Hypercapnia promotes microglial pyroptosis via inhibiting mitophagy in hypoxemic adult rats

Abstract Background Hypoxemia is a typical symptom of acute respiratory distress syndrome. To avoid pulmonary morbidity, low tidal volume ventilation is often applied. The ventilation strategy will certainly cause hypercapnia. This study aimed to explore whether hypercapnia would promote microglial pyroptosis via inhibiting mitophagy in adult rats with hypoxemia. Methods The cerebral oxygen extraction ratio (CERO2) and partial pressure of brain tissue oxygen (PbtO2) in a rat model of hypercapnia/hypoxemia were assessed. The reactive oxygen species (ROS) production and the expression of LC3‐II/I, p62, caspase‐1, gasdermin D‐N domains (GSDMD‐N), IL‐1β, and IL‐18 in microglial cells were detected. Results Hypercapnia decreased the PbtO2 levels of the hypoxic rats, which was further evidenced by the increased levels of CERO2. Expression levels of LC3‐II were reduced, while p62 expression was increased by hypercapnia in hypoxic microglia. Hypercapnia increased the production of ROS and the expression of caspase‐1, GSDMD‐N, IL‐1β, and IL‐18 in hypoxia‐activated microglia. Scavenging ROS inhibited microglial pyroptosis and expression of IL‐1β and IL‐18. Conclusions These results suggest that hypercapnia‐induced mitophagy inhibition may promote pyroptosis and enhance IL‐1β and IL‐18 release in hypoxia‐activated microglia.

inflammatory response. [10][11][12] To exert its functions, the NLRP3 inflammasome requires to be activated. The NLRP3 inflammasome can be triggered by an array of stimuli, including ATP, potassium efflux, heme, urate, and reactive oxygen species (ROS). [13][14][15][16][17] Activation of the NLRP3 inflammasome can upregulate the expression of caspase-1 and then leads to interleukin (IL)-18 and IL-1β maturation and pyroptosis activation. Pyroptosis is a proinflammatory cell death. 18 Recent studies have reported that gasdermin D (GSDMD) is the executioner of pyroptosis. 19 After being cleaved by caspase-1, gasdermin D-N domains (GSDMD-N) can form a pore in the plasma membrane. IL-1β and IL-18 are released through the pores, thereby perpetuating cascade inflammatory response. 20 Mitochondria-derived ROS is the key stimuli to trigger the activation of NLRP3 inflammasome. 21 When the levels of oxygen fall, hypoxia would damage mitochondria and induce ROS production. [22][23][24] Mitophagy can eliminate damaged mitochondria, reduce ROS production, and alleviate NLRP3 inflammasome activation. 25 It remains to be ascertained whether hypercapnia would activate the NLRP3 inflammasome through inhibiting mitophagy in hypoxia-activated microglia.
In the present study, we hypothesized that hypercapnia may promote microglial pyroptosis via inhibiting mitophagy in hypoxemic adult rats. It was surmised that hypercapnia might exert its effect through inhibiting mitophagy, inducing ROS production, activating NLRP3 inflammasome, which ultimately would lead to IL-18 and IL-1β maturation and pyroptosis activation.

| Animals and experimental groups
The Experimental Animal Care and Use Committee at Jinan University approved all animal procedure protocols (No.

| The rat model of hypercapnia/hypoxemia
All rats were fasted with access to water overnight before the experiments. The rat model of hypercapnia/hypoxemia was established as described in our previous study. 9 Briefly, the rats were anesthetized with pentobarbital sodium (30 mg/kg) followed by mechanical ventilation. The tidal volume (9 mL/kg body weight), respiratory rate (45 breaths/min), and inspiratory-to-expiratory ratio (1:1) were fixed. Sham group was exposed to the air. Hypercapnia group was exposed to 5% CO 2 to maintain the potential of hydrogen (pH) of arterial blood at 7.20-7. 25. Hypoxemia group was exposed to 16% O 2 to maintain partial pressure of arterial blood oxygen (PaO 2 ) at 55-60 mmHg. HH group was exposed to 16% O 2 mixing with 5% CO 2 to maintain PaO 2 at 55-60 mmHg and pH at 7. 20-7.25. Rats in the HH + NAC group were treated with an intraperitoneal injection of 150 mg/kg NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 minutes before being exposed to 5% CO 2 and 16% O 2 . The rats, which were used for Western blotting analysis, immunofluorescence staining and ROS evaluation, were not performed invasive manipulation except ventilation. The rats were executed immediately after hypercapnia/hypoxemia treatment.

