Chemical Hypoxia Induces Pyroptosis in Neuronal Cells by Caspase-Dependent Gasdermin Activation

Hypoxia-induced neuronal death is a major cause of neurodegenerative diseases. Pyroptosis is a type of inflammatory programmed cell death mediated by elevated intracellular levels of reactive oxygen species (ROS). Therefore, we hypothesized that hypoxia-induced ROS may trigger pyroptosis via caspase-dependent gasdermin (GSDM) activation in neuronal cells. To test this, we exposed SH-SY5Y neuronal cells to cobalt chloride (CoCl2) to trigger hypoxia and then evaluated the cellular and molecular responses to hypoxic conditions. Our data revealed that CoCl2 induced cell growth inhibition and the expression of hypoxia-inducible factor-1α in SH-SY5Y cells. Exposure to CoCl2 elicits excessive accumulation of cytosolic and mitochondrial ROS in SH-SY5Y cells. CoCl2-induced hypoxia not only activated the intrinsic (caspases-3, -7, and -9) apoptotic pathway but also induced caspase-3/GSDME-dependent and NLRP3/caspase-1/GSDMD-mediated pyroptosis in SH-SY5Y cells. Importantly, inhibition of caspase-3 and -1 using selective inhibitors ameliorated pyroptotic cell death and downregulated GSDM protein expression. Additionally, treatment with a ROS scavenger significantly suppressed caspase- and pyroptosis-related proteins in CoCl2-treated SH-SY5Y cells. Our findings indicate that hypoxia-mediated ROS production plays an important role in the activation of both apoptosis and pyroptosis in SH-SY5Y neuronal cells, thus providing a potential therapeutic strategy for hypoxia-related neurological diseases.


Introduction
Hypoxia is a condition in which the supply of oxygen is insufficient to maintain normal cellular function in cells, tissues, and organs [1].The brain consumes approximately 20 percent of the total oxygen supply to generate adenosine triphosphate (ATP) molecules that are essential for cell integrity, brain tissue viability, and electrophysiological activity [2].ATP is synthesized via the oxidative phosphorylation pathway in mitochondria under aerobic conditions [3].However, hypoxic conditions diminish mitochondrial ATP production, thus resulting in irreversible cellular dysfunction, including disturbances in ion channel homeostasis, membrane damage, impaired mitochondrial metabolism, and reactive oxygen species (ROS) production [4,5].Studies have demonstrated that hypoxia is pivotal in triggering neuronal cell death that can lead to neurodegenerative disorders, including Alzheimer's disease [6], Parkinson's disease [7], multiple sclerosis [8], Huntington's disease [9], and amyotrophic lateral sclerosis [10].
Cellular ROS are primarily generated by mitochondria during cellular respiration in the electron transport chain (ETC) where electrons leak and directly interact with oxygen to form superoxide anions, hydroxyl ions, and hydrogen peroxide in mitochondrial complexes I and III [11].However, the net production of ROS from mitochondria can be markedly elevated under various pathological conditions, including hypoxia, ischemia, reperfusion, inflammation, and genetic defects in the ETC [12,13].Hypoxia induces excessive ROS production through inefficient electron transport to the ETC in the mitochondria due to the lack of electron acceptor oxygen [14].In Alzheimer's disease, the decrease in cerebral blood flow causes cerebral hypoxia that enhances neuroinflammation, amyloid-β (Aβ) production, tau hyperphosphorylation, and mitochondrial dysfunction [15].Moreover, the Aβ aggregates activate the NADPH oxidase 2 that is highly expressed in microglial cells, thus resulting in an increase in ROS release [16].In Parkinson's disease, mitochondrial complex I deficiency is abundantly observed in dopaminergic neurons of the substantia nigra [17].
Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor, plays a central role in the cellular response to low oxygen concentrations [18].HIF-1 consists of the oxygen-sensitive HIF-1α and the constitutively stable HIF-1β, both of which are basic helix-loop-helix proteins containing a PAS domain that is responsible for dimerization and DNA binding [19].Studies have demonstrated that hypoxia enhances the levels of HIF-1α, ultimately leading to translocation to the nucleus, dimerization with HIF-1β, and binding to hypoxia-response elements for the transcription of target genes to adapt to a low oxygen environment [20].In hypoxic conditions, overexpressed HIF-1α triggers the transcription of the β-secretase 1 gene and directly interacts with the γ-secretase complex.A significant increase in βand γ-secretase activities promotes the cleavage of Aβ precursor protein and the deposition of extracellular Aβ plaques in Alzheimer's disease [21].
Cell death is an essential process for organism development, maintenance of tissue homeostasis, and host defense against pathogens [22].Cell death can typically be classified as regulated or non-regulated based on signal dependency.Unregulated cell death is caused by an unexpected cell injury.In contrast, regulated cell death, also known as programmed cell death, is regulated by intracellular signaling pathways [23].Pyroptosis is a lytic form of programmed cell death characterized by the formation of pores in the plasma membrane [24].Gasdermin (GSDM) consists of functional N-terminal and inhibitory C-terminal domains connected by a flexible interdomain linker.In response to diverse damaging signals, the linker region of gasdermin is cleaved by active caspases or granzymes, and the N-terminal fragments are translocated to the plasma membrane to form pores [25].Recent studies have reported that pyroptosis can be induced by hypoxia [26,27].Under hypoxic conditions, PD-L1 physically interacts with p-Stat3 and translocates to the nucleus, ultimately enhancing gasdermin C (GSDMC) gene and protein expression.Caspase-8 activated by macrophage-derived TNFα cleaves GSDMC, thus resulting in a switch from apoptosis to pyroptosis in cancer cells [28].Hypoxia induces ROS overproduction and promotes the NF-κB/HIF-1α signaling pathways, and the enhanced expression of cleaved caspase-1 and gasdermin D (GSDMD) further promotes pyroptosis in CoCl 2 myoblasts [29].Moreover, chronic intermittent hypoxia induces GSDMD-mediated pyroptosis via activation of the NLRP3 inflammasome and caspase-1 in renal tubular epithelial cells [30].However, the molecular mechanisms underlying hypoxia-induced pyroptosis in neurons remain largely unknown.In this study, we investigated the role of hypoxia in neuronal cell pyroptosis and the possible molecular mechanisms underlying pyroptosis under hypoxic conditions.

