The experimental protocol adhered to the national standard laboratory animal guidelines for ethical review of animal welfare (GB/T 35892-2018) and was approved by the Fuzhou University Affiliated Provincial Hospital (the original name: Shengli Clinical Medical College of Fujian Medical University) Animal Experiment Committee. Specifically, 130 C57BL/6 male mice, 18 weeks old, were procured from Shanghai Xipur-Bikai Experimental Animal Co., Ltd and maintained in a pathogen-free environment. We only used male adult mice to avoid possible interference due to the physiological cycle and female hormones in the activity of the renin-angiotensin system. Ten mice were included in each group (N = 10). In experiment I, 20 mice in total were randomly assigned to the Control and VILI groups; in experiment II, 30 mice were randomly assigned to Control, VILI, and VILI+4-phenylbutyric acid (4-PBA) groups; in experiment III, 40 mice were randomly assigned to Control, VILI, VILI+ Resorcinolnaphthalein (RES), and VILI+MLN-4760 groups; in experiment IV, 40 mice were randomly assigned to Control, VILI, VILI+ACE2, and VILI+ACE2+A779 groups. Measurements were repeated thrice for each animal.

Injectable anesthetics, ketamine (100 mg/kg, H20023609, Zhejiang Xianle Pharmaceutical Co., Ltd., Xianju, Zhejiang, China), and xylazine (10 mg/kg, B25730, Yuanye biotechnology, Shanghai, China), were administered intraperitoneally (i.p.) for anesthetizing the animals. One-quarter of the initial dose was added every 30 min to maintain anesthesia. A No. 20 catheter (Cusabio Life Science, Wuhan, Hubei, China) was sterilized and inserted into the trachea. Mechanical ventilation (MV) for 4 h was conducted using a ventilator (R500, RWD Life Science Co., Ltd., Shenzhen, Guangdong, China) with a high tidal volume of 20 mL/kg at a rate of 80 breaths per minute and a breathing ratio of 1:1 without exerting positive end-expiratory pressure [23]. Mice in the MV group underwent ventilation for 4 h, whereas mice in the control group were intubated and spontaneously anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). After the 4-h MV was completed, mice were allowed to breath spontaneously. Vital signs of the animals were monitored by evaluating the color of the mucous membrane in the mouth, limb skin color, temperature, and heart rates. At 2 hour after the 4-h MV, the bronchoalveolar lavage fluid (BALF) and lung tissue were collected from the mice.

Bronchoalveolar lavage fluid (BALF) was collected 2 h after the mice were resuscitated. A small incision was made in the midline of the neck to expose the trachea to collect BALF after the mice were fully anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). A sterile tracheal cannula was inserted and secured with a ligature. Phosphate-buffered saline (PBS) was injected into the tracheal cannula such that it entered the lungs. Controlled instillation was performed by gently pushing PBS in and out four times to dislodge and collect lung samples. The lavage fluid was aspirated into the syringe, and this instillation-aspiration process was repeated until 1.5 mL BALF was collected from each animal.

After euthanizing the animals by cervical dislocation, lung tissues were collected for subsequent experiments, including hematoxylin-eosin (H&E) staining, measuring the wet/dry (W/D) weight of lungs, immunohistochemical staining, and western blotting. The model establishment of lung injury was confirmed by significant changes in H&E staining, lung edema (lung wet/dry ratio), and concentrations of interleukin-1 beta (IL-1$\beta$) and interleukin-6 (IL-6) in BALF.