| Measurement of cerebral oxygen extraction ratio
The right femoral artery and the right jugular vein were cannulated upstream. The blood samples were collected at 3 hours after ventilation. The hemoglobin concentration (Hb), saturation of arterial blood oxygen (SaO 2 ), PaO 2 , saturation of jugular venous blood oxygen (SjVO 2 ), and pressure of jugular venous blood oxygen (PjVO 2 ) were measured using a blood gas/electrolyte analyzer (Model 5700; San Diego, CA, USA). The content of arterial blood oxygen (CaO 2 ), content of jugular venous blood oxygen (CjVO 2 ), and cerebral oxygen extraction ratio (CERO 2 ) were calculated using the following formulas:

| Measurement of the partial pressure of brain tissue oxygen (PbtO 2 )
The levels of PbtO 2 were measured at 0.5, 1, 1.5, 2, 2.5, and 3 hours after ventilation. To evaluate the PbtO 2 , a midline incision over vertex was performed after anesthesia. After this, a hole was drilled caudal to the coronal suture, 4 mm from the midline. The dura was punctured, and a microsensor for PbtO 2 was inserted into brain tissue. 26 A monitor (Integra CAMO2, Integra LifeSciences Limited, County Offaly, Ireland) was used to measure the PbtO 2 .

| ROS evaluation of brain tissue
The ROS of brain tissue was evaluated using a ROS ELISA kit were added to incubate the plate in a dark place for 15 minutes at 37°C. Then, the stop buffer was added, and the optical density (OD) was measured spectrophotometrically at a wavelength F I G U R E 1 Hypercapnia increased CERO 2 and OCR in vivo and in vitro (n = 4). (A) There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < .01). (B) Simple effects analyses show an increased CERO 2 in Hypoxemia group (*P < .05), but not in Hypercapnia group (ns P > .05) compared with Sham group. HH group has the highest CERO 2 levels as compared with Hypoxemia group (**P < .01) and Hypercapnia group (**P < .01). (C) The OCR levels are the highest in 12 h group as compared with other groups (12 h vs 0 h: P < .01; 12 h vs 6 h: P > .05; 12 h vs 24 h: P < .01). The OCR levels are increased time-dependently and achieved a stationary phase at 18 min. So the microglia cells were treated for 12 hours, and the OCR was examined at 18 min after the treatment, as shown in graph (D) and (E). (D) There is an interaction effect between 0.2% O 2 treatment and 15% CO 2 treatment (P < .01). (E) Simple effects analyses show increased levels of OCR in Hypoxia group (**P < .01), but not in HC group (ns P > .05) compared with Control group. Hypoxia + HC group has the highest levels of OCR as compared with Hypoxia group (**P < .01) and HC group (**P < .01). ns, nonsignificant; Sham group, shamoperated group; HH group, hypercapnia + hypoxemia group; HC group, high concentration of carbon dioxide group; CERO 2 , cerebral oxygen extraction rate; OCR, oxygen consumption rate. The concentrations of O 2 and CO 2 in the air are 21% and 0.03% of 450 nm. The concentrations of ROS in the samples were then determined by comparing the optical density of the samples to the standard curve.

| BV-2 microglial cell cultures and treatment
BV-2 microglial cells were purchased form CHI Scientific (cat. no.  and were cultured and treated as described in our previous study. 9  cat. no. 42F0374K) in a humidified incubator with 5% CO 2 /95% air. The microglial cells were randomly divided into five groups: Control group, high concentration of carbon dioxide group (abbreviated HC group), Hypoxia group, Hypoxia + HC group, and Hypoxia + HC +NAC group. Control group was exposed to 5% CO 2 + 20% O 2 . HC group was exposed to 15% CO 2 + 20% O 2 to maintain the pH of the supernatant at 7.20-7.25. Hypoxia group was exposed to 5% CO 2 + 0.2% O 2 to maintain partial pressure of oxygen (PO 2 ) of the supernatant at 55-60 mmHg. Hypoxia + HC group was exposed to 15% CO 2 + 0.2% O 2 to maintain PO 2 at 55-60 mmHg and pH at 7.20-7.25. The cells in the Hypoxia + HC + NAC group were treated with 2 mmol/L NAC (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215) for 30 minutes before being exposed to 15% CO 2 + 0.2% O 2 .