Effect of CoCl 2 -Induced Hypoxia on Cell Viability and Morphological Changes
Cobalt chloride (CoCl 2 ) is a well-known hypoxia-mimetic chemical compound that induces hypoxia-like conditions in vitro [31].To investigate whether CoCl 2 leads to neuronal cytotoxicity, SH-SY5Y neuronal cells were treated with different concentrations of CoCl 2, and cell viability was monitored at 24 h.As shown in Figure 1A, CoCl 2 treatment significantly induced cell death in a dose-dependent manner.In particular, a significant reduction of cell viability was observed after exposure to 100 µM CoCl 2 compared with that of the control cells.When the concentration of CoCl 2 reached 200 µM, the cell viability of SH-SY5Y decreased around 50%.Therefore, we selected CoCl 2 concentrations of 200 µM for subsequent experiments.
dose-dependent manner using phase-contrast microscopy.As shown in Figure 1B, there was no significant change in SH-SY5Y cell population and cell morphology under 50 µM CoCl2 treatment.However, SH-SY5Y cells treated with ≥100 µM CoCl2 exhibited a significant decrease in cell numbers and morphological alterations including rounding up, membrane blebbing, cell shrinkage and neurite degeneration.These results indicated that hypoxia may adversely affect neuronal cell viability as well as cell morphology.

Chemical Hypoxia Induces Accumulation of HIF-1α in SH-SY5Y Cells
To examine if CoCl2-induced hypoxia affects the protein expression of HIF-1α, we determined the expression levels of HIF-1α protein by Western blot analysis in SH-SY5Y cells treated with different CoCl2 concentrations up to 200 µM.Our results demonstrated that HIF-1α expression was absent in the untreated SH-SY5Y cells (Figure 2A).However, CoCl2 induced the increase in HIF-1α protein expression in a dose-dependent manner.In The effect of CoCl 2 in cell proliferation and cell morphology was also observed in a dose-dependent manner using phase-contrast microscopy.As shown in Figure 1B, there was no significant change in SH-SY5Y cell population and cell morphology under 50 µM CoCl 2 treatment.However, SH-SY5Y cells treated with ≥100 µM CoCl 2 exhibited a significant decrease in cell numbers and morphological alterations including rounding up, membrane blebbing, cell shrinkage and neurite degeneration.These results indicated that hypoxia may adversely affect neuronal cell viability as well as cell morphology.

