Recombinant rat LIF (NBP2-35263) was purchased from Novus Biologicals (Littleton, CO, USA) and dissolved in sterile distilled water to a concentration of 0.1 mg/mL. Dulbecco’s modified eagle medium (DMEM)/Low Glucose (11885084) was purchased from Gibco (Carlsbad, CA, USA). Chicken polyclonal glial fibrillary acidic protein (GFAP, ab4674), rabbit polyclonal GFAP (GFAP, ab7260), anti-associated speck-like protein (ASC, ab180799), and anti-gasdermin D (GSDMD, ab219800) antibodies were acquired from Abcam (Cambridge, UK). HO-1 antibody (#SPA895) was purchased from Stressgen (London, Ontario, Canada). Nrf2 (PA5-27882), NLRP3 (PA5-115661), Caspase 1 (MA5-16215), IL-1 beta (PA5-46956), IL-18 (PA5-76082) antibodies, and MitoSOX^TM red mitochondrial superoxide indicator (MP36008) were purchased from Invitrogen^TM (Carlsbad, CA, USA). Various antibodies, including rabbit anti-Phospho-Akt (#4060), anti-Akt (#4691), anti-Phospho-p44/42 MAPK (ERK1/2) (#4370), anti-p44/42 MAPK (ERK1/2) (#9102), anti-Phospho-SAPK/JNK (#4668), rabbit anti-SAPK/JNK (#9252), rabbit anti-Phospho-p38 MAPK (#4511), and rabbit anti-p38 MAPK (#9092), along with SB203580 (#5633), SP600125 (#8177), PD98059 (#9900), and LY294002 (#9901) were bought from Cell Signaling Technology (Boston, MA, USA). ZnPP was purchased from MedChen Express (Madison, WI, USA). Abcam (London, Cambridge, UK) provided Goat Anti-Chicken IgY H&L (Alexa Fluor® 488, ab150169), Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 594, ab150076), and Donkey Antibody to Goat IgG - H&L (Alexa Fluor® 594, ab150136). Proteintech (Wuhan, Hubei, China) provided the Beta Actin Mouse Monoclonal antibody (66009-1-ig), horseradish peroxidase (HRP)-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (SA00001-1), HRP-conjugated Affinipure Anti-Rabbit IgG (H+L) (SA00001-2), and Hoechst (No.33342). Protease inhibitors were purchased from Roche Diagnostics GmbH (Basel, Switzerland). The Cell Counting Kit-8 (CCK-8) (No.96992), TdT-mediated dUTP nick end labeling Kit (11684795910), Hoechst 33342/PI Double Staining Kit (orb219888), and TRizol (B511311) reagents were acquired from Sigma (St. Louis, MO, USA), Roche (Basel, Switzerland), Biorbyt (Cambridge, UK), and Shenggong (Shanghai, China), respectively. The Reactive Oxygen Species Assay Kit (S0033S) and Nuclear and Cytoplasmic Protein Extraction Kit (P0027) were bought from Beyotime (Haimen, China). The PrimeScript RT Reagent Kit and SYBR Premix Ex Taq^TM were purchased from TaKaRa (Tokyo, Japan). The remaining chemicals were of the best quality that could be purchased from stores.

In vivo internationally recognized animal models of PWMI. Healthy Sprague Dawley (SD) rat pups at post-natal day 3 (PND3) were randomly assigned to either the control (Ctrl) or PWMI group, obtained from the Center of Experimental Animals at Shengjing Hospital of China Medical University. The animals were kept in a room with controlled temperature (22–25 °C) and a 12-hour light/dark cycle and were given food and water ad libitum. All animal procedures were approved by the Experimental Animal Ethics Committee of Shengjing Hospital of China Medical University (2021PS075K), and adhered to Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All PWMI categories were created as previously outlined [27, 28]. Briefly, PND3 pups were placed in a refrigerator at –20 °C for 7–10 min. After administering cold anesthesia, the surgeon meticulously separated the right common carotid artery from the nearby tissue and tied it off, after which the injury was stitched with an 8-0 suture. The procedure was completed within 5 min. After surgery, the puppies were transferred to a warm area with a heat lamp for 10 min, reunited with their mother, and allowed to recuperate for 1 h. They were then exposed to 6% oxygen (94% nitrogen saturation) at 37 °C for 90 min in a humidified chamber. Following recovery, they were placed back in their cages to resume eating. The control group comprised rats that underwent sham surgery without hypoxic exposure. During PND6 (HI3d), damage severity (ranging from mild to severe) was assessed based on the overall animal health, neurobehavioral assessments, and histopathological examinations. Rats with moderate-to-severe injuries were used in future experiments.