| Oxygen consumption rate evaluation of BV-2 microglial cells
The Oxygen consumption rate (OCR) was evaluated using a cellulate

| ROS measurement in microglia
The ROS production in BV-2 microglial cells was evaluated using a ROS assay kit (cat. no. BB-4705-2; BestBio, Shanghai, China) following the manufacturers' instructions. Briefly, DCFH-DA was diluted with DMEM high glucose without FBS (1:1500). The coverslips with adherent BV-2 microglial cells were cultured with DMEM high glucose supplemented with 10% FBS. After the treatment, the medium was changed to diluted DCFH-DA (2 mL/well). Then, the plates were incubated for 20 minutes at 37°C, 5% CO 2 . The coverslips were washed with DMEM high glucose without FBS. Finally, the coverslips were mounted by a fluorescent mounting medium and detected using a fluorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan).

| Double immunofluorence labeling
In vivo, the rats were anesthetized with pentobarbital sodium and transcardially perfused with normal saline and 4% paraformaldehyde at 3 hours after ventilation. The brains were harvested and postfixed in 4% paraformaldehyde. This tissue was then dehydrated in graded sucrose and cut into sections of 10 μm thickness. The sections were blocked in 5% normal donkey serum for 0.5 hours at room temperature.
In vitro, the coverslips with adherent BV-2 microglial cells were fixed with 4% paraformaldehyde at 24 hours after treatment. The coverslips were blocked in 5% normal donkey serum for 0.5 hours at room temperature sequentially.
After that, the following primary antibodies were added to in- (E) In contrast, a significant decrease in p62 expression is observed in Hypoxemia group compared with Sham group (**P < .01). The levels of p62 expression in HH group are higher compared with Hypoxemia group (**P < .01). ns, nonsignificant; Sham group, sham-operated group; HH group, hypercapnia + hypoxemia group. The concentrations of O 2 and CO 2 in the air are 21% and 0.03% were washed on the following day, and the secondary antibodies

| Statistical analysis
The statistical analysis was performed by the SPSS19.0 statistical (IBM, New York, USA). All values are expressed as mean ± standard error of mean (SEM). Shapiro-Wilk test was used to assess the data distribution. All data were normally distributed in the present study.
Repeated measures one-way analysis of variance (ANOVA) was used to analyze the repeated measurement data. Factorial ANOVA was for the interaction effects. When an interaction was examined, simple effects analyses were evaluated. Differences were considered statistically significant if the P-value < .05.

| Hypercapnia increased cerebral oxygen extraction rate in hypoxemic rats
Significant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < .01) ( Figure 1A). Simple effects analyses found an increased CERO 2 in Hypoxemia group (P < .05), but not in Hypercapnia group (P > .05) compared with Sham group. HH group had the highest CERO 2 levels as compared with Hypoxemia group (P < .01) and Hypercapnia group (P < .01) ( Figure 1B).

| 15% CO 2 increased the oxygen consumption rate (OCR) in hypoxic BV-2 microglial cells
The OCR levels were the highest in 12 hours group as compared with other groups (12 hours vs 0 hour: P < .01; 12 hours vs 6 hours: P > .05; 12 hours vs 24 hours: P < .01). Besides, the OCR levels were increased time-dependently and achieved a stationary phase at 18 minutes ( Figure 1C). Because of this, microglia were treated for 12 hours, and the OCR was examined at 18 minutes after the treatment ( Figure 1D, E). Significant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < .01; Figure 1D). Simple effects analyses found increased levels of OCR in Hypoxia group (P < .01), but not in HC group (P > .05) compared with Control group. Hypoxia + HC group had the highest levels of OCR as compared with Hypoxia group (P < .01) and HC group (P < .01) ( Figure 1E).

| Hypercapnia induced overproduction of ROS in the hypoxemic hippocampus
Significant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < .05) ( Figure 5A). Simple effects analyses found increased levels of ROS in Hypoxemia group (P < .01), but not in Hypercapnia group (P > .05) compared with Sham group. HH group had the highest ROS levels as compared with Hypoxemia group (P < .01) and Hypercapnia group (P < .01) ( Figure 5B).

| 15% CO 2 induced overproduction of ROS in hypoxic BV-2 microglial cells
Enhanced ROS immunofluorescence was observed in Hypoxia group, but not in HC group compared with Control group.
Hypoxia + HC group had the strongest ROS fluorescence as compared with Hypoxia group and HC group. ROS fluorescence was obviously reduced with NAC pretreatment (2 mmol/L) in BV-2 microglial cells ( Figure 5C). In contrast, a significant decrease in p62 expression is observed in Hypoxia group compared with Control group (P < .01). The levels of p62 expression in Hypoxia + HC group are higher compared with Hypoxia group (P < .01). ns, nonsignificant; HC group, high concentration of carbon dioxide group levels of caspase-1 as compared with Hypoxemia group (P < .01) and Hypercapnia group (P < .01) ( Figure 6A, C). Additionally, the protein expression of caspase-1 was significantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < .01) ( Figure 6C). The full Western blots of the above each group were shown in Figure   S9. Double immunofluorescence was used to examine caspase-1 expression in microglia of the hippocampus ( Figure 6D). Enhanced caspase-1 immunofluorescence was observed in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group had the most intense caspase-1 fluorescence when compared with Hypoxemia group and Hypercapnia group. In rats given NAC treatment, caspase-1 fluorescence was noticeably attenuated ( Figure 6D).

| 15% CO 2 enhanced NLRP3 inflammasome activation via inducing ROS overproduction in hypoxic BV-2 microglial cells
Significant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < .01) ( Figure S1B). Simple effects analyses found increased protein expression levels of caspase-1 in Hypoxia group (P < .01), but not in HC group (P > .05) compared with Control group. Hypoxia + HC group had the highest expression levels of caspase-1 as compared with Hypoxia group (P < .01) and HC group (P < .01) ( Figure S1A, C). Additionally, the protein expression of caspase-1 was significantly suppressed with NAC pretreatment (2 mmol/L) in BV-2 microglial cells (P < .01)

F I G U R E 5 Hypercapnia induced overproduction of ROS in the hypoxemic hippocampus and BV2 cells (n = 4). (A) There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < .05). (B) Simple effects analyses show increased levels of ROS in
Hypoxemia group (**P < .01), but not in Hypercapnia group (ns P > .05) compared with Sham group. HH group has the highest ROS levels as compared with Hypoxemia group (**P < .01) and Hypercapnia group (**P < .01). (C) Immunofluorescence images showing the production of ROS (a-e, green). Enhanced ROS immunofluorescence is evident in Hypoxia group, but not in HC group compared with Control group. Hypoxia + HC group exhibits the most intense ROS fluorescence as compared with Hypoxia group and HC group. ROS fluorescence is noticeably reduced with NAC pretreatment (2 mmol/L) in BV-2 microglial cells. Scale bars: 20 μm. ns, nonsignificant; Sham group, shamoperated group; HH group, hypercapnia + hypoxemia group; ROS, reactive oxygen species; HC group, high concentration of carbon dioxide group. The concentrations of O 2 and CO 2 in the air are 21% and 0.03% ( Figure S1C). The full Western blots of the above each group were shown in Figure S10. Double immunofluorescence was used to examine caspase-1 expression in BV-2 microglial cells ( Figure S1D).
Enhanced caspase-1 immunofluorescence was observed in Hypoxia group, but not in HC group compared with Control group.
Hypoxia + HC group had the strongest caspase-1 fluorescence as compared with Hypoxia group and HC group. Of note, caspase-1 fluorescence was evidently reduced with NAC pretreatment (2 mmol/L) in BV-2 microglial cells ( Figure S1D).

| Hypercapnia increased GSDMD-N expression via inducing ROS overproduction in microglia in the hypoxic hippocampus
Significant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < .01) ( Figure S2B). Simple effects analyses found increased protein expression levels of GSDMD-N in Hypoxemia group (P < .05), but not in Hypercapnia group (P > .05) compared with Sham group. HH group showed F I G U R E 6 Hypercapnia enhanced NLRP3 inflammasome activation via inducing ROS overproduction in microglia of the hypoxic hippocampus (n = 4). (A) Immunoreactive bands of caspase-1 (10 kDa) and β-actin (42 kDa). (B) There is an interaction effect between hypoxia treatment and hypercapnia treatment (P < .01). (C) Simple effects analyses show increased protein expression levels of caspase-1 in Hypoxemia group (**P < .01), but not in Hypercapnia group (ns P > .05) compared with Sham group. HH group has the highest expression levels of caspase-1 as compared with Hypoxemia group (**P < .01) and Hypercapnia group (**P < .01). The protein expression of caspase-1 is significantly suppressed with NAC pretreatment (150 mg/kg) in rats (**P < .01). (D) Immunofluorescence images showing the expression of Iba1 + microglia (a, d, g, j, m, green), caspase-1 (b, e, h, k, n, red), and the colocalization of caspase-1 and microglia (x, f, i, l, o). Enhanced caspase-1 immunofluorescence is evident in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group emits the strongest caspase-1 fluorescence as compared with Hypoxemia group and Hypercapnia group. Caspase-1 fluorescence is markedly attenuated in rats given NAC pretreatment. Scale bars: 10 μm. ns, nonsignificant; Sham group, sham-operated group; HH group, hypercapnia + hypoxemia group. The concentrations of O 2 and CO 2 in the air are 21% and 0.03% the highest expression levels of GSDMD-N in comparison with Hypoxemia group (P < .01) and Hypercapnia group (P < .01).
Additionally, the protein expression of GSDMD-N was significantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < .01) ( Figure S2A, C). The full Western blots of the above each group were shown in Figure S11.

| Hypercapnia increased IL-1β expression via inducing ROS overproduction in microglia in the hypoxic hippocampus
Significant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < .01) ( Figure S3B). Simple effects analyses found increased protein expression levels of IL-1β in Hypoxemia group (P < .01), but not in Hypercapnia group (P > .05) compared with Sham group. HH group showed the highest expression levels of IL-1β in comparison with Hypoxemia group (P < .01) and Hypercapnia group (P < .01). Additionally, the protein expression of IL-1β was significantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < .01) ( Figure S3A,C). The full Western blots of the above each group were shown in Figure S12. Double immunofluorescence was used to examine IL-1β expression in microglia of the hippocampus ( Figure S3D). Enhanced IL-1β immunofluorescence was observed in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group had the strongest IL-1β fluorescence as compared with Hypoxemia group and Hypercapnia group. IL-1β fluorescence was evidently reduced in rats given NAC pretreatment ( Figure S3D).

| 15% CO 2 increased IL-1β expression via inducing ROS overproduction in hypoxic BV-2 microglial cells
Significant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < .01) ( Figure S4B). Simple effects analyses found increased protein expression levels of IL-1β in Hypoxia group (P < .01), but not in HC group (P > .05) compared with Control group. Hypoxia + HC group had the highest expression levels of IL-1β as compared with Hypoxia group (P < .01) and HC group (P < .01). Additionally, the protein expression of IL-1β was significantly suppressed with NAC pretreatment (2 mmol/L) in BV-2 microglial cells (P < .01) ( Figure S4A, C). The full Western blots of the above each group were shown in Figure S13. Double immunofluorescence confirmed IL-1β expression in BV-2 microglial cells ( Figure S4D). Enhanced IL-1β immunofluorescence was observed in Hypoxia group, but not in HC group compared with Control group.
Hypoxia + HC group had the strongest IL-1β fluorescence as compared with Hypoxia group and HC group. IL-1β fluorescence was markedly suppressed with NAC pretreatment (2 mmol/L) in BV-2 microglial cells ( Figure S4D).

| Hypercapnia increased IL-18 expression via inducing ROS overproduction in microglia in the hypoxic hippocampus
Significant interaction effects were observed between hypercapnia treatment and hypoxia treatment (P < .01) ( Figure S5B). Simple effects analyses found increased protein expression levels of IL-18 in Hypoxemia group (P < .01), but not in Hypercapnia group (P > .05) compared with Sham group. HH group showed the highest expression levels of IL-18 in comparison with Hypoxemia group (P < .01) and Hypercapnia group (P < .01). Additionally, the protein expression of IL-18 was significantly suppressed with NAC pretreatment (150 mg/kg) in rats (P < .01) ( Figure S5A, C). The full Western blots of the above each group were shown in Figure S14. Double immunofluorescence was used to examine IL-18 expression in microglia of the hippocampus ( Figure S5D). Enhanced IL-18 immunofluorescence was observed in Hypoxemia group, but not in Hypercapnia group compared with Sham group. HH group had the strongest IL-18 fluorescence as compared with Hypoxemia group and Hypercapnia group. IL-18 fluorescence was evidently reduced in rats given NAC pretreatment ( Figure S5D).

| 15% CO 2 increased IL-18 expression via inducing ROS overproduction in hypoxic BV-2 microglial cells
Significant interaction effects were observed between 0.2% O 2 treatment and 15% CO 2 treatment (P < .01) ( Figure S6B). Simple effects analyses found increased protein expression levels of IL-18 in Hypoxia group (P < .01), but not in HC group (P > .05) compared with Control group. Hypoxia + HC group had the highest expression levels of IL-18 as compared with Hypoxia group (P < .01) and HC group (P < .01). Additionally, the protein expression of IL-18 was significantly suppressed with NAC pretreatment (2 mmol/L) in BV-2 microglial cells (P < .01) ( Figure S6A, C). The full Western blots of the above each group were shown in Figure S15. Double immunofluorescence confirmed IL-18 expression in BV-2 microglial cells ( Figure S6D). Enhanced IL-18 immunofluorescence was observed in Hypoxia group, but not in HC group compared with Control group.
Hypoxia + HC group had the strongest IL-18 fluorescence as compared with Hypoxia group and HC group. IL-18 fluorescence was markedly suppressed with NAC pretreatment (2 mmol/L) in BV-2 microglial cells ( Figure S6D).

| D ISCUSS I ON
The present results have shown that hypercapnia promoted py- The present rat and cell models of hypercapnia/hypoxemia were established as described in our previous study. 9 In Sham group, the rats were exposed to air when the ventilator settings were fixed.
The PaCO 2 levels of the rats were maintained between 35 and 45 mmHg, and the PaO 2 levels were maintained between 90 and 100 mmHg. The normal range of human PaCO 2 and PaO 2 is 35-45 and 80-100 mmHg, respectively, which are comparable between rats and humans. In the hypercapnia/hypoxemia models, PO 2 levels of the arterial blood and culture supernatant were maintained at 55-60 mmHg and pH levels of the arterial blood and culture supernatant at 7.20-7.25. These are consistent with the change of hypoxemia and permissive hypercapnia in ARDS. [27][28][29][30] We reported in our previous study that hypercapnia could induce IL-1β overproduction in hypoxia-activated microglia. However, the underlying mechanism has remained unclear. It has been reported that the expression of caspase-1 would be upregulated after NLRP3 inflammasome being activated. Caspase-1 could lead to IL-18 and IL-1β maturation and pyroptosis activation. GSDMD-N is the executioner of pyroptosis. GSDMD-N helped to form a pore in the plasma membrane, from that IL-1β and IL-18 are released. 19,20 Under hypoxic conditions, ROS is known to be critical for NLRP3 inflammasome activation. [31][32][33] Furthermore, hippocampus is known to be critical for spatial and contextual memory. 34 And the receptor of inflammatory cytokines (ie, IL-1β) 35 are comparatively higher in the hippocampus and are tightly linked to the deficits in hippocampus-dependent memory. 36 Thus, the expression levels of caspase-1, GSDMD-N, IL-18, and IL-1β in the hippocampus microglia in this study were determined, and the production of ROS was also evaluated. Significant interaction effects were observed on ROS production, caspase-1, GSDMD-N, IL-18, and IL-1β expression between hypercapnia treatment and hypoxia treatment. More importantly, we have shown that hypercapnia upregulated the expression of caspase-1, GSDMD-N, IL-18, and IL-1β in hypoxic microglia via inducing ROS overproduction. Remarkably, caspase-1, GSDMD-N, IL-18, and IL-1β expression in hypoxic microglia was downregulated when ROS was scavenged by NAC. These results indicate that hypercapnia promotes microglial pyroptosis via inducing ROS overproduction and activating the NLRP3 inflammasome.
When the levels of oxygen fall, hypoxia would damage mitochondria and induce ROS production. [22][23][24] Mitophagy can eliminate damaged mitochondria, reduce ROS production, and then alleviate NLRP3 inflammasome activation. 25 To ascertain whether hypercapnia would activate the NLRP3 inflammasome through inhibiting mitophagy in hypoxia-activated microglia, expression of LC3-II and p62 in the microglia was determined. p62 is a multidomain protein that interacts with phagophores through the LC3-interacting domain and with the ubiquitinated protein aggregates through the ubiquitin-associated domain, 37 which promotes degradation of unwanted molecules, 38 while p62 is an autophagy substrate and is degraded by autophagy. 39 We found that the expression levels of LC3-II were reduced, while p62 expression was increased by hypercapnia in the hypoxic hippocampus and BV-2 microglia. These results indicate that hypercapnia might enhance the NLRP3 inflammasome activation in hypoxia-activated microglial cells via inhibiting mitophagy and impairing autophagy flux.
To determine the effect of hypercapnia on oxidative metabolism of the rat brain, CERO 2 and partial pressure of brain tissue oxygen (PbtO 2 ) were examined. Hypoxemia, as a typical symptom of ARDS, decreases the oxidative metabolism rate of the brain to avoid or reduce cerebral damage via preventing the PbtO 2 from dropping to a very low level. 40 In this study, we showed hypercapnia alone was not enough to increase or decrease the levels of PbtO 2 and CERO 2 .
In the first 1.5 hours, the levels of PbtO 2 were elevated by hypercapnia in the hypoxemic rats. This may be the reason why hypercapnia was neuroprotective in rats with transient global cerebral ischemia-reperfusion injury 41 and lateral fluid percussion injury. 42 However, hypercapnia significantly decreased the levels of PbtO 2 in the hypoxemic rats after 2 hours' ventilation. It was further evidenced by the increased levels of CERO 2 at 3 hours after ventilation.
These results suggest that hypercapnia is neuroprotective within a very short period (1.5 hours), but hypercapnia could aggravate neuronal functions injury over a protracted period via increasing CERO 2 and decreasing PbtO 2 . This is consistent with the expression of IL-1β and IL-18 in microglia. It was observed that hypercapnia alone may not be sufficient to increase the expression of IL-1β and IL-18, but hypercapnia may exert its effects in the presence of hypoxia. It was suggested that hypercapnia can induce ROS overproduction via intensifying hypoxia, may activate the NLRP3 inflammasome, and promote IL-1β release.
In vitro, to determine the effect of a high concentration of CO 2 on oxidative metabolism in hypoxic BV-2 microglial cells, the oxygen consumption rate (OCR) was evaluated. There was an interaction effect on the levels of OCR between 0.2% O 2 treatment and 15% CO 2 treatment. The high concentration of CO 2 treatment could increase the levels of OCR in hypoxic BV-2 microglial cells, which were consistent with that hypercapnia increased levels of CERO 2 in hypoxic rats. The results suggest that hypercapnia could intensify microglial hypoxia both in vivo and in vitro.
There are four limitations to this study. Firstly, it has been reported that NLRP3 inflammasome is expressed in astrocytes. 43 However, the lack of the NLRP3 inflammasome in astrocytes being investigated in the present study is a limitation. Secondly, the present study did not prove the connection between cerebral oxygen metabolism (CERO 2 and PbtO 2 ) and mitophagy, which is an area for future research. Thirdly, the models of hypercapnia/hypoxemia were established with certain concentrations of O 2 and CO 2 . The lack of concentration gradient of O 2 and CO 2 being investigated in the present study is another limitation. Fourthly, the study did not prove the upstream mechanism by which hypercapnia inhibits mitophagy. It is well documented that the PINK1-Parkin axis functions as the core machinery for mitophagy. 44 It was surmised that hypercapnia may exert its effect through mediating the PINK1-Parkin signaling axis.
In summary, the present results have demonstrated the underlying mechanism whereby hypercapnia can promote pyroptosis in hypoxic microglia. In this connection, hypercapnia was found to intensify the cerebral hypoxia via increasing CERO 2 and decreasing PbtO 2 . ROS was overproduced by hypercapnia in hypoxic microglial cells coupled with mitophagy inhibition and activation of NLRP3 inflammasome. Remarkedly, caspase-1, GSDMD-N, IL-18, and IL-1β expression was downregulated when ROS was scavenged. Thus, hypercapnia-induced mitophagy inhibition, NLRP3 inflammasome activation, and pyroptosis in microglia may be a potential target to mitigate neuroinflammation.

CO N FLI C T O F I NTE R E S T
The authors have declared that no conflict of interest exists.