To inhibit ERS in vivo, 4-PBA (1 mg/kg, HY-A0281, MedChemExpress, Monmouth Junction, NJ, USA) was instilled through the trachea 6 h before MV. To reduce the dosage and its effect on the whole body, 4-PBA was administrated through the trachea instead of injecting i.p. In the pilot experiments, we tested NLRP3 expression at the indicated intervals and found that, after 6 h, the expression of NLRP3 was markedly decreased (Supplementary Fig. 1). ACE2’s enzymatic function was either activated or inhibited by subcutaneous injection of resorcinolnaphthalein (RES, 20 µg/kg, HY-122445, MedChemExpress, Monmouth Junction, NJ, USA) or MLN-4760 (20 µg/kg, HY-19414, MedChemExpress, Monmouth Junction, NJ, USA), respectively, via osmotic minipumps (Alzet Osmotic Minipumps, Durect Corporation, Cupertino, CA, USA) 6 h before MV, following protocols described previously [24, 25]. The osmotic minipumps were implanted under anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg) and delivered the indicated dose over the 6 h until start of MV. The Ang (1-7) receptor, Mas, was antagonized by subcutaneous injection of A779 (80 µg/kg, HY-P0216, MedChemExpress, Monmouth Junction, NJ, USA) 6 h before MV.

To increase the protein expression of ACE2, 300 µg of pGLV-EF1a-GFP-ACE2 (GenePharma Co., Ltd., Shanghai, China) was instilled into the trachea 24 h before MV. An equal concentration of pGLV-EF1a-GFP was instilled in the trachea of animals of the other groups. In the two groups receiving tracheal instillations 6 or 24 hours before MV, the mice were anesthetized when they received tracheal instillations, then allowed to recover until the MV experiment.

For H&E staining, lung tissue sections were prepared by embedding them in paraffin. The sections underwent serial washing, starting with xylene, ethanol and distilled water. Subsequently, the sections were immersed in hematoxylin solution for 5 minutes, followed by washing with tap water and a treatment with 1% hydrochloric acid alcohol. This was followed by washing with tap water and treatment with 0.6% ammonia water. The sections were finally washed under running water. The sections were immersed in an eosin staining solution for 2 minutes, followed by serial immersion in 95% ethanol, absolute ethanol, and xylene steps for dehydration. Finally, the sections were dried and sealed with neutral gum. The staining was observed under a microscope (BX50, Olympus, Tokyo, Japan).

For IHC, lung samples were initially washed with PBS and incubated with 5% normal goat serum. Subsequently, the samples were incubated with rabbit GSDMD antibody (1:500, ab209845, Abcam, Cambridge, MA, USA) and ACE2 antibody (1:500, ab272500, Abcam, Cambridge, MA, USA) for 1–2 hours at 37 °C. Subsequently, samples were incubated with a biotin-labeled secondary antibody (1:2000, ab207996, Abcam, Cambridge, MA, USA) at 37 °C for 20 minutes, followed by washing with PBS. The samples were further incubated with horseradish enzyme-labeled streptavidin at 37 °C for 20 minutes and washed thrice with PBS. Color development was achieved using diaminobenzidine, and microscopic observation was conducted using the Olympus BX50 microscope.

For ELISA, 100 µL of the BALF supernatant was added per well in a 96-well plate. ELISA was performed following the manufacturer’s instructions (ELISA kits for IL-1$\beta$ and IL-6, Beyotime, Shanghai, China). IL-1$\beta$ and IL-6 levels were determined using a standard curve. Similar methods were employed to detect the concentrations of Ang II (EK-M27496, EK-Bioscience, Shanghai, China) and Ang (1-7) (EK-M29369, EK-Bioscience, Shanghai, China). Specifically, to measure Ang II and Ang (1-7) levels, lung tissues were homogenized after lysis with lysis buffer (R0020-100, Solarbio, Beijing, China) at 4 °C. After centrifugation at 12,000 $\times$g (4 °C, 10 min), the supernatant was collected, and the concentrations of Ang II and Ang (1-7) were measured using the aforementioned procedure.

The supernatant was collected after centrifuging BALF or culture medium at 1000 $\times$g for 10 minutes. LDH levels were determined using an LDH kit (#MX3235-500T, MKBio, Shanghai, China) following the detailed instructions. The optical density (OD) value at 450 nm was recorded for each well.

A549 (CRM-CCL-185) alveolar type II epithelial cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified eagle medium (DMEM) (#21068028, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, # A5669801, Gibco, Grand Island, NY, USA), 10 mg/mL of streptomycin, and 10,000 units/mL of penicillin (#P4333, Sigma-Aldrich, Louis, MO, USA). VILI was simulated in vitro by subjecting A549 cells to cyclic stretch (CS). A549 cells were seeded on BioFlex plates (Flexcell Int, Hillsborough, NC, USA). After reaching approximately 50% confluency, the BioFlex plate was mounted on a Flexcell cell stress loading system (FX-5000T, Flexcell Int, Hillsborough, NC, USA). The cells attached to the device were subjected to CS, with a stretch-recovery cycle of 15 times per minute for 4 h [26]. The A549 cells used in the study underwent short tandem repeats (STR) profiling to validate their authenticity and were tested to be free of mycoplasma contamination. The cells were cultured in a humidified incubator at a temperature of 37 °C, with 5% CO₂.

Ang (1-7) (HY-12403, MedChemExpress, Monmouth Junction, NJ, USA) was added to the medium at a final concentration of 100 nmol/L for pretreatment over 12 h, with continuous intervention during CS. Tunicamycin (TM, HY-A0098, MedChemExpress, Monmouth Junction, NJ, USA) was added to the medium at a final concentration of 2 µg/mL to induce ERS.

Initially, 100 µL of cells (5000 cells) were seeded into each well of a 96-well plate. Cells were cultured and treated with the indicated drugs. Subsequently, 10 µL of CCK-8 solution (#96992, Sigma-Aldrich, Louis, MO, USA) was added to each well. Blank controls comprised the cell culture medium, drug, and CCK-8 solution (without cells). Cells were incubated in an incubator for 1 h. Following incubation, absorbance at 450 nm was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

To measure the ROS production in lung tissues, following the harvesting, lungs were instantly frozen and homogenized using polytron (40 mM ice-cold Tris$\cdot$HCl and 0.1% Tween buffer, pH 7.4). Half of the homogenate was treated with 2^′,7^′-Dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen, Waltham, MA, USA, 5 µM), and the remaining half was used as a blank. After incubation for 45 min at 37 °C, the fluorescence intensity was measured using a microplate reader (PerkinElmer, Waltham, MA, USA) with excitation and emission wavelengths of 485 nm and 538 nm, respectively.

To measure ROS levels in cells, DCFH-DA (Invitrogen, Waltham, MA, USA) was diluted at 1:1000 in a serum-free culture solution. Cells were seeded in 6-well plates and treated with the indicated drugs. The culture solution was removed, and 1 mL of diluted DCFH-DA was added per well. The cells were incubated in the incubator for 20 minutes. Subsequently, the cells were washed thrice with serum-free cell culture solution to eliminate any residual DCFH-DA. Fluorescence intensity was measured using a microplate reader (PerkinElmer, Waltham, MA, USA) with excitation and emission wavelengths of 485 nm and 538 nm, respectively.

TRIzol® reagent was used to extract 1 µg of total RNA from 10 mg mouse lung tissue and reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (RR047A; Takara Bio, Inc. San Jose, CA, USA). SYBR green reagent (Takara Bio, Inc., San Jose, CA, USA) was used for qPCR. The 2^{-Δ⁢Δ⁢Cq} method was used to normalize the expression levels to those of GAPDH. The following primer sequences were used for qPCR: ACE2 forward (F), 5^′- ACTCCGATCATCAAGCGTCA-3^′ and reverse (R), 5^′-GGCTCCATTCAGTGTTCCAGA-3^′; GAPDH F, 5^′-GTCGTACCACAGGCATTGTGATGG-3^′ and R, 5^′-GCAATGCCTGGGTACATGGTGG-3^′.

The Minute™ Cytoplasmic and Nuclear Fractionation kit (SC-003, Invent Biotechnologies, Plymouth, MA, USA) was used to separate proteins from the cytoplasmic and nuclear fractions. After separation, the proteins were subjected to western blot analysis using specific antibodies, including those against ACE2 (1/5000, ab108252, Abcam, Cambridge, MA, USA), C/EBP homologous protein (CHOP) (5 µg/mL, ab11419, Abcam, Cambridge, MA, USA), glucose-regulated protein 78 (GRP78) (1 µg/mL. ab21685, Abcam, Cambridge, MA, USA), NLRP3 (1/1000, ab263899; Abcam, Cambridge, MA, USA), cleaved caspase-1 (c-caspase-1) (1/1000, ab256469, Abcam, Cambridge, MA, USA), GSDMD-N (1/1000, ab215203, Abcam, Cambridge, MA, USA), and GAPDH (1/10000, ab181602, Abcam, Cambridge, MA, USA). The membranes were incubated with goat anti-mouse IgG+IgM H&L (HRP) secondary antibody (1/5000, ab47827, Abcam, Cambridge, MA, USA). Bands were visualized using an electrogenerated chemiluminescence (ECL) kit (#PE0010, Solarbio, Beijing, China) and analyzed using Image Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA).

Data are presented as the average $\pm$ standard deviation. Statistical analysis was conducted with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests (GraphPad Prism version 7.0, GraphPad Software, San Diego, CA, USA). A p-value 0.05 was considered statistically significant.

To elucidate the association of ACE2 expression with VILI, we established a mouse model of VILI through prolonged MV. Our assessment revealed notable alveolar structural damage (Fig. 1A). Thickened alveolar walls (infiltration of inflammatory cells into the alveolar walls), hyaline membranes (the accumulation of proteinaceous material in the alveolar spaces), cellular infiltration (massive infiltration of inflammatory cells), and edema were visualized in VILI-inflicted tissues. Pulmonary edema (Fig. 1B) and an increase in IL-1$\beta$ and IL-6 levels in BALF (Fig. 1C) confirmed a successful VILI induction. ACE2 protein levels, typically abundant in alveolar cells, were substantially downregulated in the VILI tissue, as evidenced in the results of immunohistochemistry (Fig. 1D,E). Concomitantly, lung tissue homogenates showed significantly elevated Ang II levels (Fig. 1F), coupled with a marked reduction in the conversion of Ang II to Ang (1-7) (Fig. 1G). Both mRNA and protein levels of ACE2 in the VILI-inflicted lung tissue were diminished significantly (Fig. 1H–J). Taken together, there was a reduction in ACE2 level, culminating in Ang II accumulation and diminished levels of Ang (1-7) in VILI-inflicted lung tissue.

Fig. 1.

ACE2/Ang (1-7) levels are downregulated while Ang II is upregulated in mice with VILI. (A) Lung tissue damage due to MV-induced VILI. (B) Lung edema in VILI. (C) Results of inflammatory cytokine levels across BALF samples. (D,E) Expression of ACE2 in lung tissues by immunohistochemistry. (F,G) Comparison of Ang II and Ang (1-7) levels in tissues. (H–J) Comparison of protein or mRNA expressions of ACE2 in VILI. VILI, ventilator-induced lung injury; W/D, wet/dry weight ratio; IL-6, interleukin-6; IL-1$\beta$, interleukin-1$\beta$; BALF, bronchoalveolar lavage fluid; ACE2, angiotensin-converting enzyme 2; Ang, angiotensin. Scale bar = 100 µm. ***p 0.001 vs. control. N = 10; repeats = 3.

In VILI-inflicted lung tissue, a significant elevation in the levels of CHOP and GRP78 proteins was found, along with the increase in ROS levels, suggesting ERS induction (Fig. 2A–C). Alveolar cells in the VILI group displayed an augmentation in GSDMD, as shown by immunohistochemistry analysis (Fig. 2D,E). An elevation in LDH secretion into BALF was found (Fig. 2F). The levels of NLRP3, c-caspase-1, and GADMD-N proteins in lung tissues were increased significantly (Fig. 2G,H), suggesting ERS induction in VILI and increased pyroptosis in alveolar cells.

Fig. 2.

ERS and pyroptosis are induced in VILI. (A,B) Changes of ERS-related proteins in VILI. (C) Results of ROS levels in tissues. (D,E) Changes of expression of the GSDMD protein. (F) Results of LDH concentrations in BALF samples. (G,H) The levels of pyroptosis-related proteins, NLRP3 and c-caspase-1. VILI, ventilator-induced lung injury; ERS, endoplasmic reticulum stress; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; ROS, reactive oxygen species; BALF, bronchoalveolar lavage fluid; LDH, lactate dehydrogenase; NLRP3, NOD-like receptor protein 3 inflammasome; GSDMD, gasdermin D. Scale bar = 100 µm. ***p 0.001 vs. control. N = 10; repeats = 3.

Heightened ROS levels due to ERS activation led to the stimulation of NLRP3, a primary mechanism underlying pyroptosis. To further elucidate the regulatory mechanism of ERS and pyroptosis in VILI, we employed 4-PBA to inhibit ERS and performed MV. Mice were randomly divided into three groups: control, VILI, and VILI+4-PBA. Intervention with 4-PBA significantly alleviated alterations in alveolar structure, lung edema, and inflammation, all induced by VILI (Fig. 3A–C). Moreover, lung tissue of mice of the VILI+4-PBA group displayed substantially lower levels of CHOP and GRP78 proteins compared to those in the VILI group, along with marked reductions in marker proteins of pyroptosis and LDH levels (Fig. 3D–H). Thus, ERS inhibition by 4-PBA significantly alleviated VILI-induced pyroptosis, suggesting that ERS plays a role in inducing pyroptosis in VILI.

Fig. 3.

Inhibition of ERS alleviates pyroptosis in VILI in vivo. (A) Effect of 4-PBA (ERS inhibitor) intervention on VILI. (B) Effect of ERS inhibition on VILI-inflicted lung edema. (C) Effect of ERS inhibition on inflammatory cytokine levels in BALF of mice with VILI. (D–G) Comparison of ERS and pyroptosis-related protein levels in each group. (H) Effect of ERS inhibition on LDH concentrations in BALF samples from mice with VILI. VILI, ventilator-induced lung injury; ERS, endoplasmic reticulum stress; IL-6, interleukin-1; IL-1$\beta$, interleukin-1$\beta$; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; NLRP3, NOD-like receptor protein 3 inflammasome; GSDMD, gasdermin D; LDH, lactate dehydrogenase; BALF, bronchoalveolar lavage fluid. Scale bar = 100 µm. ***p 0.001 vs. control, ^#⁢#p 0.01 vs. VILI. N = 10; repeats = 3.

ACE2’s primary role is to facilitate the conversion of Ang II to Ang (1-7). To elucidate whether the inhibitory effects of Ang (1-7) on pyroptosis in VILI are associated with ERS, VILI was emulated in vitro by CS, and TM was employed to induce ERS. Consequently, A549 cells were divided into the following four groups: control, CS, CS+Ang (1-7), and CS+Ang (1-7) +TM. Prolonged in vitro CS significantly reduced cell viability, and Ang (1-7) markedly restored the compromised cell viability induced by CS. Notably, cell viability in the CS+Ang (1-7) +TM group was significantly lower compared to the CS+Ang (1-7) group (Fig. 4A). CS in vitro upregulated the protein expressions of CHOP and GRP78 in A549 cells. Ang (1-7) substantially mitigated ERS during CS, and TM effectively induced ERS in vitro (Fig. 4B,C). In line with the trend of ERS, in vitro CS enhanced LDH efflux, NLRP3 activation, and GSDMD-N levels. Ang (1-7) significantly ameliorated CS-induced pyroptosis, with its levels being markedly higher in the CS+Ang (1-7) +TM group compared to the CS+Ang (1-7) group (Fig. 4D–F). Ang (1-7) could alleviate ERS and pyroptosis during CS. Moreover, upon continuous ERS, the inhibitory effect of Ang (1-7) on pyroptosis was impeded, suggesting that Ang (1-7) mitigates alveolar pyroptosis in VILI through its ERS-inhibiting role.

Fig. 4.

Ang (1-7) inhibits pyroptosis by alleviating endoplasmic reticulum stress (ERS) under CS in vitro. (A) Effect of Ang (1-7) and ERS activator on the viability of alveolar cells under CS in vitro. (B,C) Comparison of protein expressions of ERS markers in cells of each group. (D) Comparison of LDH secretion in each group. (E,F) Effect of Ang (1-7) and ERS activator on the expression of pyroptosis-related proteins in alveolar cells under CS. CS, cyclic stretch; Ang, angiotensin; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; NLRP3, NOD-like receptor protein 3 inflammasome; GSDMD, gasdermin D; LDH, lactate dehydrogenase. ***p 0.001 vs. control, ^#⁢#p 0.01 vs. CS, ^^^p 0.001/^^p 0.01 vs. CS+Ang (1-7). N = 10; repeats = 3. TM, tunicamycin.

To elucidate the effects of ACE2 in VILI, we employed RES to activate ACE2’s function, while MLN-4760 was used to inhibit it. VILI was induced in the model. RES alleviated lung injury significantly in the mouse model of VILI, evidenced by reduced lung edema (W/D) and lowered IL-6 and IL-1$\beta$ levels in BALF. In contrast, MLN-4760 exacerbated lung damage, edema, and inflammation, as shown by the H&E staining, lung edema (W/D), and IL-6 and IL-1$\beta$ levels in BALF samples (Fig. 5A–C). Upon ACE2 activation, Ang II elevation induced by VILI was mitigated, and low levels of Ang (1-7) were effectively restored. Conversely, ACE2 inhibition promoted Ang II accumulation and suppressed its conversion to Ang (1-7) (Fig. 5D,E). ACE2’s role in converting Ang II to Ang (1-7) is thus intricately involved in the induction of VILI.

Fig. 5.

ACE2 is important in promoting Ang (1-7) production and lung injury in mice with VILI. (A) Effect of activator (RES) and inhibitor (MLN-4760) of ACE2 on lung injury in mice with VILI demonstrated by H&E staining. (B,C) Effect of activation or inhibition of ACE2 on lung edema and inflammation in VILI. (D,E) Comparison of Ang II and Ang (1-7) levels in the lung tissue. VILI, ventilator-induced lung injury; RES, resorcinolnaphthalein; W/D, wet/dry weight ratio; IL-6, interleukin-6; IL-1$\beta$, interleukin-1$\beta$; BALF, bronchoalveolar lavage fluid; Ang, angiotensin. Scale bar = 100 µm. ***p 0.001 vs. control, ^#⁢#p 0.01 vs. VILI. N = 10; repeats = 3.

We examined the effects of RES and MLN-4760 on ERS and pyroptosis in VILI. RES significantly suppressed the expression of CHOP and GRP78 proteins in VILI, and ACE2 inhibition further aggravated ERS (Fig. 6A,B). ACE2 activation attenuated ROS accumulation and LDH efflux in lung tissue and BALF with VILI induction, whereas ACE2 inhibition exacerbated pyroptosis (Fig. 6C,D). GSDMD expression in lung tissues was significantly inhibited by RES but enhanced remarkably by MLN-4760, as shown by the results of immunohistochemistry (Fig. 6E,F). Taken together, ACE2 reduction inhibits the conversion of Ang II to Ang (1-7), contributing further to ERS and pyroptosis induction in VILI.

Fig. 6.

ACE2 mitigates ERS and pyroptosis in VILI by promoting Ang (1-7) production. (A,B) Effects of ACE2 activator and inhibitor (MLN-4760) on ERS in lung tissues of mice with VILI. (C) ROS levels in lung tissues. (D) Levels of LDH secretion in BALF. (E,F) Effects of activation or inhibition of ACE2 on GSDMD expression in VILI. ERS, endoplasmic reticulum stress; VILI, ventilator-induced lung injury; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; RES, resorcinolnaphthalein. Scale bar = 100 µm. ***p 0.001 vs. control, ^#p 0.05 vs. VILI, ^#⁢#p 0.01 vs. VILI. N = 10; repeats = 3.

To further substantiate the mechanism through which ACE2 mitigates ERS and pyroptosis in VILI, rescue experiments were performed. ACE2 protein was overexpressed by transfection, while A779 was used to antagonize Mas, the receptor for Ang (1-7). The mice were categorized into the four following groups: control, VILI, VILI+ACE2, VILI+ACE2+A779. Augmenting ACE2 protein expression effectively curbed lung tissue damage, ameliorated lung edema (W/D), and attenuated inflammation. Notably, while A779 had no significant effect on the ACE2 protein expression, it markedly reduced the ameliorative effects of ACE2 overexpression on VILI (Fig. 7A–C). A779 did not significantly change the ACE2 overexpression in lung tissues, as shown by the results of immunohistochemistry (Fig. 7D,E). Furthermore, in vivo overexpression of ACE2 reduced the accumulation of Ang II in lung tissues while promoting the production of Ang (1-7). The Mas inhibitor had no statistically discernible effect on Ang II and Ang (1-7) (Fig. 7F,G). The substantial attenuation of VILI by ACE2 protein was markedly hindered upon blocking of the Ang (1-7) receptor. ACE2’s mechanism for inhibiting VILI is intrinsically linked to the catalytic generation of Ang (1-7).

Fig. 7.

Inhibition of the Ang (1-7) receptor blocks ACE2’s effects on lung injury in mice with VILI. (A) Effect of ACE2 overexpression and inhibitor of the Ang (1-7) receptor, MAS1 (A779) on lung injury in mice with VILI. (B,C) Comparison of lung edema and inflammation in each group. (D,E) Comparison of ACE2 protein levels in the lung tissue. (F,G) Comparison of Ang II and Ang (1-7) levels in the lung tissue. VILI, ventilator-induced lung injury; ACE2, angiotensin-converting enzyme 2; RES, resorcinolnaphthalein; W/D, Wet/Dry weight ratio; IL-6, interleukin-1; IL-1$\beta$, interleukin-1$\beta$; BALF, bronchoalveolar lavage fluid; Ang, angiotensin. Scale bar = 100 µm. ***p 0.001 vs. control, ^#⁢#p 0.01 vs. VILI, ^^p 0.01 vs. VILI+ACE2. N = 10; repeats = 3.

The effects of overexpression of ACE2 protein and inhibition of the Ang (1-7) receptor on ERS and pyroptosis were assessed. With enhanced ACE2 protein levels, the expression of CHOP and GRP78 proteins and ROS production were substantially suppressed. ERS in the VILI+ACE2+A779 group was notably higher than that in the VILI+ACE2 group (Fig. 8A–C). Furthermore, ACE2 overexpression effectively reduced LDH efflux into BALF and suppressed NLRP3 activation, along with the generation of GSDMD-N. The level of pyroptosis in the VILI+ACE2+A779 group was significantly higher than that in the VILI+ACE2 group (Fig. 8D–F). Ang (1-7) receptor blockade significantly inhibited the alleviation of ERS and pyroptosis by ACE2 protein in VILI. ACE2’s mechanism for inhibiting ERS and pyroptosis in VILI is closely associated with its catalytic generation of Ang (1-7).

Fig. 8.

Ang (1-7) receptor antagonist blocks the inhibitory effects of ACE2 on ERS and pyroptosis in VILI mice. (A–C) Effect of overexpression of ACE2 and inhibition of the Ang (1-7) receptor, MAS1 (A779) on ERS and ROS levels in mice with VILI. (D) Levels of LDH efflux in BALF. (E,F) Effect of ACE2 overexpression and MAS1 inhibitor (A779) on pyroptosis. ERS, endoplasmic reticulum stress; CHOP, C/EBP homologous protein; GRP78, glucose-regulated protein 78; VILI, ventilator-induced lung injury; ACE2, angiotensin-converting enzyme 2; ROS, reactive oxygen species; LDH, lactate dehydrogenase; BALF, bronchoalveolar lavage fluid; NLRP3, NOD-like receptor protein 3 inflammasome; GSDMD, gasdermin D. ***p 0.001 vs. control, ^#⁢#p 0.01 vs. VILI, ^^p 0.01 vs. VILI+ACE2. N = 10; repeats = 3.