Chemical Hypoxia Induces Accumulation of HIF-1α in SH-SY5Y Cells
To examine if CoCl 2 -induced hypoxia affects the protein expression of HIF-1α, we determined the expression levels of HIF-1α protein by Western blot analysis in SH-SY5Y cells treated with different CoCl 2 concentrations up to 200 µM.Our results demonstrated that HIF-1α expression was absent in the untreated SH-SY5Y cells (Figure 2A).However, CoCl 2 induced the increase in HIF-1α protein expression in a dose-dependent manner.In particular, the protein expression of HIF-1α was significantly increased at the 200 µM con-centration of CoCl 2 compared to the levels in response to the other CoCl 2 doses (p < 0.001) (Figure 2B).These results suggested that CoCl 2 mimicked hypoxia in SH-SY5Y cells.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 4 of 15 particular, the protein expression of HIF-1α was significantly increased at the 200 µM concentration of CoCl2 compared to the levels in response to the other CoCl2 doses (p < 0.001) (Figure 2B).These results suggested that CoCl2 mimicked hypoxia in SH-SY5Y cells.

Chemical Hypoxia Enhances Cytosolic and Mitochondrial ROS
Hypoxic conditions promote increased intracellular oxidative stress, particularly in the form of ROS [32].To explore if CoCl2 treatment induces oxidative stress, we assessed the cytosolic and mitochondrial ROS generation in CoCl2-exposed SH-SY5Y cells (Figure 2C).Cytosolic ROS production induced by CoCl2 was determined by 2′-7′dichlorofluorescin diacetate (DCFH-DA) assay.Results indicate that the cytosolic ROS level was

Chemical Hypoxia Enhances Cytosolic and Mitochondrial ROS
Hypoxic conditions promote increased intracellular oxidative stress, particularly in the form of ROS [32].To explore if CoCl 2 treatment induces oxidative stress, we assessed the cytosolic and mitochondrial ROS generation in CoCl 2 -exposed SH-SY5Y cells (Figure 2C).Cytosolic ROS production induced by CoCl 2 was determined by 2 ′ -7 ′ dichlorofluorescin diacetate (DCFH-DA) assay.Results indicate that the cytosolic ROS level was significantly increased in 100 or 200 µM CoCl 2 -treated SH-SY5Y cells.As ROS generated by mito-chondrial Complex III under hypoxia stabilizes the HIF-1α protein [33], mitochondrial superoxide anion (O 2 − ) levels were determined using MitoSOX.In agreement with changes in intracellular ROS levels, treatment with 200 µM CoCl 2 markedly increased mitochondrial ROS levels compared to normoxic controls (Figure 2D).These results indicate that CoCl 2 -induced hypoxia increased cytosolic and mitochondrial superoxide generation in SH-SY5Y cells.

Chemical Hypoxia Triggers Intrinsic Apoptotic Pathways in SH-SY5Y Cells
Oxidative stress plays a crucial role in initiating apoptosis [34].To evaluate the effect of CoCl 2 on the B cell lymphoma-2 (Bcl-2) family in SH-SY5Y cells, Western blotting was performed to detect the expression of Bax and Bcl-2 proteins and the Bax/Bcl-2 ratio.The results revealed that treatment with CoCl 2 significantly increased the pro-apoptotic Bax protein level in 100 or 200 µM CoCl 2 -treated SH-SY5Y cells, while levels of antiapoptotic Bcl-2 protein were markedly decreased at 200 µM CoCl 2 compared to levels in normoxic cells (Figure 3A).The Bax/Bcl-2 ratio was significantly increased following treatment with 100 or 200 µM CoCl 2 (p < 0.05), and this correlated with intracellular ROS generation (Figure 3B).levels compared to normoxic controls (Figure 2D).These results indicate that CoCl2-induced hypoxia increased cytosolic and mitochondrial superoxide generation in SH-SY5Y cells.

Chemical Hypoxia Triggers Intrinsic Apoptotic Pathways in SH-SY5Y Cells
Oxidative stress plays a crucial role in initiating apoptosis [34].To evaluate the effect of CoCl2 on the B cell lymphoma-2 (Bcl-2) family in SH-SY5Y cells, Western blotting was performed to detect the expression of Bax and Bcl-2 proteins and the Bax/Bcl-2 ratio.The results revealed that treatment with CoCl2 significantly increased the pro-apoptotic Bax protein level in 100 or 200 µM CoCl2-treated SH-SY5Y cells, while levels of antiapoptotic Bcl-2 protein were markedly decreased at 200 µM CoCl2 compared to levels in normoxic cells (Figure 3A).The Bax/Bcl-2 ratio was significantly increased following treatment with 100 or 200 µM CoCl2 (p < 0.05), and this correlated with intracellular ROS generation (Figure 3B).

