HIF-1α/YAP induces interleukin 1β autocrine in hepatocytes, leading to hepatocytic fibrosis: adaptive fibrosis in nonalcoholic fatty liver

HIF-1α/YAP induces interleukin 1β autocrine in hepatocytes, leading to hepatocytic fibrosis: adaptive fibrosis in nonalcoholic fatty liver | 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 HIF-1α/YAP induces interleukin 1β autocrine in hepatocytes, leading to hepatocytic fibrosis: adaptive fibrosis in nonalcoholic fatty liver Jian-Gang Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6538340/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nonalcoholic fatty-liver disease (NAFLD) has become the most common type of chronic liver disease and a global public-health problem. Fibrosis is always secondary to nonalcoholic steatohepatitis (NASH), an inflammatory fibrotic process in which hepatic stellate cells (HSCs), macrophages, and hepatic sinusoidal endothelial cells (HSECs) may all play roles. However, steatosis-associated fibrosis ( i.e. , NAFL fibrosis) and the key role of hepatocytes in liver fibrosis are not well understood. In this study, we explored the mechanisms of high-lipid microenvironment (HLME)-induced NAFL hepatocytic fibrosis and interleukin 1β (IL-1β) regulation through the hypoxia-inducible factor 1α (HIF-1α)/yes-associated protein (YAP) signaling pathway. C57BL/6J mice were fed a high-fat diet (HFD) for 1–3 months. Moreover, Hep G2 and Hepa 1-6 cells were treated with palmitic acid (PA) for 24 h to establish a NAFL model. Furthermore, we performed immunohistochemical (IHC) staining, western blot (WB) analysis, real-time quantitative polymerase chain reaction (RT-qPCR), silencing, and co-immunoprecipitation (Co-IP) experiments. The results showed that a HFD or PA induced high expression of collagen type I α1 chain and type III α1 chain (COL1A1, COL3A1), matrix metalloproteinase 2 (MMP-2), HIF-1α, YAP, and IL-1β in hepatocytes compared with the control group. IL-1β receptor was highly expressed in hepatocytes, and HIF-1α/YAP regulated the expression of IL-1β. In conclusion, hepatocytes contributed to NAFL fibrosis by IL-1β autocrine regulation via HIF-1α/YAP signaling in a HLME. Health sciences/Molecular medicine Health sciences/Medical research/Experimental models of disease Health sciences/Pathogenesis/Inflammation nonalcoholic fatty liver IL-1β autocrine hepatocytic fibrosis HIF-1α/YAP HLME Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Points Fibrosis occurred at the NAFL stage and originated from hepatocytes rather than hepatic stellate cells. High-lipid, hypoxic, and mechanical stress was present, with lipid deposition in hepatocytes. High lipid levels were an independent factor. A high-fat diet or palmitic acid induced high expression of COL1A1, COL3A1, MMP-2, HIF-1α, YAP, and IL-1β in hepatocytes. HIF-1α/YAP regulated expression of IL-1β, the receptor of which was highly expressed in hepatocytes; IL-1β in turn regulated hepatocytic fibrosis via autocrine regulation. Introduction Economic development and lifestyle changes have contributed to nonalcoholic fatty-liver disease (NAFLD), increasingly becoming a major global-health problem [ 1 , 2 ]. NAFLD generally refers to liver damage with histological changes similar to those seen in alcoholic fatty liver, but its cause is related to metabolic dysfunction; therefore, it is also referred as to metabolic dysfunction–associated fatty liver disease (MAFLD) [ 3 , 4 ]. High-fat diets (HFDs), obesity, and insulin resistance (metabolic syndrome [MetS]) are the main causes of NAFLD [ 2 , 5 – 7 ]. Pathologically, NAFLD progresses from nonalcoholic fatty liver (NAFL; also called steatosis) to nonalcoholic steatohepatitis (NASH), and then to fibrosis, which occurs secondary to NASH [ 2 , 8 ]. Lipid deposition causes oxidative stress (OS) and peroxidation in mitochondria [ 9 , 10 ], leading to hepatocytic damage. This endogenous injury further activates Kupffer cells to release inflammatory factors such as transforming growth factor-β (TGF-β), inducing inflammatory response (NASH) [ 11 ] and further activating hepatic stellate cells (HSCs) as myofibroblasts to secrete extracellular matrix (ECM), leading to fibrosis [ 12 – 14 ]. HSCs are considered the main source of ECM and play a crucial role in the formation of NASH fibrosis [ 15 – 17 ]. The TGF-β/Smad signaling pathway is a key mechanism in HSC activation [ 18 – 21 ]. However, little is known about fibrosis during the NAFL stage, which is absent in oxidative injury, inflammatory-cell reactions, and HSC activation. