Prostaglandin E2 alleviates acetaminophen-induced liver injury through DDIT4-enhanced autophagy regulated by circLima1/miR- 486

Prostaglandin E2 alleviates acetaminophen-induced liver injury through DDIT4-enhanced autophagy regulated by circLima1/miR- 486 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Prostaglandin E2 alleviates acetaminophen-induced liver injury through DDIT4-enhanced autophagy regulated by circLima1/miR- 486 Chao Chen, Jun Guan, Shanshan Wu, Meng Hong, Yanli Ren, Guodi Wu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8110558/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Studies indicate that acetaminophen (APAP) overdose induces autophagy, thereby attenuating APAP-induced hepatocyte death. Prostaglandin E2 (PGE2) has been shown to reduce serum transaminase levels and prevent mortality in APAP-induced liver injury(AILI) in mice, and PGE2 downregulation reduces autophagy in skeletal muscle tissue of young mice. However, the hepatoprotective mechanisms of PGE2 have not been elucidated. Methods High-throughput sequencing profiled circRNA, miRNA, and mRNA expression. Differentially expressed mRNAs were analyzed via protein-protein interaction networks and integrated with single-cell sequencing data from GEO. A circRNA/miRNA/mRNA network was computationally predicted (miRanda) and validated through miRNA mimic transfection and dual-luciferase assays. The role of Autophagy in PGE2-mediated protection was assessed by dual-fluorescent LC3 tracking and mitochondrial flux analysis. Results In the PGE2 group, a marked upregulation of circLima1 expression was detected, concomitant with downregulation of miR-486a-3p and miR-486b-3p. Transcriptomic analysis revealed significant DDIT4 upregulation in both APAP versus CON and PGE2 versus APAP comparisons. miRNA mimic transfection and dual-luciferase reporter assays were applied to confirm the regulatory axis involving circLima1/miR-486/DDIT4. Furthermore, we demonstrated that APAP + PGE2 enhances autophagic activity in hepatocytes, as shown by biochemical autophagic flux assays and LC3 dual-fluorescent lentivirus tracking. Pharmacological inhibition of autophagy with 3-methyladenine abolished the protective effects of dmPGE2 in AILI mice. Conclusion These results elucidate the role of PGE2 in alleviating AILI by enhancing autophagy through the circLima1/miR-486/DDIT4 regulatory axis network and provide a preliminary understanding of the molecular mechanisms underlying the hepatoprotective effects of dmPGE2 in AILI mice. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology Prostaglandin E2 Acetaminophen circRNA miRNA DNA damage induced transcript 4 Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction PGE2, a bioactive lipid synthesized from arachidonic acid (AA) via the sequential action of cyclooxygenase (COX) and prostaglandin E synthase (PGES)[ 1 ], is one of the most abundant prostaglandins in the human body and exerts diverse biological effects[ 2 ]. The downregulation of PGE2 in skeletal muscle tissue with overexpression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) leads to muscle atrophy and reduction of autophagy in young mice [ 3 ]. Injection of PGE2 reverses aged muscle stem cell dysfunction, leading to increased regeneration and strength[ 4 ]. Prior research has shown that dmPGE2, a metabolically stable PGE2 analog with physiological effects comparable to endogenous PGE2, exhibits hepatoprotective properties in animal models of Acetaminophen(APAP)-induced liver injury(AILI)[ 5 ]. Research shows that APAP overdose induces autophagy, which attenuates APAP-induced liver cell death by removing damaged mitochondria and APAP-protein adducts (APAP-ADs)[ 6 ]. However, the precise mechanisms underlying the alleviation effect of PGE2 on acute liver injury remain unclear. PGE2 might protect hepatocytes by inducing autophagy and restoring mitochondrial function. The circRNA/miRNA/mRNA network is reported to play an essential role in AILI[ 7 ]. CircRNAs are a class of highly conserved noncoding RNAs characterized by covalently closed-loop structures lacking polyadenylated tail and 5’-to-3’ polarity[ 8 ]. These molecules, formed through back-splicing, demonstrate greater stability than linear RNAs due to their resistance to exonuclease activity and are increasingly recognized as potential diagnostic biomarkers for various diseases[ 9 , 10 ]. Functionally, circRNAs modulate gene expression through mechanisms such as miRNA sequestration, interactions with RNA-binding proteins, and limited peptide translation[ 11 ]. CircRNAs bind to complementary miRNAs and inhibit their regulatory effects on target genes [ 12 , 13 ]. This mechanism is facilitated by the presence of complementary sequences within circRNAs specific to miRNAs, which enables the formation of stable RNA-miRNA complexes that exhibit resistance to degradation [ 8 , 14 ]. Consequently, by acting as miRNA sponges, circRNAs sequester miRNAs and prevent their interaction with target genes, thereby resulting in target gene derepression [ 15 ]. Thus, through this miRNA sponge mechanism, circRNAs post-transcriptionally regulate miRNA function and are consequently pivotal regulators of gene expression in cellular processes [ 16 ]. In AILI, circ-CBFB has been identified as a miR-185-5p sponge, modulating p66Shc expression and mitochondrial dynamics[ 17 ]. miRNAs also play pivotal roles in AILI. As key regulators of APAP metabolism, cytochrome P450 enzymes (CYP450s) were regulated by specific miRNAs. For instance, miR-27b binds to the 3’-untranslated region (3’-UTR) of CYP3A4, downregulating its expression in HEK293 cells[ 18 ]. Serum levels of miR-122-5p, miR-378a-5p, miR-125b-5p, and miR-27b-3p are elevated in children with APAP overdose compared to healthy controls. Papageorgiou et al. further demonstrated that miR-375 modulates UGT1A1 expression, which increased the risk for APAP-induced ALF[ 19 ]. However, the circRNA/miRNA/mRNA networks involved in PGE2-mediated mitigation of AILI remain unexplored. Collectively, while the link between noncoding RNAs and liver diseases is increasingly acknowledged, the precise regulatory functions of circRNA and miRNA in dmPGE2-mediated alleviation of AILI remain unclear. To investigate the role of it underlying PGE2-mediated alleviation of AILI, we performed sequencing of circRNAs, miRNAs, and mRNAs in liver tissues from mice treated with APAP and dmPGE2. Through high-throughput sequencing, we aimed to elucidate the role of circRNA/miRNA/mRNA interactions in the mechanism by which dmPGE2 alleviates AILI. 2. Materials and Methods 2.1 Model establishment Male C57BL/6J mice (wild-type, WT), aged 8 weeks, were procured from SLAC Animal (Shanghai, China) and housed in a specific pathogen free (SPF) environment for 1 week before experimentation, with an average weight of 20 ± 2 g. The mice were fasted for 15h and then injected intraperitoneally with either 500 mg/kg APAP or dmPGE2 (MCE, HY-106420) in saline. Control groups received equivalent volumes of saline. Based on published articles[ 20 , 21 ], our prior studies demonstrate optimal toxicity/protection of PGE2 and APAP (Figure S1 ). dmPGE2 was administered 1h before APAP, and samples were collected 6h post-APAP administration. The Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University approved all experimental procedures involving mice. The animal studies were conducted following ARRIVE guidelines 2.0 (Animal Research: Reporting of In vivo Experiments). The Hepa1-6 mouse hepatoma cell line (ATCC, CRL-1830) and the Hep3B hepatoma cell line (ATCC, HB-8064) were treated with 10 mM APAP for 24h, with 2 µM PGE2 (MCE, HY-101952s) added 1h before APAP treatment to investigate its potential protective effects. Doses of PGE2 and APAP were selected based on prior studies demonstrating optimal toxicity/protection. The Hepa1-6 mouse hepatoma cell line was used for cell-based experiments of the CircRNA/miRNA/mRNA axis, including Fig. 5 E,F,G. The Hep3B hepatoma cell line was used for Seahorse and autophagic puncta quantification, including Fig. 6 D, E, F, Fig. 7 A, B, C, D, F, G, H. The number of replicates for cell-based experiments was 3. 2.2 Histology Samples from the left lobe of the liver were fixed in 4% formaldehyde, embedded in paraffin, sectioned, and stained with H&E. Necrotic area was measured with Image Pro Plus through the Irregular AOI option manually by a blinded veterinary pathologist. 5 slides from each sample were used to calculate the percent of necrotic area. 2.3 RNA sequencing Total RNA was extracted using RNAiso Plus (Takara, Japan, 9108) with RNase-free DNase I (Tiangen, China, RT411), and 5 µg total RNA from each sample was utilized for mRNA, miRNA, and circRNA library construction. Three mice of each group were used to establish RNA libraries. Raw reads with more than 10% unidentified base or 30% low-quality base(sQ < = 20) were removed to make clean reads in the quality control process. Differential expression analysis was performed using the DESeq2 R package with an adjusted P-value threshold of less than 0.05. High-throughput sequencing data for circRNA, miRNA, and mRNA have been deposited in the GEO database under accession numbers GSE241509, GSE241510, and GSE241511. Detailed information about novel circRNAs is provided in Dataset S1, with their sequences included in Dataset S2. The identification of circLima1(novel_circ_0004240,chr15:99819493–99819958(-)), circSlc25a15(novel_circ_0013536, chr8:22383206–22395827(-)), circLsm14a (novel_circ_0013005, chr7:34351285–34351517(-)) were based on CIRCpedia v3. The details were listed in CIRCpedia v3 database ( https://bits.fudan.edu.cn/circpediav3/convert ) and tables S2. An overview of circRNAs, miRNAs, and mRNAs is presented in supplemental tables S3-5. cDNA amplification was conducted using specific primers and the SYBR Green PCR Master Mix (Solarbio, China) on the QuantStudio Dx (Thermo Scientific, USA). The ''pheatmap'' and ''ggplot2'' packages were used to plot the heat maps and volcano maps, respectively. In addition, the clusterProfiler package was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of DEmRNAs. KEGG contains numerous signaling pathways[ 22 – 24 ]. Significantly enriched GO and KEGG terms were identified under the screening conditions of P < 0.05. The primers used for qRT-PCR are listed in Table S6 . The protein–protein interaction (PPI) network was constructed using the top 10 differentially expressed mRNAs ranked by maximal clique centrality (MCC) score via the cytoHubba plugin in Cytoscape [25] . The circRNA/miRNA/mRNA networks were predicted using the miRanda tool with thresholds set at a total score of 140 and a total energy of less than 17 kcal/mol. The siRNA and miRNA mimics used are listed in Table S7 . 