Chemical Hypoxia Activates Caspase-3/GSDME-Mediated Pyroptosis
Recent studies have identified that caspase-3 plays a key executor protein in both apoptotic and pyroptotic pathways [37].Activated caspase-3 cleaves gasdermin E (GSDME) to generate a N-terminal fragment of GSDME (GSDME-NT) that induces pyroptosis by forming membrane pores [38,39].To investigate the effect of CoCl 2 -induced hypoxia on the GSDME-mediated pyroptosis in SH-SY5Y cells, we assessed GSDME protein expression by Western blotting (Figure 4A).The results revealed that the level of GSDME-NT was significantly increased when SH-SY5Y cells were treated with 200 µM of CoCl 2 , and this was consistent with the increased levels of cleaved caspase-3 (Figure 4B,C).These results suggested that CoCl 2 induced GSDME-dependent pyroptosis through caspase-3 activation.

Chemical Hypoxia Activates Caspase-3/GSDME-Mediated Pyroptosis
Recent studies have identified that caspase-3 plays a key executor protein in both apoptotic and pyroptotic pathways [37].Activated caspase-3 cleaves gasdermin E (GSDME) to generate a N-terminal fragment of GSDME (GSDME-NT) that induces pyroptosis by forming membrane pores [38,39].To investigate the effect of CoCl2-induced hypoxia on the GSDMEmediated pyroptosis in SH-SY5Y cells, we assessed GSDME protein expression by Western blotting (Figure 4A).The results revealed that the level of GSDME-NT was significantly increased when SH-SY5Y cells were treated with 200 µM of CoCl2, and this was consistent with the increased levels of cleaved caspase-3 (Figure 4B,C).These results suggested that CoCl2 induced GSDME-dependent pyroptosis through caspase-3 activation.

ROS Activation Contributed to Hypoxia-Mediated Pyroptosis
Recent studies indicated that ROS play important roles in the progression of caspasemediated pyroptosis [41,42].The present study demonstrated that CoCl 2 treatment elevates intracellular ROS levels.To further verify the role of ROS in hypoxia-mediated pyroptosis, we investigated if the ROS scavenger N-acetyl-L-cysteine attenuated the upregulation of pyroptosis-related proteins in SH-SY5Y cells.As presented in Figure 7A, N-acetyl-Lcysteine pretreatment significantly suppressed the expression of cleaved caspase-3 and the cleavage of GSDME in CoCl 2 -treated SH-SY5Y cells.However, there was no significant difference in the expression of cleaved caspase-3 after N-acetyl-L-cysteine treatment in the CoCl 2 -treated cells.Furthermore, N-acetyl-L-cysteine reduced the expression of cleaved caspase-1 protein and the formation of GSDMD-NT compared to levels in CoCl 2 -treated SH-SY5Y cells (Figure 7B).These findings suggest that ROS contributes to the activation of pyroptosis-associated proteins under hypoxic conditions in SH-SY5Y cells.

ROS Activation Contributed to Hypoxia-Mediated Pyroptosis
Recent studies indicated that ROS play important roles in the progression of caspasemediated pyroptosis [41,42].The present study demonstrated that CoCl2 treatment elevates intracellular ROS levels.To further verify the role of ROS in hypoxia-mediated pyroptosis, we investigated if the ROS scavenger N-acetyl-L-cysteine attenuated the upregulation of pyroptosis-related proteins in SH-SY5Y cells.As presented in Figure 7A, N-acetyl-L-cysteine pretreatment significantly suppressed the expression of cleaved caspase-3 and the cleavage of GSDME in CoCl2-treated SH-SY5Y cells.However, there was no significant difference in the expression of cleaved caspase-3 after N-acetyl-L-cysteine treatment in the CoCl2treated cells.Furthermore, N-acetyl-L-cysteine reduced the expression of cleaved caspase-1 protein and the formation of GSDMD-NT compared to levels in CoCl2-treated SH-SY5Y cells (Figure 7B).These findings suggest that ROS contributes to the activation of pyroptosis-associated proteins under hypoxic conditions in SH-SY5Y cells.

Discussion
In the present study, we assessed the effects and mechanisms of hypoxia-mediated pyroptosis in SH-SY5Y cells.We observed that CoCl2-induced hypoxia effectively induced ROS accumulation and activation of two cell death pathways (apoptosis and pyroptosis) in SH-SY5Y cells.We also demonstrated that pre-treatment with caspase inhibitors reduced the cleavage of GSDMD and GSDME at the protein level.Additionally, the results from the ROS scavenging assay demonstrated that hypoxia-induced ROS play a key role as executors of pyroptosis.
HIF-1 regulates the gene expression involved in cell survival, angiogenesis, and glucose metabolism in response to cellular oxygen conditions [43].HIF-1α is unstable and rapidly degraded by the ubiquitin-dependent proteasome system under normoxic conditions [44].How-

Discussion
In the present study, we assessed the effects and mechanisms of hypoxia-mediated pyroptosis in SH-SY5Y cells.We observed that CoCl 2 -induced hypoxia effectively induced ROS accumulation and activation of two cell death pathways (apoptosis and pyroptosis) in SH-SY5Y cells.We also demonstrated that pre-treatment with caspase inhibitors reduced the cleavage of GSDMD and GSDME at the protein level.Additionally, the results from the ROS scavenging assay demonstrated that hypoxia-induced ROS play a key role as executors of pyroptosis.
HIF-1 regulates the gene expression involved in cell survival, angiogenesis, and glucose metabolism in response to cellular oxygen conditions [43].HIF-1α is unstable and rapidly degraded by the ubiquitin-dependent proteasome system under normoxic conditions [44].However, under hypoxic conditions HIF-1α is stabilized against degradation after dimerization with HIF-1β [19].CoCl 2 artificially induces hypoxia-like conditions by blocking HIF-1α degradation under normoxic conditions [45].Our results revealed that CoCl 2 significantly increased cell death as well as the protein expression of HIF-1α in SH-SY5Y neuronal cells.We also confirmed an increase in cytosolic and mitochondrial ROS generation induced by CoCl 2 in SH-SY5Y cells.In general, ROS are considered major mediators of apoptotic signaling pathways [46].Apoptosis is executed by caspases, and their activation is mediated by Bcl-2 family proteins, including Bax and Bak [47].Upon apoptotic stimuli, Bax and Bak are activated and trigger mitochondrial outer membrane permeabilization (MOMP) that leads to the release of proapoptotic proteins, including cytochrome c, from the mitochondrial intermembrane space into the cytoplasm.Cytochrome c interacts with apoptotic protease-activating factor-1 (Apaf-1) to form the Apaf-1 apoptosome that subsequently activates the initiator caspase-9 [48].Active caspase-9 then cleaves and activates effector caspase-3 and caspase-7.Consistent with these reports, we observed increased levels of Bax protein and cleaved forms of caspase-9, -3, and -7 after stimulation with CoCl 2 in SH-SY5Y cells.
Hypoxia-induced ROS activate the NLRP3 inflammasome.Previous studies have demonstrated that NLRP3 inflammasome activation contributes to various types of cell death, including apoptosis, necroptosis, ferroptosis, and pyroptosis [52].In the canonical inflammasome pathway, the NLRP3 inflammasome recruits and activates the caspase-1 that cleaves the cytosolic GSDMD to form a lytic membrane pore and induces secretion of mature IL-1β and IL-18 [29].In line with these findings, our results demonstrated that CoCl 2 not only increased the levels of NLRP3, cleaved caspase-1, and cleaved GSDME, but also increased the expression of IL-1β and IL-18 (Figure 5A,B).We also observed that caspase-1 inhibitor pretreatment improved cell proliferation and attenuated the levels of cleaved GSDME protein compared to those in CoCl 2 -treated neuronal cells.
ROS are crucial for tissue homeostasis, cell survival, and cell signaling.However, excessive ROS levels damage cellular components, including membranes, nucleic acids, and organelles [53].Recent studies have demonstrated that ROS are important mediators of various nervous system diseases.ROS can damage neurons via mitochondrial DNA mutations, MOMP alterations, and homeostasis disruption, ultimately leading to neuronal degeneration and apoptotic cell death [54].Few studies have associated hypoxia-induced ROS production with pyroptosis in neuronal cells under hypoxic conditions.In the current study, treatment with ROS scavengers decreased the levels of cleaved caspase-3 and GSDME activation and inhibited the expression of cleaved caspase-1 and cleaved GSDMD in CoCl 2treated SH-SY5Y neuronal cells.These results demonstrate that hypoxia can induce both apoptosis and pyroptosis or the apoptosis-to-pyroptosis switch through ROS accumulation in SH-SY5Y cells.The limitation of the present study is that the experiments were conducted using only one cell line.Thus, additional comparative studies are needed using normal brain cells, including primary neurons, microglia, and astrocytes.
In conclusion, we have demonstrated that CoCl 2 -induced hypoxia significantly increased cell death and HIF-1α expression in SH-SY5Y cells, led to the accumulation of cytosolic and mitochondrial ROS, and activated ROS-mediated pyroptosis under hypoxic conditions (Figure 8).These effects were inhibited by caspase-specific inhibitors and attenuated by ROS scavengers in CoCl 2 -treated SH-SY5Y cells.Therefore, we propose that a reduction in ROS levels may be the underlying mechanism of the protective or therapeutic effects of pyroptosis-induced neuronal cell damage during hypoxia.
GSDME activation and inhibited the expression of cleaved caspase-1 and cleaved GSDMD in CoCl2-treated SH-SY5Y neuronal cells.These results demonstrate that hypoxia can induce both apoptosis and pyroptosis or the apoptosis-to-pyroptosis switch through ROS accumulation in SH-SY5Y cells.The limitation of the present study is that the experiments were conducted using only one cell line.Thus, additional comparative studies are needed using normal brain cells, including primary neurons, microglia, and astrocytes.
In conclusion, we have demonstrated that CoCl2-induced hypoxia significantly increased cell death and HIF-1α expression in SH-SY5Y cells, led to the accumulation of cytosolic and mitochondrial ROS, and activated ROS-mediated pyroptosis under hypoxic conditions (Figure 8).These effects were inhibited by caspase-specific inhibitors and attenuated by ROS scavengers in CoCl2-treated SH-SY5Y cells.Therefore, we propose that a reduction in ROS levels may be the underlying mechanism of the protective or therapeutic effects of pyroptosisinduced neuronal cell damage during hypoxia.

Cell Viability Assay
Cell viability was determined using the cell counting kit-8 (CCK-8, DoJinDo Molecular Technology Inc., Kumamoto, Japan) according to the manufacturer's protocol.Cells were seeded into 96-well plates at 1 × 10 4 cells per well in 100 µL of standard culture medium and incubated overnight.After the cells were treated with CoCl 2 at the indicated concentrations, 10 µL of CCK-8 reagent was added to each well and incubated in a tissue culture incubator at 37 • C for an additional hour to enable the reaction of WST-8.The absorbance was measured at 450 nm using a SpectraMax ABS Plus plate reader (Molecular Devices, San Jose, CA, USA).All experiments were independently repeated three times.The viability of CoCl 2 -treated cells is presented as a percentage of that of untreated cells in parallel experiments.

Cell Morphology
SH-SY5Y cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well in standard culture medium.Cells were treated with CoCl 2 (0, 50, 100, or 200 µM) for 24 h at 37 • C under a humidified atmosphere of 5% CO 2 and 95% air.Cell growth, growth conditions, and morphological changes were observed using a phase-contrast microscope (CKX31; Olympus, Tokyo, Japan).

ROS Production Assay
Detection of intracellular and mitochondrial ROS in CoCl 2 -treated SH-SY5Y cells was performed using confocal fluorescence microscopy and flow cytometry.Intracellular ROS was detected using the oxidation-sensitive fluorescent probe DCFH-DA (Cat# ab113851; Abcam).For confocal fluorescence microscopy, SH-SY5Y cells were seeded on glass coverslips and incubated for 24 h and then treated with CoCl 2 (0, 50, 100, or 200 µM) for 24 h at 37 • C in 5% CO 2 .To determine the amount of intercellular ROS, the culture medium was discarded, and the cells were rinsed with Dulbecco's phosphate-buffered saline (DPBS) and incubated with 10 µM DCFH-DA in Hank's balanced salt solution (HBSS) at 37 • C for 30 min in darkness.After washing with HBSS three times, cells were fixed in 4% paraformaldehyde (PFA) for 5 min.Cell nuclei were counterstained with 4 ′ ,6-diamidino-2-phenylindole (DAPI) and stained cells were observed under a Zeiss LSM-800 confocal microscope (Zeiss, Oberkochen, Germany).Imaging analysis was performed using ZEN 2010 image software (version 3.3, Zeiss).For flow cytometry analysis, SH-SY5Y cells were seeded in 6-well plates and treated with CoCl 2 (0, 50, 100, or 200 µM) for 24 h at 37 • C in 5% CO 2 .After washing with HBSS, cells were stained at 37 • C for 20−30 min with 10 µM DCFH-DA.The cells were washed again with DPBS and detached using a 0.25% trypsin/0.53mM EDTA solution.The cells were then transferred in the FACS tube using growth medium and centrifuged at 200× g at 4 • C for 10 min.Samples were resuspended using DPBS and examined by using an FACSAria™ III (Becton Dickinson, Franklin Lakes, NJ, USA), and data were analyzed using FACSDiva (version 6.0, Becton Dickinson).
MitoSOX™ Red superoxide indicator (Cat# M36008; Invitrogen, Waltham, MA, USA) was used to assess mitochondrial ROS levels in CoCl 2 -treated SH-SY5Y cells.The incubation procedure was identical to that used for the DCFH-DA probe.After CoCl 2 treatment, SH-SY5Y cells were incubated with 5 µM MitoSOX™ Red reagent in HBSS buffer for 10 min at 37 • C in darkness and were then washed twice with PBS and fixed with 4% paraformaldehyde for 15 min.The cell nuclei were labeled with DAPI (1 mg/mL) for 4 min.Fluorescence images were captured using a Zeiss LSM-800 confocal microscope (Zeiss).The fluorescence intensity was measured using a flow cytometer as described above.