The mice were euthanized by Inhalant Anesthesia Overdose (5% isoflurane mixed with oxygen). Brain tissue was extracted from rats sacrificed every 3 days following PWMI induction and immersed in 4% paraformaldehyde for 24 h. Subsequently, the brains underwent several alcohol and xylene treatments before being encased in paraffin blocks. Brain samples were sliced with a rotary microtome (Leica RM 2135, Leica Instruments, Nussloch, Germany) to produce consecutive 4 µm thick sections in a coronal orientation from the paraffin-embedded tissue block for GFAP/NLRP3 dual immunofluorescence labeling or ASC/Caspase-1 immunohistochemical analysis. The posterior two-thirds of the brain tissue was used for western-blot analysis.

CTX TNA2 rat astrocytes were acquired from ACTT (Rockefeller, MD, USA), and all cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma. The cell was grown in low glucose DMEM with 10% fetal bovine serum, 50 U/mL penicillin, 50 µg/mL streptomycin, and 1% L-glutamine, after which the culture medium was discarded. Briefly, the cell layer was rinsed with phosphate-buffered saline (PBS) to remove all traces of serum containing the trypsin inhibitor. Subsequently, 0.25% Trypsin-0.53 millimolar EDTA solution (1 mL) was transferred into the flask, and the cells were monitored using an inverted microscope (TS2, Nikon Corporation, Tokyo, Japan) until the cell layer was evenly spread out, typically within 3 to 5 min. Full growth solution (6–8 mL) was added, and the cells were carefully removed using a pipette. Appropriate portions of the cell mixture were then transferred into fresh culture containers, which were maintained at 37 °C.

Cells were stained with Hoechst 33342/PI using the Double Staining Kit per manufacturer instructions. Hoechst 33342/PI Chromogen (10 µL) were equally added to 2 mL Dilution Buffer and mixed (final density, 5 µg/mL). Subsequently, the staining solution was applied to the cells and incubated in the dark for 5 min at 30 °C. The cells were then rinsed three times with PBS and observed under a fluorescence microscope (M40D, Wanheng Precision Instrument Co. LTD., Shanghai, China).

Following the standard removal of wax and water from the brain tissue sections, they were treated with 10% serum for 20 min at 37 °C, and incubated overnight at 4 °C with Chicken polyclonal GFAP and Rabbit polyclonal NLRP3 antibodies (both 1:500). Tissue slices were rinsed three times with PBS-TritonX-100. Alexa Flour@488 (green fluorescence) and Alexa Flour@594 (red fluorescence) secondary antibodies were used at a 1:200 dilution for 2 h at 37 °C. Rat astrocytes were seeded into 6-well plates, fixed in 4% paraformaldehyde for 15 min, and washed thrice with PBS (5 min each). The cells were then treated with 0.1% TritonX-100, left at room temperature for 30 min, subjected to three 5-minute PBS washes, blocked with 10% goat serum for 15 minutes at 37 °C, and incubated overnight at 4 °C with either HO-1 or Nrf2 antibody (both 1:200). After three PBS washes, cells were incubated with Alexa Flour@594 secondary antibodies (1:200) 1 h at 37 °C. Next, the tissue sections and cells were rinsed thrice with PBS, stained with DAPI (1:2000) for 2 min, rinsed again, and imaged using a confocal laser scanning microscope (LSM880, Zeiss, Germany).

Following deparaffinization and rehydration, brain samples were exposed to 50 µL of 0.1% TritonX-100 (diluted with 0.1% sodium citrate) and preserved at room temperature for 10 min, rinsed thrice with PBS (5 min each), treated with 3% H₂O₂ at room temperature for 10 min to inhibit peroxidase activity, and washed three more times with PBS for (5 min each). Subsequently, tissues were blocked using 10% goat serum diluted for 20 min at 37 °C, followed by overnight incubation at 4 °C with either Anti-ASC or Caspase 1 antibody (both 1:500). After cleaning, sections were incubated with biotinylated secondary antibodies (diluted to 1:200) for 1 h at 37 °C, followed by treatment with avidin-biotin-peroxidase (1:200) for 45 min at 37 °C. Staining was performed using 0.05% 3,3’-diaminobenzidine with 0.03% H₂O₂, followed by counterstaining with Mayer’s hematoxylin. Experiments were conducted without primary antibodies, showing no positive staining. Sections were finally analyzed using a light microscope (E100, Nikon Corporation, Tokyo, Japan).

An in vitro OGD/R model was established as previously described [25, 29]. After a brief wash with glucose-free culture medium, primary astrocyte cultures were replenished with fresh glucose-free medium without serum. To induce OGD, cells were incubated in a humidified Tri-gas chamber (Thermo Scientific, Waltham, MA, USA) under hypoxic conditions for 24 h, with a gas mixture of 94% N₂, 5% CO₂, and 1% O₂. After 24 h, OGD exposure was stopped by changing the medium to serum-free 80% DMEM (containing 7.5 mM glucose) and adding LIF stock solution to the culture medium at concentrations of 1, 3, 10, 30, and 60 ng/mL. The cells were then incubated at 37 °C with 5% CO₂ for another 24 hours under normal oxygen conditions. Control cells, which were included in all experiments, underwent the same treatment except for OGD exposure. For inhibitor experiments, cells were pretreated with/without OGD/R and then treated with various concentrations of PD98059, SP600125, SB203580, and LY294002 for 1 h, followed by treatment with LIF (60 ng/mL) for another hour. Cell lysates were immunoblotted and pretreated with the indicated inhibitors for 2 h, followed by treatment with LIF (60 ng/mL) for another 24 h, after which cell lysates were immunoblotted to assess HO-1 expression.

RNA was isolated from Ctrl and OGD/R groups of CTX TNA2 cells using the Ribo-Zero™ rRNA Removal Kit (Epicenter, New Zealand), with three parallel replicates per group. Next, six samples (three per group) were shipped to Novogene Corporation (Beijing, China) for RNA-seq analysis using the Illumina Nova-Seq 6000 platform. Differentially expressed genes (DEGs) between the two groups were identified using DEG-seq and analyzed using the KEGG database for pathway analysis. The Novo-gene network platform was used to analyze the data (https://www.novogene.com).

A diacetyl dichloro fluorescein (DCFH-DA) probe was used to detect intercellular ROS. In summary, cells were seeded in 6-well dishes and treated with OGD/R and/or LIF. After removing the medium, 1 mL of medium containing the DCFH-DA (diluted 1:1000 in serum-free medium) was added to each well. Following a 40-minute incubation period, the medium was removed, and the cells were rinsed thrice with PBS. A fluorescence microscope was used to observe the cells, and absorbance was measured using a fluorescence spectrophotometer (NanoVne, GE Healthcare, Chicago, IL, USA).

Cells were loaded with 5 µM MitoSOX™Red (Thermo Scientific, MP36008) in Hank’s Balanced Salt Solution (HBSS) for 10 min at 37 °C, washed twice with PBS, and imaged using a confocal microscope (LSM880, Zeiss, Oberkochen, Germany) (excitation/emission: 510/580 nm). Fluorescence intensity was quantified using ImageJ v1.53 (National Institute of MentalHealth, Bethesda, MD, USA).

CTX TNA2 rat astrocytes were stained using the in situ cell death detection kit per manufacturer instructions, as previously described [25].

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Sigma, #96992). Cells were seeded in 96-well plates (5 $\times$ 10³ cells/well) and subjected to experimental treatments. After interventions, 10 µL CCK-8 reagent was added to each well, followed by incubation at 37 °C for 2 h. Absorbance was measured at 450 nm using a SpectraMax M5 microplate reader (Molecular Devices). Data were normalized to the control group (untreated cells = 100% viability). Triplicate technical replicates and five biological replicates were performed for each condition.

Cellular RNA was extracted using the TRizol reagent, and reverse transcription was conducted as previously described. Real-time PCR was conducted using a multicolor detection system (Light Cycler 480Ⅱ, Roche, Basel, Switzerland), followed by melting curve analysis to examine the melting temperature of the product. The RNA underwent reverse transcription to produce cDNA utilizing the Prime Script RT Reagent Kit under reaction conditions of 37 °C for 15 min followed by 85 °C for 5 s. The resulting cDNA was diluted in water by a factor of 10 and stored at –20 °C. Quantitative PCR (qPCR) was performed to measure LIFR and $\beta$-actin mRNA levels in a PCR setup using SYBR Premix Ex Taq^TM. Thermo cycling was conducted with the following parameters: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s, using $\beta$-actin as the internal reference gene. The specific primer sequences used were as follows:

NLRP3 F: 5^′-GCCTTGAAGAGGAGTGGATAG-3^′;

NLRP3 R: 5^′-TGGGTGTAGCGTCTGTTGAG-3^′;

ASC F: 5^′-CTCAGGGCACAGCCAGAACAG-3^′;

ASC R: 5^′-GCCATACAGAGCATCCAGCAA-3^′;

GSDMD F: 5^′-TGCGGGAGTGGTCAAGAA-3^′;

GSDMD R: 5^′-TGCTCAGGAGGCAGTAGGG-3^′;

caspase1 F: 5^′-TGGATTGCTGGATGAACTT-3^′;

caspase1 R: 5^′-GCTGATGGACCTGACTGA-3^′;

IL-1$\beta$ F: 5^′-TTCAAATCTCACAGCAGCAT-3^′;

IL-1$\beta$ R: 5^′-CACGGGCAAGACATAGGTAG-3^′;

IL-18 F: 5^′-GCAGTAATACGGAGCATAAA-3^′;

IL-18 R: 5^′-ATCCTTCACAGATAGGGTCA-3^′;

$\beta$-actin F: 5^′-GGAGATTACTGCCCTGGCTCCTAGC-3^′;

$\beta$-actin R: 5^′-GGCCGGACTCATCGTACTCCTGCTT-3^′.

Cytoplasmic and nuclear proteins were extracted and isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (WLA020a, WanleiBio, Shenyang, Liaoning, China), transferred to a microcentrifuge tube, and centrifuged at 500 $\times$g for 3 min to form a pellet. The supernatant was then discarded, leaving the cell pellet as dry as possible. Ice-cold Cytoplasmic Extraction Reagent I (CER I) was added to the cell pellets and thoroughly mixed. Following a 10-minute incubation of the tube containing fully suspended cells on ice, ice-cold CER II was added to the cells and left on ice for an additional minute. The tubes were centrifuged for 5 minutes at maximum speed in a microcentrifuge (–16,000 $\times$g). The cytoplasmic extract was promptly transferred to a fresh pre-chilled tube. The insoluble pellet fraction was mixed with an ice-cold Nuclear Extraction Reagent (NER), vortexed for 15 s, and placed on ice. Vortexing was repeated every 10 minutes for a total of 40 minutes. Following centrifugation of the tube at the highest speed (–16,000 $\times$g) in a microcentrifuge for 10 min, the supernatant was immediately transferred to a fresh, pre-chilled tube and stored at –80 °C until needed.

Rat astrocytes from the CTX TNA2 cell line were processed for western blotting following a previously outlined protocol [25]. After separation on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, proteins (30 µg) were transferred to a polyvinylidene difluoride membrane using electro-blotting. The proteins were examined using primary antibodies and then treated with HRP-conjugated secondary anti-rabbit IgG antibodies to prevent possible errors in loading and transfer between samples in various lanes. Bands for phospho-ERK, Akt, JNK, and p38 MAPK were removed and retested using antibodies against total ERK, Akt, JNK, and p38 MAPK, and the blots were developed using an electrochemiluminescence western blotting detection reagent.

Data are presented as the mean $\pm$ standard deviation (SD). Statistical analysis was performed via a one-way analysis of variance, followed by Student’s t-test using GraphPad Prism software (version 7.0; GraphPad Software, Inc., San Diego, CA, USA). Mean values were considered statistically significant at p 0.05.

We used an OGD/R model to mimic the pathological processes in CTX TNA2 rat astrocytes during PWMI. CTX TNA2 cells were treated with OGD for 0–48 h, followed by 24-h reoxygenation to construct an in vitro ischemia-reperfusion (I/R) model. OGD/R-induced cytotoxicity was first tested by the CCK-8 assay. CTX TNA2 cells exhibited substantial damage after 12–48 h of OGD, followed by 24-h reoxygenation. Cell death after OGD exposure for 12, 24, and 48 h increased in a time-dependent manner (Fig. 1A). Therefore, OGD24 h/R24 h was used for further studies, and the results of phase-contrast microscopy were consistent with those of the CCK-8 assay (Fig. 1B). Hoechst 33342/PI double staining was used to further evaluate the effects of OGD/R-induced cell injury in CTX TNA2 cells. As shown in Fig. 1C, most cells had normal nuclear morphology with uniform blue nuclei in the control group. After exposure to OGD/R, more cells with bright red nuclei were observed. To further validate the effect of OGD/R, TUNEL was used to measure the effect of OGD/R, showing that the OGD/R group had elevated apoptosis levels (Fig. 1D). We investigated intracellular ROS production by monitoring DCFH₂-DA conversion into DCF. Cell exposure to OGD/R significantly increased DCF fluorescence (Fig. 1E). Next, we assessed the ROS levels using MitoSOX staining. Consistent with our DCFH₂-DA staining results, ROS levels increased in CTX TNA2 cells exposed to OGD/R (Fig. 1F). Altogether, these data suggest that OGD/R induces oxidative stress-dependent cell injury in CTX TNA2 cells.

We conducted a genome-wide RNA-seq to examine how OGD/R affects CTX TNA2 cell viability and injury. Total RNAs were isolated from the TNA2 cells exposed to OGD/R and sequenced. A total of 5569 genes were differentially expressed upon OGD/R exposure (fold change $\ge$2, p 0.05), including 2274 upregulated and 3295 downregulated mRNAs (Fig. 1G,H). KEGG pathway enrichment analysis indicated that several biological pathways were enriched. The pathways most significantly correlated with OGD/R were involved in the PI3K/Akt, MAPK, and NOD-like receptor signaling pathways (Fig. 1I,J). The top gene upregulated by OGD/R was NLRP3, suggesting a role of OGD/R in NLRP3-mediated pyroptosis. Further validation confirmed that OGD/R significantly increased NLRP3-dependent pyroptosis-related gene expression at the mRNA level (Fig. 1K). Collectively, these findings indicate that OGD/R induces pyroptosis in CTX TNA2 rat astrocytes.

We further confirmed that OGD/R induced pyroptosis in CTX TNA2 rat astrocytes using western blotting and immunofluorescence. Consistent with our qRT-PCR results, western blot results showed OGD/R increased pyroptosis-related protein expression, including NLRP3, ASC, GSDMD, caspase-1, IL-1$\beta$, and IL-18 (Fig. 2A,B). Further immunofluorescence analysis revealed higher levels of pyroptosis, as indicated by increased caspase-1 (Fig. 2C), NLRP3 (Fig. 2D), ASC (Fig. 2E), and GSDMD (Fig. 2F) in CTX TNA2 rat astrocytes exposed to OGD/R. Altogether, these results suggest that OGD/R induces pyroptosis in CTX TNA2 rat astrocytes.

We performed further analyses using in vivo models of HI-induced PWMI in PND3 rats. We validated that HI induces pyroptosis in PWMI rat models, showing that pyroptosis-related protein levels, including NLRP3, GSDMD, ASC, caspase-1, IL-1$\beta$, and IL-18 were all significantly upregulated by HI (Fig. 3A,B). In addition, in line with our western blot findings, immunofluorescence analysis revealed NLRP3 upregulation in astrocytes (Fig. 3C), along with caspase-1 and ASC expression (Fig. 3D) by immunohistochemistry studies in PND3 rats, 3 days after PWMI.

Previous studies revealed that LIF has neuroprotective effects against PWMI [25, 26]. However, the mechanisms through which LIF mediates its therapeutic effects remain unclear. Nrf2/HO-1 is important for protecting neurons against OGD/R and HI-induced injury [29, 30, 31]. We first investigated the possibility that LIF activates the Nrf2/HO-1 pathway by treating CTX TNA2 rat astrocytes with various LIF concentrations for 24 h. As shown in Fig. 4A, LIF (1–60 ng/mL) induced HO-1 expression in a concentration-dependent manner. Treatment of CTX TNA2 rat astrocytes with LIF resulted in a time-dependent increase in HO-1 protein expression (Fig. 4A). Consistent with the western blot findings, confocal immunofluorescence analysis revealed that LIF induced HO-1 upregulation (Fig. 4B).

Nrf2 is an important transcription factor that regulates antioxidant response element (ARE)-driven HO-1 expression. We then determined whether LIF could activate Nrf2. CTX TNA2 rat astrocytes were treated with LIF (60 ng/mL), and Nrf2 protein levels in the cytosol and nucleus were determined by western blotting. As shown in Fig. 4C, LIF promotes Nrf2 accumulation in the nucleus compared to control cells. Confocal immunofluorescence analysis confirmed the western blotting results, as indicated by the increased nuclear localization of Nrf2 (Fig. 4D). Collectively, these results suggest that LIF activates the Nrf2/HO-1 pathway.

Many signal transduction pathways, such as the MAPK and PI3K/AKT, are required for the regulation of Nrf2 expression and nuclear translocation, thereby leading to HO-1 upregulation [32]. We tested whether LIF activates the MAPK and PI3K/Akt pathway. We examined the total and phosphorylated protein levels of MAPK and PI3K/AKT in CTX TNA2 rat astrocytes treated with LIF by western blotting and found that LIF activated ERK, p38MAPK, JNK, and PI3K/Akt. LIF treatment (60 ng/mL) induced a time-related increase in the phosphorylation of ERK1/2 and PI3K/Akt (Fig. 5A), and JNK and p38MAPK (Fig. 5B). The maximal activation of both PI3K/Akt and MAPK was observed 1 h after LIF stimulation. LIF activated the MAPK and PI3K/Akt cascade at 1h after LIF treatment (Fig. 5C,D).These data indicate that LIF activates the MAPK and Akt kinase pathways in CTX TNA2 rat astrocytes.

We then explored the role of the MAPK and PI3K/Akt pathways in LIF-induced HO-1 upregulation. We examined the effect of PD98059 (an ERK inhibitor), SP600125 (a JNK inhibitor), LY294002 (a PI3K/AKT inhibitor), and SB203580 (a p38 inhibitor). LIF-mediated increase in MAPK and PI3K/Akt phosphorylation was blocked by PD98059, SP600125, SB203580, and LY294002 (Fig. 5E–H). To further confirm the roles of MAPK and PI3K/Akt in the HO-1 upregulation, we tested whether pathway inhibitors could block HO-1 upregulation. As shown in Fig. 5I, PD98059, SP600125, SB203580, and LY294002 reversed LIF-induced HO-1 expression, suggesting that LIF upregulates HO-1 expression by activating AMPK and AKT signaling in CTX TNA2 rat astrocytes.

Given that pyroptosis plays a role in OGD/R and that LIF upregulates HO-1 expression by activating AMPK and AKT signaling in CTX TNA2 rat astrocytes, we hypothesized that LIF may exert a neuroprotective effect in OGD/R-induced cell injury by inhibiting pyroptosis-dependent neurotoxicity through HO-1 upregulation in CTX TNA2 rat astrocytes. LIF treatment significantly increased cell viability (Fig. 6A,B) and decreased OGD/R-induced apoptosis (Fig. 6C) and ROS production (Fig. 6D). NLRP3 inflammasome-related genes (ASC, NLRP3, active caspase 1, IL-1$\beta$, and IL-18) were all suppressed compared to those of the OGD/R group at mRNA (Fig. 6E) and protein level (Fig. 6F). To further confirm the roles of HO-1 in the neuroprotective effect of LIF against OGD/R-induced cell injury, we used HO-1 inhibitor ZnPP. Co-treatment of LIF and ZnPP (10 µM) (Fig. 6G) partially abolished the LIF-mediated viability enhancement in CTX TNA2 rat astrocytes, indicating that HO-1 induction contributes to the neuroprotective effect of LIF against OGD/R-induced cell injury. Together, these results demonstrated that LIF mitigates OGD/R-induced pyroptosis-dependent neurotoxicity by inducing HO-1 expression in CTX TNA2 rat astrocytes.