2.4 Single-cell sequencing For single-cell sequencing, data were obtained from the GEO dataset GSE166178 and analyzed using the SingleR package (version 1.0.5) and Seurat (version 5.2.1) [26, 27] . Cell type specific marker genes included hepatocyte markers (Alb, Apoa2, Apoc3, and Mup3). 2.5 Dual-Luciferase reporter assay 293T (ATCC, CRL-3216) cells were seeded into 24-well plates and incubated for 24h to reach 60%–70% confluence. Wild-type and mutant reporter plasmids containing the Ddit4 3'-UTR were constructed before the assay. The binding sites of plasmids and miRNA mimics are shown in Fig. S8 C. The 293T cells were transiently co-transfected with miRNA mimics and 0.1 µg of either the Ddit4-3'-UTR wild-type or mutant reporter plasmids using Lipofectamine 2000. Luminescence was recorded using a VICTOR Nivo Multimode Plate Reader (PerkinElmer, USA). 2.6 Immunoblotting Total protein extracts were prepared using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Samples were run on 12% acrylamide gels and transferred onto nitrocellulose membranes (0.45µm, Merck, MA, USA). The antibodies used include mTOR (1:2000, Proteintech, 66888), p-mTOR (1:2000, Proteintech, 67778), DDIT4 (1:2000, CST, 2516S), p62 (1:2000, CST, 39749), LC3 (1:2000, Proteintech, 14600), and GAPDH (1:5000, Proteintech, 60004). Protein bands were detected using an enhanced chemiluminescence assay (ECL, Biosharp, China, BL520) and quantitatively analyzed by ImageJ software (v1.53). The original results were provided in supplemental Figure S9 . 2.7 Quantification of tandem fluorescent-tagged LC3 The tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) system provides a robust method for monitoring autophagic flux. Hepatocytes were transduced with a dual fluorescent lentiviral construct encoding mRFP-EGFP-LC3 (Sangon Biotech, China, GPL2001). This system utilizes Stub-RFP (588 nm) and Sens-GFP (488 nm) fused to LC3, with Sens-GFP fluorescence selectively quenched in acidic autolysosomal compartments. Autophagosomes were identified by yellow fluorescence (Sens-GFP + /Stub-RFP + ), whereas autolysosomes exhibited red fluorescence (Sens-GFP - /Stub-RFP + ), enabling dynamic assessment of autophagic flux. Fluorescent puncta were quantified hourly at 40×magnification using the Operetta CLS™ high-content imaging system(PerkinElmer, USA). Nuclei were identified via Hoechst 33342 staining (2 µg/mL), and puncta quantification was performed using Harmony software (v4.9). Screening criteria included a radius ≤ 5 pixels, contrast > 0.1, and an uncorrected spot-to-region intensity ratio > 1.5. 2.8 Mitochondrial function assessment Hep3B cells were seeded into XF96 microplates (Agilent Technologies, USA) at a density of 1×10⁴ cells per well and cultured to ~ 90% confluence before mitochondrial functional analysis. Cellular metabolic parameters, including extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), were measured using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA, 103015-100) according to the manufacturer’s protocol. 2.9 Statistical analysis Data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by post-hoc Student-Newman-Keuls (SNK) tests for multiple comparisons. Correlations between variables were assessed using Spearman’s rank correlation coefficient (two-tailed). A significance threshold of P < 0.05 was applied for all analyses. 3. Result 3.1 dmPGE2 Alleviates AILI In Vivo Histopathological analysis via H&E staining demonstrated vacuolization in both APAP and APAP + dmPGE2 treatment groups(Fig. 1 A). Notably, neither group exhibited significant immune cell infiltration. High dose dmPGE2 administration markedly reduced necrotic area (Fig. 1 B), indicative of hepatoprotective activity. Consistently, AST and ALT levels, established biomarkers of liver damage, were significantly reduced (Fig. 1 C), further supporting dmPGE2’s hepatoprotective role. And dmPGE2 administration 2h after APAP injection still markedly ameliorated liver injury in AILI mice (Fig. 1 D). Critically, dmPGE2 treatment did not significantly alter hepatic GSH or GSSG + GSH levels (Fig. 1 E), excluding modulation of GSH metabolism as a primary mechanism. 3.2 Differential Expression Analysis of circRNAs Between AILI Mice Treated with dmPGE2 In this study, we conducted a comprehensive analysis of circRNAs following sequencing in three groups: saline (CON), APAP (APAP), and dmPGE2 + APAP (PGE2). Sequencing yielded approximately 177.08 million (CON), 153.98 million (APAP), and 152.09 million (PGE2) high-quality reads. The identified circRNAs were distributed across 20 chromosomes (Fig. 2 A, Dataset S1). Notably, 94.88% of circRNAs originated from exonic sequences, with only 0.97% in intronic and 4.14% in intergenic regions (Fig. 2 B). The majority of circRNAs ranged from 201 to 400 base pairs (Fig. 2 C), reflecting a predominant size range. Hierarchical clustering of 243 differentially expressed circRNAs (DEcircRNAs) revealed distinct expression patterns (Fig. 2 D). Specifically, 89 circRNAs were upregulated and 63 downregulated in PGE2 vs. APAP (Fig. 2 E). Gene Ontology (GO) and KEGG analyses highlighted enriched pathways, including 94 in APAP vs. CON, 133 in PGE2 vs. CON, and 135 in PGE2 vs. APAP (Fig. 2 F-G), underscoring dmPGE2’s role in modulating liver injury pathways. 3.3 Differential Expression Analysis of miRNAs Between AILI Mice Treated with dmPGE2 miRNA sequencing yielded an average of 0.68 million (CON), 0.58 million (APAP), and 0.66 million (PGE2) high-quality reads(Table S4 ). Alignment rates of small RNA sequences to the mouse genome were 38.56% (CON), 36.81% (APAP), and 33.91% (PGE2) (Table S11 ). Small RNA lengths ranged from 18 to 35 nucleotides, with 22 nt representing the most abundant size class (Fig. 3 A). Transcripts per million (TPM) distributions for miRNAs were comparable across groups (Fig. 3 B). Hierarchical clustering of 243 differentially expressed miRNAs (DEmiRNAs) revealed distinct expression profiles across the CON, APAP, and PGE2 groups (Fig. 3 C). Differential expression analysis identified 49 DEmiRNAs in APAP vs. CON, 44 in PGE2 vs. CON, and 26 in PGE2 vs. APAP comparisons (Fig. 3 D). GO analysis associated APAP vs. CON target genes with cellular components, whereas PGE2 vs. APAP genes were enriched in single-organism cellular processes (Fig. 3 E). KEGG pathway analysis demonstrated comparable enrichment intensities across comparisons despite pathway specificity (Fig. 3 F). 3.4 Differential Expression Analysis of mRNAs Between AILI Mice Treated with dmPGE2 Comparative mRNA analysis identified 1,437 DEmRNAs (1,000 upregulated, 437 downregulated) in APAP vs. CON, whereas only 119 DEmRNAs (47 upregulated, 72 downregulated) were detected in PGE2 vs. APAP (Fig. 4 A, Table S5 ). GO term enrichment for biological and molecular processes was markedly higher in APAP vs. CON than it in PGE2 vs. APAP (Fig. 4 B). KEGG analysis implicated MAPK signaling, cytokine-cytokine receptor interaction, and neuroactive ligand-receptor interaction as central to APAP-induced pathogenesis (Fig. 4 C). 12 genes were differentially expressed across all comparisons (Fig. 4 D), including upregulated Ddit4, Retreg1, and Gadd45g (Fig. 4 E) and downregulated Pdk4, Cidec, and Slc7a11, etc. in PGE2 groups (Fig. 4 F). Protein interaction networks of APAP vs. CON DEmRNAs identified 38 co-expressed hub genes (e.g., Hsp90ab1, Hspa1a; Fig. 4 G). Notably, the top 10 hub genes showed no expression changes in APAP + dmPGE2 vs. APAP (Fig. 4 H), suggesting dmPGE2-mediated protection operates independently of APAP-induced transcriptional upregulation. 3.5 Construction and Validation of the circRNA/miRNA/mRNA Coexpression Network While count of DEcircRNA or DEmiRNA were conserved in APAP vs. CON, PGE2 vs. APAP or PGE2 vs. CON comparisons, abruptly low DEmRNA was observed in PGE2 vs. APAP(Fig. 5 A). Logarithmic analysis of DEmiRNA/DEmRNA interactions further emphasized these disparities (Fig. 5 B). In APAP vs. CON, inverse correlations between miRNA and circRNA expression failed to account for mRNA changes (Fig. 5 C). Conversely, in PGE2 vs. APAP, miR-486a-3p and miR-486b-3p were downregulated contrasting their APAP vs. CON upregulation via opposing regulatory effects of circLima1 and circSlc25a15 (Fig. 5 D). The expression of DDIT4、miR-486a-3p、miR-486b-3p、circLima1 and circSlc25a15 in dmPGE2-treated AILI mice were confirm by QPCR(Fig. S10 A-C). circLima1-siRNA (siRNA targeted circLima1) reversed the upregulation of Ddit4 induced by PGE2, while this siRNA downregulated circLima1 and upregulated miRNA-486a/b (Fig. S8 A-B).The mimics of miR-486a/b-3p suppressed Ddit4 expression significantly(P < 0.01, Fig. 5 F). Dual luciferase reporter assay showed that miR-486a/b-3p significantly suppressed the relative reporter activity of wild-type 3 ʹ UTR Ddit4(p < 0.01) compared to the rest of the control groups (Fig. 5 G, Fig. S8 C). At the same time, it did not affect the relative reporter activity of mutant 3 ʹ UTR Ddit4, confirming that Ddit4 is the target of miR-486a/b-3p regulated by circLima1. 3.6 PGE2 alleviates APAP-induced hepatocyte injury by upregulating Ddit4 Flow cytometry analysis of immune cells from APAP or APAP + dmPGE2-treated mice revealed no changes in B cells, CD4 + T cells, CD8 + T cells, or Kupffer cells among CD45 + cells. However, neutrophil accumulation increased post-dmPGE2 treatment (Fig. 6 A-B). Single-cell RNA sequencing from the GSE dataset demonstrated elevated Ddit4 expression in APAP-treated hepatocytes but not in immune or stromal cells (Fig. 6 C). qPCR confirmed Ddit4 upregulation in APAP-treated Hep3B cells, further enhanced by dmPGE2 (Fig. 6 D). Ddit4 knockdown via siRNA abolished PGE2’s protective effects against APAP-induced injury (Fig. 6 E). ECAR levels of 40mins from Neg-siRNA APAP + PGE2 group is higher than it from Neg-siRNA APAP group(P 0.05). Seahorse analysis revealed impaired mitochondrial function in Ddit4-siRNA-transfected cells, with no recovery upon PGE2 treatment, unlike Neg-siRNA-transfected cells (Fig. 6 F). These results indicate that blocking the upregulation of Ddit4 impaired the restoration of mitochondrial function of PGE2 after APAP treatment. 3.7 PGE2 alleviated AILI by enhancing autophagy via DDIT4 Following transfection with a lentiviral construct encoding mRFP-EGFP-LC3, Hep3B cells exhibited distinct cytoplasmic yellow puncta (indicative of early autophagosomes) after 12h rapamycin treatment (Fig. 7 A). Co-treatment with APAP and rapamycin induced pronounced accumulation of late-stage autophagic vesicles (red puncta), whereas APAP combined with PGE2 predominantly enhanced early-stage autophagic activity (yellow puncta) (Fig. 7 B). Autophagic puncta were quantified using high-content imaging analysis, with yellow puncta defined by GFP/mRFP colocalization (Fig. 7 C). Quantitative analysis demonstrated elevated autophagic flux in both APAP + rapamycin and APAP + PGE2 groups relative to controls. Specifically, APAP + PGE2 treatment preferentially increased early-stage puncta (yellow). At the same time, APAP + rapamycin promoted late-stage autophagosome maturation (red) (Fig. 7 D). Western blot analysis revealed significant suppression of phosphorylated mTOR (p-mTOR), an increasement in the LC3-II/LC3-I ratio, and upregulation of DDIT4 following PGE2 treatment, supporting a mechanistic model in which PGE2-mediated DDIT4 induction suppresses mTOR phosphorylation to activate autophagy (Fig. 7 E). Genetic silencing of DDIT4 abrogated PGE2-driven autophagic enhancement, as shown by comparable yellow puncta counts in APAP + PGE2 and APAP-only groups under DDIT4 knockdown conditions (Fig. 7 F-G). Co-administration of the autophagy inhibitor 3-methyladenine (3-MA) with dmPGE2 markedly exacerbated APAP-induced hepatotoxicity, evidenced by elevated serum AST and ALT levels in AILI mice (Fig. 7 H). Kaplan-Meier survival analysis of mice subjected to a lethal APAP dose confirmed dmPGE2-mediated protection, which was abolished by 3-MA co-treatment (Fig. 7 I), underscoring the hepatoprotective role of dmPGE2-dependent autophagy. 4. Discussion In the present study, we employed AILI animal models and cell models to identify the key gene DDIT4, which exhibited a continuous increase in expression following APAP or APAP + PGE2/dmPGE2 treatment. We propose that PGE2 induces DDIT4 via the regulatory axis network involving circLima1, miR-486, and DDIT4. The upregulation of DDIT4 in hepatocytes, in turn, inhibits downstream mTORC1, thereby enhancing autophagy. Consequently, APAP-ADs, which play a critical role in APAP-induced hepatocyte necrosis [ 28 , 29 ] by impaired mitochondrial function, can be removed through autophagy induced by DDIT4[ 30 ]. APAP is considered safe for alleviating fever and pain at therapeutic doses, but APAP overdose represents the leading cause of acute liver failure [ 31 ]. APAP is metabolized by cytochrome P-450 2E1 (CYP2E1) in hepatocytes to generate N-acetyl-p-benzoquinoneimine (NAPQI)[ 32 ] while excess NAPQI covalently binds to intracellular proteins, forming APAP-ADs, which contribute to hepatocyte damage[ 33 ]. In clinical practice, N-acetylcysteine (NAC) serves as the antidote for APAP overdose by acting as a precursor for glutathione (GSH) synthesis, thereby facilitating the detoxification of excess NAPQI[ 34 ]. Notably, NAC demonstrates maximal efficacy when administered early following APAP overdose, as it does not reverse existing APAP-ADs formation. Recent studies using a zebrafish model of APAP toxicity have shown that co-administration of NAC with PGE2 extends the therapeutic window, attenuates hepatocyte apoptosis, normalizes serum transaminase levels, and significantly reduces mortality[ 35 ]. This study provides a more comprehensive understanding of the cytoprotective mechanisms of dmPGE2 in AILI mice. APAP primarily undergoes II phase metabolism, forming inactive metabolites, while a small fraction is metabolized into NAPQI[ 36 ], leading to APAP-ADs formation[ 37 ] and hepatotoxicity[ 38 – 40 ]. DDIT4, a 232-amino-acid protein, is cytoplasmic[ 41 ] under normal conditions but can also localize to the cell membrane and mitochondria[ 42 , 43 ], which typically responds to stressors like hypoxia[ 44 ], ischemia[ 45 ], DNA damage[ 41 ], and ROS activation[ 46 ]. mTORC1, the primary target of DDIT4, is a crucial regulator of intracellular synthesis and metabolism, including enhancing protein production [ 47 ], promoting lipid synthesis [ 48 ], impeding the formation of autophagosomes [ 49 ], and curtailing autophagy [ 50 ]. Induction of DDIT4 suppressed mTORC1 activity to induce autophagy by preserving the function of the tuberous sclerosis protein 1/2 complex (TSC1/2) [ 51 ]. Autophagy can eliminate damaged mitochondria and maintain mitochondrial homeostasis[ 52 ], as in liver injury induced by alcohol or APAP[ 53 ]. In our research, the induction of DDIT4 is observed in mice treated with APAP. DDIT4 is upregulated by various hormones and growth factors, including glucocorticoids [ 54 ], insulin [ 55 ], catecholamines [ 56 ], aldosterone [ 57 ], growth hormones [ 58 ], and melatonin [ 59 ]. The study shows that the downregulation of PGE2 in skeletal muscle tissue with overexpression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) leads to muscle atrophy and reduction of autophagy in young mice [ 3 ], indicating that the relationship between PGE2, DDIT4, autophagy, and muscle atrophy requires further investigation.s Our findings also represent a significant step toward identifying new therapeutic targets in AILI. As the only clinically approved antidote against APAP overdose, NAC supports hepatic GSH synthesis and prevents APAP-ADs formation but does not act directly on NAPQI nor remove APAP-ADs[ 34 , 60 ]. Research shows that APAP overdose induces autophagy, which attenuates AILI by removing damaged mitochondria and APAP-ADs[ 6 ]. We found that dmPGE2 induced autophagy in the liver from AILI mice, indicating that targeting the PGE2/circLima1/miR-486/DDIT4 axis might induce selective autophagy, particularly in liver tissue impaired by APAP overdose. However, our research still has limitations. DDIT4 is upregulated under stimulation such as DNA damage and ROS activation caused by overdose of APAP [ 44 , 56 , 59 ]. Our data showed that Ddit4 was upregulated by circLima1 /miRNA-486a/b in the dmPGE2 + APAP group, but both DDIT4 and miRNA-486a/b were upregulated in the APAP group(Fig. 5 E & Fig. S8 A-B). Moreover, dmPGE2 enhanced the upregulation of DDIT4 in AILI mice. However, it did not significantly increase the expression of DDIT4 in normal mice (Fig. 6 D). So we supposed that DDIT4 is also regulated by other molecules, which is remains unverified in the present work. In AILI mice treated with dmPGE2, downregulated miRNA-486a/b through circLima1 relived the suppression of upregulation of DDIT4 induced by APAP. In normal mice, there is no suppression of DDIT4 by miRNA-486a/b. So, the dmPGE2 did not upregulate DDIT4 directly. The mechanism for the regulation of DDIT4 will be clarified in our future research. Our research did not confirm sufficiency or exclusivity of DDIT4 in PGE2 protection on AILI mice, while other pathways might be involved in PGE2-induced autophagy enhancement. In vivo knockdown or overexpression experiments would better confirm the roles of DDIT4 or circLima1, which we will do in the future. Moreover, only 119 DEmRNAs are identified in PGE2 vs. APAP, compared to 1,437 in APAP vs. CON, which indicates PGE2 alleviates liver injury significantly but relatively few changes in gene expression. The APAP-induced liver injury involves multiple pathways and genes. DmPGE2 influences downstream pathways such as autophagy instead of upstream pathway such as GSH metabolism. So there are few changes in gene expression of dmPGE2 + APAP vs. APAP. Further research is required to clarify its mechanism. In summary, this study investigated the protective effects of PGE2 against APAP-induced acute liver injury. We observed a significant increase in DDIT4 expression in both the APAP model and following PGE2 treatment. Notably, DDIT4 was found to induce autophagy by inhibiting mTORC1 activity, with its regulation mediated through the interaction of circLima1 and miRNA-486. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate The animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University. The animal studies were conducted according to the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). The euthanasia process strictly complied with the AVMA Guidelines for the Euthanasia of Animals and ARRIVE 2.0 requirements for methodological detail and reproducibility. Mice were anesthetized in an induction chamber with 5%-7% isoflurane (oxygen flow rate: 1–2 L/min) and monitored every 10 seconds until loss of righting reflex, slowed regular breathing, and absence of tactile response (to avoid over-anesthesia). Euthanasia was performed via cervical dislocation under anesthesia, with confirmation of cessation of breathing/cardiac activity and absence of eyelid reflex (reverified after 30 seconds). All methods were carried out in accordance with relevant guidelines and regulations. The authors declare that they have not use AI-generated work in this manuscript. Fundings This work was financially supported by Science and Technology Major Projects of Zhejiang Province (NO. 2018C04016) and National Science and Technology Major Project of China (NO. 2018ZX10302206). Author Contribution C.C. designed the whole study. C.C., G.J. and G.XY. conducted animal experiments and analyzed the data. W.J. and W.SS. made relevant edits to the manuscript. H.M., W.GD. and R.YL. revised the manuscript. C.Z. and Z.HH. supervised and reviewed the manuscript. All authors contributed to the article and approved the submitted version. Acknowledgement Not applicable Data Availability Data is provided within the manuscript or supplementary information files. High-throughput sequencing data for circRNA, miRNA, and mRNA have been deposited in the GEO database under accession numbers GSE241509, GSE241510, and GSE241511. The Single-cell sequencing data was sourced from the GEO dataset GSE166178. References Park, J. Y., Pillinger, M. H. & Abramson, S. B. Prostaglandin E2 synthesis and secretion: the role of PGE2 synthases. Clin. Immunol. 119 , 229–240 (2006). Cheng, H. et al. Role of prostaglandin E2 in tissue repair and regeneration. Theranostics 11 , 8836–8854 (2021). 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Supplementary Files S1DosesselectionofAPAPandPGE2.jpg S2SupplementalDataset.circRNAs.csv S3SupplementalDataset.novelcircRNAseqmerged.csv S4SupplementalTable.OverviewofcircRNAsequencingdata.docx S5SupplementalTable.OverviewofmiRNAsequencingdata.docx S6SupplementalTable.OverviewofmRNAsequencingdata.docx S7SupplementalTable.PrimerList.docx S8SupplementalFigure.ExpressionofcircRNAandmiRNAincellstransfectedwithCIRCoredia36195siRNAandbingdingsitesofdualluciferase.jpg S9SupplementalTable.siRNAandmiRNAmimics.docx S10SupplementalFigure.originalWBresults.pptx S11SupplementalFigure.expressionofkeyDEcircRNADEmiRNAandDEmRNAinlivertissuefromAILImice.jpg.jpg.jpg S12SupplementalTable.StatisticsofsmallRNAreadsmappedtogenome.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 01 Jan, 2026 Reviewers agreed at journal 22 Dec, 2025 Reviews received at journal 17 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 02 Dec, 2025 Editor invited by journal 02 Dec, 2025 Submission checks completed at journal 27 Nov, 2025 First submitted to journal 27 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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(A) Histological analysis (H\u0026amp;E staining) of liver tissue from AILI mice treated with varying doses of dmPGE2 (n = 5). Images were centered on the central hepatic vein, showing vacuolized hepatocytes resulting from APAP toxicity. (B) Quantification of necrotic area in liver tissue from AILI mice treated with dmPGE2 (n = 5). Necrotic areas were characterized by ballooning degeneration and lytic necrosis of hepatocytes centered around the central vein. (C) Serum AST and ALT levels in AILI mice treated with varying doses of dmPGE2 (n = 5). (D) Serum AST and ALT levels in AILI mice treated with dmPGE2 at different time points (n = 3). dmPGE2 (-1h), dmPGE2 (1h), dmPGE2 (2h), and dmPGE2 (3h) indicate administration time relative to APAP modeling, 1 h before or 1 h, 2 h, and 3 h after APAP administration. (E) Total glutathione (GSH + GSSG) and reduced GSH concentrations in liver tissue of mice treated with APAP for 0 h, 1 h, or 6 h (n = 5). Data were presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/8bc901aecd5cf52a5ad1ccd4.jpg"},{"id":97669553,"identity":"a5d550fa-8aff-4571-a140-5766f3881f12","added_by":"auto","created_at":"2025-12-08 09:28:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3628063,"visible":true,"origin":"","legend":"\u003cp\u003ecircRNA expression profiling in the liver of AILI mice following dmPGE2 treatment (n=3). (A) Genomic distribution of circRNAs across 20 chromosomes in male mice. (B) Proportion of exon-, intron-, and intergenic-derived circRNAs across experimental groups. (C) circRNA counts categorized by length (100 bp intervals) across experimental groups. (D) Heatmap of DEcircRNAs among CON, APAP, and APAP+dmPGE2 groups. (E) Volcano Plots of DEcircRNAs in APAP vs. CON, APAP vs. PGE2, and PGE2 vs. CON. The red dots indicated upregulated circRNAs, and the blue dots indicated downregulated circRNAs. (F) Go term of the top 5 enriched pathways of DEcircRNAs in biological progress, cellular component and molecular function from APAP vs. CON and PGE2 vs. APAP. (G) KEGG of the Top 8 enriched pathways of DEcircRNAs in APAP vs. CON, APAP vs. PGE2, and PGE2 vs. CON analyzed.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/594a3b6c0be039df489fd7d4.jpg"},{"id":97473680,"identity":"7a984232-938a-49f1-b6f1-b8b541540a64","added_by":"auto","created_at":"2025-12-04 18:22:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4743962,"visible":true,"origin":"","legend":"\u003cp\u003emiRNA expression profiling in the liver of AILI mice following dmPGE2 treatment (n=3). (A) Frequency percentages of miRNAs of varying lengths identified across experimental groups. (B) Transcripts per million (TPM) distribution of miRNAs across experimental groups. (C) Heatmap of differentially expressed DEmiRNAs among CON, APAP, and APAP+dmPGE2 groups. (D) Numbers of DEmiRNAs stratified by upregulation or downregulation among CON, APAP, and APAP+dmPGE2 groups. Upregulation of APAPvsCON standard for the gene was upregulated in the APAP group compared to CON. (E) GO term of the top 5 enriched pathways associated with DEmiRNAs for APAP vs. CON and PGE2 vs. APAP comparisons. (F) KEGG pathway of the top 8 enriched pathways associated with DEmiRNAs in APAP vs. CON, PGE2 vs. APAP, and PGE2 vs. CON comparisons.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/9ed385b10c2cd31a1f2e0fdd.jpg"},{"id":97668881,"identity":"eb6d7eb2-47d3-4a56-ae5b-f712958af7b0","added_by":"auto","created_at":"2025-12-08 09:26:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1552898,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression profiling in the liver of AILI mice following dmPGE2 treatment (n=3). (A) Volcano plots of DEmRNAs in APAP vs. CON and PGE2 vs. APAP comparisons. Red dots indicate upregulated mRNAs, blue dots indicate downregulated mRNAs. (B) GO terms of DEmRNAs for APAP vs. CON and PGE2 vs. APAP comparisons. (C) KEGG pathway of DEmRNAs in APAP vs. CON, PGE2 vs. APAP, and PGE2 vs. CON comparisons. (D) Venn diagram of DEmRNAs among APAP vs. CON, PGE2 vs. APAP, and PGE2 vs. CON comparisons. (E) Standardized fragments per kilobase million (FPKM) values of genes upregulated in APAP vs. CON and further upregulated in PGE2 vs. APAP. (F) Standardized FPKM values of genes upregulated in APAP vs. CON but downregulated in PGE2 vs. APAP. (G) Protein-protein interaction (PPI) network of DEmRNAs in APAP vs. CON predicted by iRegulon (Cytoscape, confidence score = 0.7). The top 10 DEmRNAs ranked by maximal clique centrality (MCC) using CytoHubba were color-coded from red (highest score) to orange (lowest score). (H) Standardized FPKM values of the top 10 genes. Data were presented as mean ± SEM; *P \u0026lt; 0.05, ns P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/8760d917fbde6a636a1a6df3.jpg"},{"id":97669033,"identity":"8e65f698-bb0a-459f-83f5-5b5fc4718570","added_by":"auto","created_at":"2025-12-08 09:27:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1938013,"visible":true,"origin":"","legend":"\u003cp\u003eConstruction and Validation of the circRNA/miRNA/mRNA Coexpression Network. (A) Number of DeCircRNA, DemiRNA, and DemRNA expression in PGE2 vs. APAP, PGE2 vs. CON, and APAP vs. CON comparisons. (B) Numbers of pairs of matched DecircRNA/DemiRNA and DemiRNA/DemRNA in PGE2 vs. CON and APAP vs. CON comparison. (C) Network of CircRNA/miRNA/mRNA in APAP vs. CON comparison. Red standard for the upregulated expression of circRNA, miRNA, or mRNA in the APAP group compared to the control group. Blue standard for downregulation. Deeper color standard for higher fold change of APAP vs. CON or PGE2 vs.CON comparison. (D) Network of CircRNA/miRNA/mRNA in PGE2 vs. APAP comparison. (E)The Ddit4 expression of Hepa1-6 cells treated with APAP, PGE2, and circRNA siRNA. (F)The Ddit4 expression of Hepa1-6 cells treated with APAP, PGE2, and miRNA mimics. (G) Dual luciferase assay of Ddit4. NC, negative control mimics; Mimics, Ddit4 mimics; UTR-NC, Plasmid with negative control 3'UTR; UTR-WT, Plasmid with wild-type Ddit4 3'UTR; UTR-Mut. Plasmid with mutant Ddit4 3'UTR. * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001, ns P\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/2d9903580edb35a27314c3af.jpg"},{"id":97670602,"identity":"352e697e-de6a-4450-a4f7-e50c4676edea","added_by":"auto","created_at":"2025-12-08 09:31:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1062930,"visible":true,"origin":"","legend":"\u003cp\u003ePGE2 alleviates APAP-induced hepatocyte injury by upregulating Ddit4. (A) Flow cytometry plot of immune cell populations. (B) Percentages of B cells, CD4+ T cells, CD8+ T cells, neutrophils, and Kupffer cells within CD45+ leukocytes. (C) Ddit4 expression levels in mice treated with APAP or saline, stratified by cell type (T cells, Kupffer cells, endothelial cells, biliary epithelial cells, NK cells, hepatocytes, neutrophils, dendritic cells, and B cells). Single-cell RNA sequencing (scRNA-seq) data were obtained from the GSE dataset. (D) Relative Ddit4 expression in Hep3B cells treated with APAP or APAP+dmPGE2, measured by quantitative PCR (qPCR). (E) Relative viability of Hep3B cells transfected with Neg-siRNA or Ddit4-siRNA and treated with APAP or APAP+dmPGE2, assessed by CCK-8 assay. (F) PGE2-mediated restoration of mitochondrial function in APAP-damaged hepatocytes via Ddit4 upregulation. *, ECAR levels of 40mins from Neg-siRNA APAP+PGE2 group is higher than it from Neg-siRNA APAP group(P \u0026lt; 0.05). ns, ECAR levels of 40mins from Ddit4-siRNA APAP+PGE2 group shows no differences from Ddit4-siRNA APAP group(P \u0026gt; 0.05). Oxygen consumption rate (OCR; left) and extracellular acidification rate (ECAR; right) were measured to assess mitochondrial respiration and glycolytic flux, respectively. Data were presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ns P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/90764f738e41fac5d29c3d3d.jpg"},{"id":97670646,"identity":"f9c1309f-3a87-468e-b172-1a0c2d77037d","added_by":"auto","created_at":"2025-12-08 09:31:06","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3179063,"visible":true,"origin":"","legend":"\u003cp\u003ePGE2 alleviates APAP-induced hepatocyte injury through enhancing autophagy. (A) Hep3B cells infected with a lentiviral construct encoding mRFP-EGFP-LC3 and treated with 10 μM rapamycin. Images were captured at 1 h intervals. (B) Representative images of mRFP-EGFP-LC3 hepatocytes treated with APAP, APAP+rapamycin, or APAP+dmPGE2. Yellow and red spots denote early and late autophagosomes, respectively. (C) Autophagic puncta quantified by high-content imaging analysis, with yellow puncta defined by GFP/mRFP colocalization. Spot detection criteria: radius ≤5 pixels, contrast \u0026gt;0.1, and spot-to-region intensity ratio \u0026gt;1.5. (D) Quantification of yellow (early) and red (late) autophagosomes treated with APAP, APAP+rapamycin, or APAP+dmPGE2. (E) The protein expressions of mTOR, phospho-mTOR (p-mTOR), GAPDH, DDIT4, p62, and LC3 in Hep3B cells treated with APAP, APAP+dmPGE2, or APAP+rapamycin. (F) Temporal changes in autophagosome formation (yellow/red spots) in APAP+dmPGE2-treated cells transfected with Ddit4-siRNA or Neg-siRNA. (G) Quantification of autophagosomes in Ddit4-siRNA or Neg-siRNA-transfected Hep3B cells treated with APAP or APAP+dmPGE2. (H) Serum AST and ALT levels in AILI mice treated with APAP, APAP+dmPGE2, or APAP+3-MA (n = 5). (I) Survival rates of mice treated with APAP, APAP+dmPGE2, or APAP+3-MA (n = 10). Data were presented as mean ± SEM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ns P\u0026gt;0.05.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/3ac83ea390d43e5d6423bce5.jpg"},{"id":97892997,"identity":"634d7847-fb68-4fc1-854d-b85b028f4d98","added_by":"auto","created_at":"2025-12-10 15:25:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22461030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/7bb3fea7-4817-4895-9bea-b6c2466f8049.pdf"},{"id":97473673,"identity":"a4f735df-59b3-4add-b91d-6909a5314581","added_by":"auto","created_at":"2025-12-04 18:22:14","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":125348,"visible":true,"origin":"","legend":"","description":"","filename":"S1DosesselectionofAPAPandPGE2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/077c5d46b069bb9885f3781a.jpg"},{"id":97473676,"identity":"4bb8271e-e48e-4a5b-b1fa-b4db719500e4","added_by":"auto","created_at":"2025-12-04 18:22:14","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1936227,"visible":true,"origin":"","legend":"","description":"","filename":"S2SupplementalDataset.circRNAs.csv","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/802c99ad2aec1ba749cd3dfd.csv"},{"id":97669186,"identity":"4dd7bddc-cad3-443e-b578-47b1793bdd49","added_by":"auto","created_at":"2025-12-08 09:27:32","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6947129,"visible":true,"origin":"","legend":"","description":"","filename":"S3SupplementalDataset.novelcircRNAseqmerged.csv","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/7cb705d721dff58137f8053f.csv"},{"id":97473684,"identity":"d08d4bee-fa48-4c2d-b8b4-b2bd0b45ea37","added_by":"auto","created_at":"2025-12-04 18:22:14","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14458,"visible":true,"origin":"","legend":"","description":"","filename":"S4SupplementalTable.OverviewofcircRNAsequencingdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/6843a1864368fb40d0391a32.docx"},{"id":97473696,"identity":"4fd89348-8dc9-4cce-b62a-e6391f864253","added_by":"auto","created_at":"2025-12-04 18:22:15","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14569,"visible":true,"origin":"","legend":"","description":"","filename":"S5SupplementalTable.OverviewofmiRNAsequencingdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/297f54455efbcd583ca684e0.docx"},{"id":97669912,"identity":"15127e73-49b2-468f-b526-ad6ef2fb6031","added_by":"auto","created_at":"2025-12-08 09:29:22","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14921,"visible":true,"origin":"","legend":"","description":"","filename":"S6SupplementalTable.OverviewofmRNAsequencingdata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/f357c7e0a6ceb4a65a35270b.docx"},{"id":97668893,"identity":"b0219926-c837-4ec7-92bf-a129341dc067","added_by":"auto","created_at":"2025-12-08 09:26:29","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":15896,"visible":true,"origin":"","legend":"","description":"","filename":"S7SupplementalTable.PrimerList.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/c7052881ebad0f9b58418ffa.docx"},{"id":97473703,"identity":"fdca874e-8b6f-4799-bdae-6cf22876d3e9","added_by":"auto","created_at":"2025-12-04 18:22:15","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":169262,"visible":true,"origin":"","legend":"","description":"","filename":"S8SupplementalFigure.ExpressionofcircRNAandmiRNAincellstransfectedwithCIRCoredia36195siRNAandbingdingsitesofdualluciferase.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/de65c7680fa1cc919778b89d.jpg"},{"id":97473694,"identity":"ed41b2fd-c6b4-45a0-9ac1-e8f408775338","added_by":"auto","created_at":"2025-12-04 18:22:14","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14720,"visible":true,"origin":"","legend":"","description":"","filename":"S9SupplementalTable.siRNAandmiRNAmimics.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/b38d750054d00f0008426665.docx"},{"id":97473708,"identity":"6562213a-f7f1-42ae-bf7f-3943be874ad5","added_by":"auto","created_at":"2025-12-04 18:22:15","extension":"pptx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1443785,"visible":true,"origin":"","legend":"","description":"","filename":"S10SupplementalFigure.originalWBresults.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/41b74a0c632e2f949b7c59e9.pptx"},{"id":97473700,"identity":"37c53246-2527-48c1-b8a6-fdb3b8801c26","added_by":"auto","created_at":"2025-12-04 18:22:15","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":134286,"visible":true,"origin":"","legend":"","description":"","filename":"S11SupplementalFigure.expressionofkeyDEcircRNADEmiRNAandDEmRNAinlivertissuefromAILImice.jpg.jpg.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/5999ab9d67cf6907e520337c.jpg"},{"id":97668979,"identity":"bfcf648d-28e1-4acf-b3ba-c2c6b8d3ad9e","added_by":"auto","created_at":"2025-12-08 09:26:53","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":13131,"visible":true,"origin":"","legend":"","description":"","filename":"S12SupplementalTable.StatisticsofsmallRNAreadsmappedtogenome.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110558/v1/c7bbf17342467440cc124bc3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prostaglandin E2 alleviates acetaminophen-induced liver injury through DDIT4-enhanced autophagy regulated by circLima1/miR- 486","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePGE2, a bioactive lipid synthesized from arachidonic acid (AA) via the sequential action of cyclooxygenase (COX) and prostaglandin E synthase (PGES)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], is one of the most abundant prostaglandins in the human body and exerts diverse biological effects[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The downregulation of PGE2 in skeletal muscle tissue with overexpression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) leads to muscle atrophy and reduction of autophagy in young mice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Injection of PGE2 reverses aged muscle stem cell dysfunction, leading to increased regeneration and strength[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Prior research has shown that dmPGE2, a metabolically stable PGE2 analog with physiological effects comparable to endogenous PGE2, exhibits hepatoprotective properties in animal models of Acetaminophen(APAP)-induced liver injury(AILI)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Research shows that APAP overdose induces autophagy, which attenuates APAP-induced liver cell death by removing damaged mitochondria and APAP-protein adducts (APAP-ADs)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the precise mechanisms underlying the alleviation effect of PGE2 on acute liver injury remain unclear. PGE2 might protect hepatocytes by inducing autophagy and restoring mitochondrial function.\u003c/p\u003e\u003cp\u003eThe circRNA/miRNA/mRNA network is reported to play an essential role in AILI[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CircRNAs are a class of highly conserved noncoding RNAs characterized by covalently closed-loop structures lacking polyadenylated tail and 5\u0026rsquo;-to-3\u0026rsquo; polarity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These molecules, formed through back-splicing, demonstrate greater stability than linear RNAs due to their resistance to exonuclease activity and are increasingly recognized as potential diagnostic biomarkers for various diseases[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Functionally, circRNAs modulate gene expression through mechanisms such as miRNA sequestration, interactions with RNA-binding proteins, and limited peptide translation[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CircRNAs bind to complementary miRNAs and inhibit their regulatory effects on target genes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This mechanism is facilitated by the presence of complementary sequences within circRNAs specific to miRNAs, which enables the formation of stable RNA-miRNA complexes that exhibit resistance to degradation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, by acting as miRNA sponges, circRNAs sequester miRNAs and prevent their interaction with target genes, thereby resulting in target gene derepression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, through this miRNA sponge mechanism, circRNAs post-transcriptionally regulate miRNA function and are consequently pivotal regulators of gene expression in cellular processes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In AILI, circ-CBFB has been identified as a miR-185-5p sponge, modulating p66Shc expression and mitochondrial dynamics[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. miRNAs also play pivotal roles in AILI. As key regulators of APAP metabolism, cytochrome P450 enzymes (CYP450s) were regulated by specific miRNAs. For instance, miR-27b binds to the 3\u0026rsquo;-untranslated region (3\u0026rsquo;-UTR) of CYP3A4, downregulating its expression in HEK293 cells[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Serum levels of miR-122-5p, miR-378a-5p, miR-125b-5p, and miR-27b-3p are elevated in children with APAP overdose compared to healthy controls. Papageorgiou et al. further demonstrated that miR-375 modulates UGT1A1 expression, which increased the risk for APAP-induced ALF[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the circRNA/miRNA/mRNA networks involved in PGE2-mediated mitigation of AILI remain unexplored.\u003c/p\u003e\u003cp\u003eCollectively, while the link between noncoding RNAs and liver diseases is increasingly acknowledged, the precise regulatory functions of circRNA and miRNA in dmPGE2-mediated alleviation of AILI remain unclear. To investigate the role of it underlying PGE2-mediated alleviation of AILI, we performed sequencing of circRNAs, miRNAs, and mRNAs in liver tissues from mice treated with APAP and dmPGE2. Through high-throughput sequencing, we aimed to elucidate the role of circRNA/miRNA/mRNA interactions in the mechanism by which dmPGE2 alleviates AILI.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Model establishment\u003c/h2\u003e\u003cp\u003eMale C57BL/6J mice (wild-type, WT), aged 8 weeks, were procured from SLAC Animal (Shanghai, China) and housed in a specific pathogen free (SPF) environment for 1 week before experimentation, with an average weight of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g. The mice were fasted for 15h and then injected intraperitoneally with either 500 mg/kg APAP or dmPGE2 (MCE, HY-106420) in saline. Control groups received equivalent volumes of saline. Based on published articles[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], our prior studies demonstrate optimal toxicity/protection of PGE2 and APAP (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). dmPGE2 was administered 1h before APAP, and samples were collected 6h post-APAP administration. The Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University approved all experimental procedures involving mice. The animal studies were conducted following ARRIVE guidelines 2.0 (Animal Research: Reporting of In vivo Experiments).\u003c/p\u003e\u003cp\u003eThe Hepa1-6 mouse hepatoma cell line (ATCC, CRL-1830) and the Hep3B hepatoma cell line (ATCC, HB-8064) were treated with 10 mM APAP for 24h, with 2 \u0026micro;M PGE2 (MCE, HY-101952s) added 1h before APAP treatment to investigate its potential protective effects. Doses of PGE2 and APAP were selected based on prior studies demonstrating optimal toxicity/protection. The Hepa1-6 mouse hepatoma cell line was used for cell-based experiments of the CircRNA/miRNA/mRNA axis, including Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE,F,G. The Hep3B hepatoma cell line was used for Seahorse and autophagic puncta quantification, including Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E, F, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, C, D, F, G, H. The number of replicates for cell-based experiments was 3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Histology\u003c/h2\u003e\u003cp\u003eSamples from the left lobe of the liver were fixed in 4% formaldehyde, embedded in paraffin, sectioned, and stained with H\u0026amp;E. Necrotic area was measured with Image Pro Plus through the Irregular AOI option manually by a blinded veterinary pathologist. 5 slides from each sample were used to calculate the percent of necrotic area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 RNA sequencing\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted using RNAiso Plus (Takara, Japan, 9108) with RNase-free DNase I (Tiangen, China, RT411), and 5 \u0026micro;g total RNA from each sample was utilized for mRNA, miRNA, and circRNA library construction. Three mice of each group were used to establish RNA libraries. Raw reads with more than 10% unidentified base or 30% low-quality base(sQ\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;20) were removed to make clean reads in the quality control process. Differential expression analysis was performed using the DESeq2 R package with an adjusted P-value threshold of less than 0.05. High-throughput sequencing data for circRNA, miRNA, and mRNA have been deposited in the GEO database under accession numbers GSE241509, GSE241510, and GSE241511. Detailed information about novel circRNAs is provided in Dataset S1, with their sequences included in Dataset S2. The identification of circLima1(novel_circ_0004240,chr15:99819493\u0026ndash;99819958(-)), circSlc25a15(novel_circ_0013536, chr8:22383206\u0026ndash;22395827(-)), circLsm14a (novel_circ_0013005, chr7:34351285\u0026ndash;34351517(-)) were based on CIRCpedia v3. The details were listed in CIRCpedia v3 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bits.fudan.edu.cn/circpediav3/convert\u003c/span\u003e\u003cspan address=\"https://bits.fudan.edu.cn/circpediav3/convert\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and tables S2. An overview of circRNAs, miRNAs, and mRNAs is presented in supplemental tables S3-5. cDNA amplification was conducted using specific primers and the SYBR Green PCR Master Mix (Solarbio, China) on the QuantStudio Dx (Thermo Scientific, USA). The ''pheatmap'' and ''ggplot2'' packages were used to plot the heat maps and volcano maps, respectively. In addition, the clusterProfiler package was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses of DEmRNAs. KEGG contains numerous signaling pathways[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Significantly enriched GO and KEGG terms were identified under the screening conditions of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The primers used for qRT-PCR are listed in Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe protein\u0026ndash;protein interaction (PPI) network was constructed using the top 10 differentially expressed mRNAs ranked by maximal clique centrality (MCC) score via the cytoHubba plugin in Cytoscape\u003csup\u003e[25]\u003c/sup\u003e. The circRNA/miRNA/mRNA networks were predicted using the miRanda tool with thresholds set at a total score of 140 and a total energy of less than 17 kcal/mol. The siRNA and miRNA mimics used are listed in Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Single-cell sequencing\u003c/h2\u003e\u003cp\u003eFor single-cell sequencing, data were obtained from the GEO dataset GSE166178 and analyzed using the SingleR package (version 1.0.5) and Seurat (version 5.2.1) \u003csup\u003e[26, 27]\u003c/sup\u003e. Cell type specific marker genes included hepatocyte markers (Alb, Apoa2, Apoc3, and Mup3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Dual-Luciferase reporter assay\u003c/h2\u003e\u003cp\u003e293T (ATCC, CRL-3216) cells were seeded into 24-well plates and incubated for 24h to reach 60%\u0026ndash;70% confluence. Wild-type and mutant reporter plasmids containing the Ddit4 3'-UTR were constructed before the assay. The binding sites of plasmids and miRNA mimics are shown in Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eC. The 293T cells were transiently co-transfected with miRNA mimics and 0.1 \u0026micro;g of either the Ddit4-3'-UTR wild-type or mutant reporter plasmids using Lipofectamine 2000. Luminescence was recorded using a VICTOR Nivo Multimode Plate Reader (PerkinElmer, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Immunoblotting\u003c/h2\u003e\u003cp\u003eTotal protein extracts were prepared using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Samples were run on 12% acrylamide gels and transferred onto nitrocellulose membranes (0.45\u0026micro;m, Merck, MA, USA). The antibodies used include mTOR (1:2000, Proteintech, 66888), p-mTOR (1:2000, Proteintech, 67778), DDIT4 (1:2000, CST, 2516S), p62 (1:2000, CST, 39749), LC3 (1:2000, Proteintech, 14600), and GAPDH (1:5000, Proteintech, 60004). Protein bands were detected using an enhanced chemiluminescence assay (ECL, Biosharp, China, BL520) and quantitatively analyzed by ImageJ software (v1.53). The original results were provided in supplemental Figure \u003cspan refid=\"MOESM9\" class=\"InternalRef\"\u003eS9\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Quantification of tandem fluorescent-tagged LC3\u003c/h2\u003e\u003cp\u003eThe tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) system provides a robust method for monitoring autophagic flux. Hepatocytes were transduced with a dual fluorescent lentiviral construct encoding mRFP-EGFP-LC3 (Sangon Biotech, China, GPL2001). This system utilizes Stub-RFP (588 nm) and Sens-GFP (488 nm) fused to LC3, with Sens-GFP fluorescence selectively quenched in acidic autolysosomal compartments. Autophagosomes were identified by yellow fluorescence (Sens-GFP\u003csup\u003e+\u003c/sup\u003e/Stub-RFP\u003csup\u003e+\u003c/sup\u003e), whereas autolysosomes exhibited red fluorescence (Sens-GFP\u003csup\u003e-\u003c/sup\u003e/Stub-RFP\u003csup\u003e+\u003c/sup\u003e), enabling dynamic assessment of autophagic flux. Fluorescent puncta were quantified hourly at 40\u0026times;magnification using the Operetta CLS\u0026trade; high-content imaging system(PerkinElmer, USA). Nuclei were identified via Hoechst 33342 staining (2 \u0026micro;g/mL), and puncta quantification was performed using Harmony software (v4.9). Screening criteria included a radius\u0026thinsp;\u0026le;\u0026thinsp;5 pixels, contrast\u0026thinsp;\u0026gt;\u0026thinsp;0.1, and an uncorrected spot-to-region intensity ratio\u0026thinsp;\u0026gt;\u0026thinsp;1.5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Mitochondrial function assessment\u003c/h2\u003e\u003cp\u003eHep3B cells were seeded into XF96 microplates (Agilent Technologies, USA) at a density of 1\u0026times;10⁴ cells per well and cultured to ~\u0026thinsp;90% confluence before mitochondrial functional analysis. Cellular metabolic parameters, including extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), were measured using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA, 103015-100) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by post-hoc Student-Newman-Keuls (SNK) tests for multiple comparisons. Correlations between variables were assessed using Spearman\u0026rsquo;s rank correlation coefficient (two-tailed). A significance threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was applied for all analyses.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 dmPGE2 Alleviates AILI In Vivo\u003c/h2\u003e\u003cp\u003eHistopathological analysis via H\u0026amp;E staining demonstrated vacuolization in both APAP and APAP\u0026thinsp;+\u0026thinsp;dmPGE2 treatment groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Notably, neither group exhibited significant immune cell infiltration. High dose dmPGE2 administration markedly reduced necrotic area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicative of hepatoprotective activity. Consistently, AST and ALT levels, established biomarkers of liver damage, were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), further supporting dmPGE2\u0026rsquo;s hepatoprotective role. And dmPGE2 administration 2h after APAP injection still markedly ameliorated liver injury in AILI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Critically, dmPGE2 treatment did not significantly alter hepatic GSH or GSSG\u0026thinsp;+\u0026thinsp;GSH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), excluding modulation of GSH metabolism as a primary mechanism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Differential Expression Analysis of circRNAs Between AILI Mice Treated with dmPGE2\u003c/h2\u003e\u003cp\u003eIn this study, we conducted a comprehensive analysis of circRNAs following sequencing in three groups: saline (CON), APAP (APAP), and dmPGE2\u0026thinsp;+\u0026thinsp;APAP (PGE2). Sequencing yielded approximately 177.08\u0026nbsp;million (CON), 153.98\u0026nbsp;million (APAP), and 152.09\u0026nbsp;million (PGE2) high-quality reads. The identified circRNAs were distributed across 20 chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Dataset S1). Notably, 94.88% of circRNAs originated from exonic sequences, with only 0.97% in intronic and 4.14% in intergenic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The majority of circRNAs ranged from 201 to 400 base pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), reflecting a predominant size range. Hierarchical clustering of 243 differentially expressed circRNAs (DEcircRNAs) revealed distinct expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Specifically, 89 circRNAs were upregulated and 63 downregulated in PGE2 vs. APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Gene Ontology (GO) and KEGG analyses highlighted enriched pathways, including 94 in APAP vs. CON, 133 in PGE2 vs. CON, and 135 in PGE2 vs. APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G), underscoring dmPGE2\u0026rsquo;s role in modulating liver injury pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Differential Expression Analysis of miRNAs Between AILI Mice Treated with dmPGE2\u003c/h2\u003e\u003cp\u003emiRNA sequencing yielded an average of 0.68\u0026nbsp;million (CON), 0.58\u0026nbsp;million (APAP), and 0.66\u0026nbsp;million (PGE2) high-quality reads(Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Alignment rates of small RNA sequences to the mouse genome were 38.56% (CON), 36.81% (APAP), and 33.91% (PGE2) (Table \u003cspan refid=\"MOESM11\" class=\"InternalRef\"\u003eS11\u003c/span\u003e). Small RNA lengths ranged from 18 to 35 nucleotides, with 22 nt representing the most abundant size class (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Transcripts per million (TPM) distributions for miRNAs were comparable across groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Hierarchical clustering of 243 differentially expressed miRNAs (DEmiRNAs) revealed distinct expression profiles across the CON, APAP, and PGE2 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Differential expression analysis identified 49 DEmiRNAs in APAP vs. CON, 44 in PGE2 vs. CON, and 26 in PGE2 vs. APAP comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). GO analysis associated APAP vs. CON target genes with cellular components, whereas PGE2 vs. APAP genes were enriched in single-organism cellular processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). KEGG pathway analysis demonstrated comparable enrichment intensities across comparisons despite pathway specificity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Differential Expression Analysis of mRNAs Between AILI Mice Treated with dmPGE2\u003c/h2\u003e\u003cp\u003eComparative mRNA analysis identified 1,437 DEmRNAs (1,000 upregulated, 437 downregulated) in APAP vs. CON, whereas only 119 DEmRNAs (47 upregulated, 72 downregulated) were detected in PGE2 vs. APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). GO term enrichment for biological and molecular processes was markedly higher in APAP vs. CON than it in PGE2 vs. APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). KEGG analysis implicated MAPK signaling, cytokine-cytokine receptor interaction, and neuroactive ligand-receptor interaction as central to APAP-induced pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). 12 genes were differentially expressed across all comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), including upregulated Ddit4, Retreg1, and Gadd45g (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and downregulated Pdk4, Cidec, and Slc7a11, etc. in PGE2 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Protein interaction networks of APAP vs. CON DEmRNAs identified 38 co-expressed hub genes (e.g., Hsp90ab1, Hspa1a; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Notably, the top 10 hub genes showed no expression changes in APAP\u0026thinsp;+\u0026thinsp;dmPGE2 vs. APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), suggesting dmPGE2-mediated protection operates independently of APAP-induced transcriptional upregulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Construction and Validation of the circRNA/miRNA/mRNA Coexpression Network\u003c/h2\u003e\u003cp\u003eWhile count of DEcircRNA or DEmiRNA were conserved in APAP vs. CON, PGE2 vs. APAP or PGE2 vs. CON comparisons, abruptly low DEmRNA was observed in PGE2 vs. APAP(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Logarithmic analysis of DEmiRNA/DEmRNA interactions further emphasized these disparities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In APAP vs. CON, inverse correlations between miRNA and circRNA expression failed to account for mRNA changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Conversely, in PGE2 vs. APAP, miR-486a-3p and miR-486b-3p were downregulated contrasting their APAP vs. CON upregulation via opposing regulatory effects of circLima1 and circSlc25a15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The expression of DDIT4、miR-486a-3p、miR-486b-3p、circLima1 and circSlc25a15 in dmPGE2-treated AILI mice were confirm by QPCR(Fig.\u003cspan refid=\"MOESM10\" class=\"InternalRef\"\u003eS10\u003c/span\u003eA-C). circLima1-siRNA (siRNA targeted circLima1) reversed the upregulation of Ddit4 induced by PGE2, while this siRNA downregulated circLima1 and upregulated miRNA-486a/b (Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eA-B).The mimics of miR-486a/b-3p suppressed Ddit4 expression significantly(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Dual luciferase reporter assay showed that miR-486a/b-3p significantly suppressed the relative reporter activity of wild-type 3 \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003eʹ\u003c/span\u003e UTR Ddit4(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the rest of the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eC). At the same time, it did not affect the relative reporter activity of mutant 3 \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003eʹ\u003c/span\u003e UTR Ddit4, confirming that Ddit4 is the target of miR-486a/b-3p regulated by circLima1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 PGE2 alleviates APAP-induced hepatocyte injury by upregulating Ddit4\u003c/h2\u003e\u003cp\u003eFlow cytometry analysis of immune cells from APAP or APAP\u0026thinsp;+\u0026thinsp;dmPGE2-treated mice revealed no changes in B cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, or Kupffer cells among CD45\u003csup\u003e+\u003c/sup\u003e cells. However, neutrophil accumulation increased post-dmPGE2 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Single-cell RNA sequencing from the GSE dataset demonstrated elevated Ddit4 expression in APAP-treated hepatocytes but not in immune or stromal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). qPCR confirmed Ddit4 upregulation in APAP-treated Hep3B cells, further enhanced by dmPGE2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Ddit4 knockdown via siRNA abolished PGE2\u0026rsquo;s protective effects against APAP-induced injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). ECAR levels of 40mins from Neg-siRNA APAP\u0026thinsp;+\u0026thinsp;PGE2 group is higher than it from Neg-siRNA APAP group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) while ECAR levels of 40mins from Ddit4-siRNA APAP\u0026thinsp;+\u0026thinsp;PGE2 group shows no differences from Ddit4-siRNA APAP group(P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Seahorse analysis revealed impaired mitochondrial function in Ddit4-siRNA-transfected cells, with no recovery upon PGE2 treatment, unlike Neg-siRNA-transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results indicate that blocking the upregulation of Ddit4 impaired the restoration of mitochondrial function of PGE2 after APAP treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7 PGE2 alleviated AILI by enhancing autophagy via DDIT4\u003c/h2\u003e\u003cp\u003eFollowing transfection with a lentiviral construct encoding mRFP-EGFP-LC3, Hep3B cells exhibited distinct cytoplasmic yellow puncta (indicative of early autophagosomes) after 12h rapamycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Co-treatment with APAP and rapamycin induced pronounced accumulation of late-stage autophagic vesicles (red puncta), whereas APAP combined with PGE2 predominantly enhanced early-stage autophagic activity (yellow puncta) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Autophagic puncta were quantified using high-content imaging analysis, with yellow puncta defined by GFP/mRFP colocalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Quantitative analysis demonstrated elevated autophagic flux in both APAP\u0026thinsp;+\u0026thinsp;rapamycin and APAP\u0026thinsp;+\u0026thinsp;PGE2 groups relative to controls. Specifically, APAP\u0026thinsp;+\u0026thinsp;PGE2 treatment preferentially increased early-stage puncta (yellow). At the same time, APAP\u0026thinsp;+\u0026thinsp;rapamycin promoted late-stage autophagosome maturation (red) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Western blot analysis revealed significant suppression of phosphorylated mTOR (p-mTOR), an increasement in the LC3-II/LC3-I ratio, and upregulation of DDIT4 following PGE2 treatment, supporting a mechanistic model in which PGE2-mediated DDIT4 induction suppresses mTOR phosphorylation to activate autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Genetic silencing of DDIT4 abrogated PGE2-driven autophagic enhancement, as shown by comparable yellow puncta counts in APAP\u0026thinsp;+\u0026thinsp;PGE2 and APAP-only groups under DDIT4 knockdown conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-G). Co-administration of the autophagy inhibitor 3-methyladenine (3-MA) with dmPGE2 markedly exacerbated APAP-induced hepatotoxicity, evidenced by elevated serum AST and ALT levels in AILI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Kaplan-Meier survival analysis of mice subjected to a lethal APAP dose confirmed dmPGE2-mediated protection, which was abolished by 3-MA co-treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI), underscoring the hepatoprotective role of dmPGE2-dependent autophagy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, we employed AILI animal models and cell models to identify the key gene DDIT4, which exhibited a continuous increase in expression following APAP or APAP\u0026thinsp;+\u0026thinsp;PGE2/dmPGE2 treatment. We propose that PGE2 induces DDIT4 via the regulatory axis network involving circLima1, miR-486, and DDIT4. The upregulation of DDIT4 in hepatocytes, in turn, inhibits downstream mTORC1, thereby enhancing autophagy. Consequently, APAP-ADs, which play a critical role in APAP-induced hepatocyte necrosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] by impaired mitochondrial function, can be removed through autophagy induced by DDIT4[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAPAP is considered safe for alleviating fever and pain at therapeutic doses, but APAP overdose represents the leading cause of acute liver failure [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. APAP is metabolized by cytochrome P-450 2E1 (CYP2E1) in hepatocytes to generate N-acetyl-p-benzoquinoneimine (NAPQI)[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] while excess NAPQI covalently binds to intracellular proteins, forming APAP-ADs, which contribute to hepatocyte damage[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In clinical practice, N-acetylcysteine (NAC) serves as the antidote for APAP overdose by acting as a precursor for glutathione (GSH) synthesis, thereby facilitating the detoxification of excess NAPQI[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Notably, NAC demonstrates maximal efficacy when administered early following APAP overdose, as it does not reverse existing APAP-ADs formation. Recent studies using a zebrafish model of APAP toxicity have shown that co-administration of NAC with PGE2 extends the therapeutic window, attenuates hepatocyte apoptosis, normalizes serum transaminase levels, and significantly reduces mortality[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis study provides a more comprehensive understanding of the cytoprotective mechanisms of dmPGE2 in AILI mice. APAP primarily undergoes II phase metabolism, forming inactive metabolites, while a small fraction is metabolized into NAPQI[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], leading to APAP-ADs formation[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and hepatotoxicity[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. DDIT4, a 232-amino-acid protein, is cytoplasmic[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] under normal conditions but can also localize to the cell membrane and mitochondria[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which typically responds to stressors like hypoxia[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], ischemia[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], DNA damage[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and ROS activation[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. mTORC1, the primary target of DDIT4, is a crucial regulator of intracellular synthesis and metabolism, including enhancing protein production [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], promoting lipid synthesis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], impeding the formation of autophagosomes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and curtailing autophagy [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Induction of DDIT4 suppressed mTORC1 activity to induce autophagy by preserving the function of the tuberous sclerosis protein 1/2 complex (TSC1/2) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Autophagy can eliminate damaged mitochondria and maintain mitochondrial homeostasis[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], as in liver injury induced by alcohol or APAP[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In our research, the induction of DDIT4 is observed in mice treated with APAP. DDIT4 is upregulated by various hormones and growth factors, including glucocorticoids [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], insulin [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], catecholamines [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], aldosterone [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], growth hormones [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and melatonin [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The study shows that the downregulation of PGE2 in skeletal muscle tissue with overexpression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) leads to muscle atrophy and reduction of autophagy in young mice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], indicating that the relationship between PGE2, DDIT4, autophagy, and muscle atrophy requires further investigation.s\u003c/p\u003e\u003cp\u003eOur findings also represent a significant step toward identifying new therapeutic targets in AILI. As the only clinically approved antidote against APAP overdose, NAC supports hepatic GSH synthesis and prevents APAP-ADs formation but does not act directly on NAPQI nor remove APAP-ADs[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Research shows that APAP overdose induces autophagy, which attenuates AILI by removing damaged mitochondria and APAP-ADs[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. We found that dmPGE2 induced autophagy in the liver from AILI mice, indicating that targeting the PGE2/circLima1/miR-486/DDIT4 axis might induce selective autophagy, particularly in liver tissue impaired by APAP overdose.\u003c/p\u003e\u003cp\u003eHowever, our research still has limitations. DDIT4 is upregulated under stimulation such as DNA damage and ROS activation caused by overdose of APAP [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Our data showed that Ddit4 was upregulated by circLima1 /miRNA-486a/b in the dmPGE2\u0026thinsp;+\u0026thinsp;APAP group, but both DDIT4 and miRNA-486a/b were upregulated in the APAP group(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE \u0026amp; Fig.\u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eA-B). Moreover, dmPGE2 enhanced the upregulation of DDIT4 in AILI mice. However, it did not significantly increase the expression of DDIT4 in normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). So we supposed that DDIT4 is also regulated by other molecules, which is remains unverified in the present work. In AILI mice treated with dmPGE2, downregulated miRNA-486a/b through circLima1 relived the suppression of upregulation of DDIT4 induced by APAP. In normal mice, there is no suppression of DDIT4 by miRNA-486a/b. So, the dmPGE2 did not upregulate DDIT4 directly. The mechanism for the regulation of DDIT4 will be clarified in our future research. Our research did not confirm sufficiency or exclusivity of DDIT4 in PGE2 protection on AILI mice, while other pathways might be involved in PGE2-induced autophagy enhancement. In vivo knockdown or overexpression experiments would better confirm the roles of DDIT4 or circLima1, which we will do in the future. Moreover, only 119 DEmRNAs are identified in PGE2 vs. APAP, compared to 1,437 in APAP vs. CON, which indicates PGE2 alleviates liver injury significantly but relatively few changes in gene expression. The APAP-induced liver injury involves multiple pathways and genes. DmPGE2 influences downstream pathways such as autophagy instead of upstream pathway such as GSH metabolism. So there are few changes in gene expression of dmPGE2\u0026thinsp;+\u0026thinsp;APAP vs. APAP. Further research is required to clarify its mechanism.\u003c/p\u003e\u003cp\u003eIn summary, this study investigated the protective effects of PGE2 against APAP-induced acute liver injury. We observed a significant increase in DDIT4 expression in both the APAP model and following PGE2 treatment. Notably, DDIT4 was found to induce autophagy by inhibiting mTORC1 activity, with its regulation mediated through the interaction of circLima1 and miRNA-486.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e The animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University. The animal studies were conducted according to the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). The euthanasia process strictly complied with the AVMA Guidelines for the Euthanasia of Animals and ARRIVE 2.0 requirements for methodological detail and reproducibility. Mice were anesthetized in an induction chamber with 5%-7% isoflurane (oxygen flow rate: 1\u0026ndash;2 L/min) and monitored every 10 seconds until loss of righting reflex, slowed regular breathing, and absence of tactile response (to avoid over-anesthesia). Euthanasia was performed via cervical dislocation under anesthesia, with confirmation of cessation of breathing/cardiac activity and absence of eyelid reflex (reverified after 30 seconds). All methods were carried out in accordance with relevant guidelines and regulations. The authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFundings\u003c/h2\u003e\u003cp\u003eThis work was financially supported by Science and Technology Major Projects of Zhejiang Province (NO. 2018C04016) and National Science and Technology Major Project of China (NO. 2018ZX10302206).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.C. designed the whole study. C.C., G.J. and G.XY. conducted animal experiments and analyzed the data. W.J. and W.SS. made relevant edits to the manuscript. H.M., W.GD. and R.YL. revised the manuscript. C.Z. and Z.HH. supervised and reviewed the manuscript. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files. 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Zonated induction of autophagy and mitochondrial spheroids limits acetaminophen-induced necrosis in the liver. \u003cem\u003eRedox Biol.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 427\u0026ndash;432 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Prostaglandin E2, Acetaminophen, circRNA, miRNA, DNA damage induced transcript 4, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-8110558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8110558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eStudies indicate that acetaminophen (APAP) overdose induces autophagy, thereby attenuating APAP-induced hepatocyte death. Prostaglandin E2 (PGE2) has been shown to reduce serum transaminase levels and prevent mortality in APAP-induced liver injury(AILI) in mice, and PGE2 downregulation reduces autophagy in skeletal muscle tissue of young mice. However, the hepatoprotective mechanisms of PGE2 have not been elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eHigh-throughput sequencing profiled circRNA, miRNA, and mRNA expression. Differentially expressed mRNAs were analyzed via protein-protein interaction networks and integrated with single-cell sequencing data from GEO. A circRNA/miRNA/mRNA network was computationally predicted (miRanda) and validated through miRNA mimic transfection and dual-luciferase assays. The role of Autophagy in PGE2-mediated protection was assessed by dual-fluorescent LC3 tracking and mitochondrial flux analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn the PGE2 group, a marked upregulation of circLima1 expression was detected, concomitant with downregulation of miR-486a-3p and miR-486b-3p. Transcriptomic analysis revealed significant DDIT4 upregulation in both APAP versus CON and PGE2 versus APAP comparisons. miRNA mimic transfection and dual-luciferase reporter assays were applied to confirm the regulatory axis involving circLima1/miR-486/DDIT4. Furthermore, we demonstrated that APAP\u0026thinsp;+\u0026thinsp;PGE2 enhances autophagic activity in hepatocytes, as shown by biochemical autophagic flux assays and LC3 dual-fluorescent lentivirus tracking. Pharmacological inhibition of autophagy with 3-methyladenine abolished the protective effects of dmPGE2 in AILI mice.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese results elucidate the role of PGE2 in alleviating AILI by enhancing autophagy through the circLima1/miR-486/DDIT4 regulatory axis network and provide a preliminary understanding of the molecular mechanisms underlying the hepatoprotective effects of dmPGE2 in AILI mice.\u003c/p\u003e","manuscriptTitle":"Prostaglandin E2 alleviates acetaminophen-induced liver injury through DDIT4-enhanced autophagy regulated by circLima1/miR- 486","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 18:22:09","doi":"10.21203/rs.3.rs-8110558/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T15:42:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-01T16:26:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176991493307253490848933188277249436392","date":"2025-12-22T22:22:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T23:44:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234429305365959541806908652267694237661","date":"2025-12-02T22:32:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T21:18:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-02T21:14:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-02T06:02:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-28T01:59:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-28T01:53:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"347c0c3a-af4c-48c6-987c-237119beabbf","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58989989,"name":"Biological sciences/Cell biology"},{"id":58989990,"name":"Health sciences/Diseases"},{"id":58989991,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-30T18:54:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 18:22:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8110558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8110558","identity":"rs-8110558","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}