Western Blot Analysis
SH-SY5Y cells were harvested and lysed with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) on ice to obtain protein extracts.Lysates were centrifuged at 4 • C for 20 min at 14,000× g.The Pierce™ BCA Protein Assay Kit (Cat# 23225, Thermo Fisher Scientific) was used to measure the protein concentration.A total of 20 µg of denatured

Figure 1 .
Figure 1.Effect of chemical hypoxia on SH-SY5Y cell proliferation and cellular morphology.(A) SH-SY5Y cells were treated with 0−1000 µM CoCl2 as indicated for 24 h and detected using a CCK-8 assay.Data are expressed as the percentage of cell viability versus unexposed control cells.Data represent mean ± standard deviation from at least three independent experiments.Statistical analysis was performed by Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells.(B) Representative bright-field images of SH-SY5Y cells treated with CoCl2 (50, 100, or 200 µM) or vehicle for 24 h.Red arrowheads indicate the damaged SH-SY5Y cells.Images were obtained using a routine inverted light microscope at ×400 magnification.

Figure 1 .
Figure 1.Effect of chemical hypoxia on SH-SY5Y cell proliferation and cellular morphology.(A) SH-SY5Y cells were treated with 0−1000 µM CoCl 2 as indicated for 24 h and detected using a CCK-8 assay.Data are expressed as the percentage of cell viability versus unexposed control cells.Data represent mean ± standard deviation from at least three independent experiments.Statistical analysis was performed by Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells.(B) Representative bright-field images of SH-SY5Y cells treated with CoCl 2 (50, 100, or 200 µM) or vehicle for 24 h.Red arrowheads indicate the damaged SH-SY5Y cells.Images were obtained using a routine inverted light microscope at ×400 magnification.

Figure 2 .
Figure 2. Effect of chemical hypoxia on HIF-1α expression and ROS generation on SH-SY5Y.(A) Protein expression levels of HIF-1α were detected by Western blot in SH-SY5Y cells in induced hypoxia with CoCl2 (0, 50, 100, or 200 µM) for 24 h.α-tubulin levels are presented as a loading control.(B) Relative levels of HIF-1α protein expression following treatment with CoCl2.Data are presented as the mean ± standard deviation of three independent experiments, and results normalized to untreated control cells.*** p < 0.001 vs. the unexposed control cells according to Student's t-tests.(C) Flow cytometric analysis of cytosolic ROS generation using DCFH-DA and mitochondrial superoxide radicals using MitoSOX in SH-SY5Y cells treated with CoCl2 (0, 50, 100, or 200 µM) for 24 h.(D) Representative fluorescent images of DCFH-DA (green) staining and MitoSOX (red) staining in SH-SY5Y cells treated with 200 µM CoCl2 were obtained using confocal microscopy.Non-treated SH-SY5Y cells were used as negative controls.×200 magnification.

Figure 2 .
Figure 2. Effect of chemical hypoxia on HIF-1α expression and ROS generation on SH-SY5Y.(A) Protein expression levels of HIF-1α were detected by Western blot in SH-SY5Y cells in induced hypoxia with CoCl 2 (0, 50, 100, or 200 µM) for 24 h.α-tubulin levels are presented as a loading control.(B) Relative levels of HIF-1α protein expression following treatment with CoCl 2 .Data are presented as the mean ± standard deviation of three independent experiments, and results normalized to untreated control cells.*** p < 0.001 vs. the unexposed control cells according to Student's t-tests.(C) Flow cytometric analysis of cytosolic ROS generation using DCFH-DA and mitochondrial superoxide radicals using MitoSOX in SH-SY5Y cells treated with CoCl 2 (0, 50, 100, or 200 µM) for 24 h.(D) Representative fluorescent images of DCFH-DA (green) staining and MitoSOX (red) staining in SH-SY5Y cells treated with 200 µM CoCl 2 were obtained using confocal microscopy.Non-treated SH-SY5Y cells were used as negative controls.×200 magnification.

