriptional regulation require further elucidation.

Remimazolam mitigates TBI-ALI by suppressing post-traumatic ATF3 upregulation, thereby reducing NLRP3 inflammasome activation. This attenuates alveolar epithelial pyroptosis, preserves junctional protein integrity and BAB function, and ultimately ameliorates pulmonary pathology. These findings position remimazolam as a key therapeutic agent for neurotrauma-induced secondary organ dysfunction.

Abbreviations

ALI

Acute lung injury

ATP

Adenosine triphosphate

ATF3

Activating transcription factor 3

BAB

Blood-air barrier

BALF

Bronchoalveolar lavage fluid

CCI

Controlled cortical impact

ELISA

Enzyme-Linked Immunosorbent Assay

GSDMD

Gasdermin D

H&E

Hematoxylin and eosin

IL-1β

Interleukin-1β

IL-6

Interleukin-6

LPS

Lipopolysaccharide

NLRP3

NOD-like receptor protein 3

TBI

Traumatic brain injury

TBI-ALI

TBI-induced acute lung injury

TNF-α

Tumor necrosis factor-alpha

W/D weight ratio

Wet-to-dry weight ratio

Rem

Remimazolam

Authors’ contributions

**Chang Sun: ** Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. **Yi Zhang: ** Conceptualization, Investigation, Writing - original draft, Writing - review & editing. **Jiahan Wang: ** Investigation, Software, Visualization. **Bailun Wang: ** Investigation, Data curation. **Angran Gu: ** Investigation, Data curation. **Yuelan Wang: ** Writing - review & editing. **Changping Gu: ** Conceptualization, Resources, Supervision, Writing - review & editing. All authors participated in manuscript writing and approved the final version of the manuscript.

Funding

This work was supported by Natural Science Foundation of Shandong Province (ZR2023MH076).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Contributions

Ethics approval and consent to participate

All animal experiments conducted in this study were approved by the Laboratory Animal Ethics Committee of Shandong First Medical University (Approval No. W202406120616).

Consent for publication

All authors gave their consent for publication.

Competing interests

The authors declare no competing interests.