Figure 3 .
Figure 3.Effect of chemical hypoxia on apoptosis related proteins in CoCl2-treated SH-SY5Y cells.Cells were treated with the indicated concentrations (0, 50, 100, or 200 µM) of CoCl2 for 24 h.(A) Western blotting was used to observe Bax, Bcl-2, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-9, cleaved caspase-9, PARP, and cleaved PARP protein expression in SH-SY5Y cells.(B) The relative gray values of Bax and Bcl-2 were quantified.The Bax/Bcl-2 ratio was calculated, and α-tubulin was used as the internal protein loading control.(C-F) The relative intensities of the proteins were calculated by comparing them to the intensity of α-tubulin.Data are presented as the mean ± standard deviation of three independent experiments and the results are normalized to untreated control cells.Statistical analysis was performed by Student's t-test, * p < 0.05, *** p < 0.001 vs. untreated control cells.

Figure 3 .
Figure 3.Effect of chemical hypoxia on apoptosis related proteins in CoCl 2 -treated SH-SY5Y cells.Cells were treated with the indicated concentrations (0, 50, 100, or 200 µM) of CoCl 2 for 24 h.(A) Western blotting was used to observe Bax, Bcl-2, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7, caspase-9, cleaved caspase-9, PARP, and cleaved PARP protein expression in SH-SY5Y cells.(B) The relative gray values of Bax and Bcl-2 were quantified.The Bax/Bcl-2 ratio was calculated, and α-tubulin was used as the internal protein loading control.(C-F) The relative intensities of the proteins were calculated by comparing them to the intensity of α-tubulin.Data are presented as the mean ± standard deviation of three independent experiments and the results are normalized to untreated control cells.Statistical analysis was performed by Student's t-test, * p < 0.05, *** p < 0.001 vs. untreated control cells.

Figure 4 .
Figure 4.Chemical hypoxia triggers GSDME-mediated pyroptosis in SH-SY5Y cells.(A) Protein expression levels of caspase-3, cleaved caspase-3, GSDME, and GSDME-NT were detected using Western blotting analysis in SH-SY5Y cells treated with CoCl2 at different concentrations (0, 50, 100, or 200 µM) for 24 h, and the relative protein expressions of (B) cleaved caspase-3 and (C) GSDME-NT were quantified.α-tubulin was used as the internal protein loading control.Data are presented as the mean ± standard deviation of three independent experiments, and the results are normalized to untreated control cells.** p < 0.01, *** p < 0.001 vs. untreated control cells using Student's t-tests.

Figure 4 .
Figure 4.Chemical hypoxia triggers GSDME-mediated pyroptosis in SH-SY5Y cells.(A) Protein expression levels of caspase-3, cleaved caspase-3, GSDME, and GSDME-NT were detected using Western blotting analysis in SH-SY5Y cells treated with CoCl 2 at different concentrations (0, 50, 100, or 200 µM) for 24 h, and the relative protein expressions of (B) cleaved caspase-3 and (C) GSDME-NT were quantified.α-tubulin was used as the internal protein loading control.Data are presented as the mean ± standard deviation of three independent experiments, and the results are normalized to untreated control cells.** p < 0.01, *** p < 0.001 vs. untreated control cells using Student's t-tests.

Figure 5 .
Figure 5.Chemical hypoxia contributes to GSDMD-mediated pyroptosis in SH-SY5Y cells.(A) Western blotting was used to observe NLPR3, caspase-1, cleaved caspase-1, GSDMD, GSDME-NT, IL-1β and IL-18 protein expression in SH-SY5Y cells treated with various concentrations of CoCl2 (0, 50, 100, or 200 µM) for 24 h.(B-F) Relative gray values of each group were quantified by densitometry.α-tubulin was used as the internal protein loading control.Data are presented as the mean ± standard deviation of three independent experiments, and the results are normalized to untreated control cells.Statistical analysis was performed by Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells.

Figure 5 .
Figure 5.Chemical hypoxia contributes to GSDMD-mediated pyroptosis in SH-SY5Y cells.(A) Western blotting was used to observe NLPR3, caspase-1, cleaved caspase-1, GSDMD, GSDME-NT, IL-1β and IL-18 protein expression in SH-SY5Y cells treated with various concentrations of CoCl 2 (0, 50, 100, or 200 µM) for 24 h.(B-F) Relative gray values of each group were quantified by densitometry.α-tubulin was used as the internal protein loading control.Data are presented as the mean ± standard deviation of three independent experiments, and the results are normalized to untreated control cells.Statistical analysis was performed by Student's t-test, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells.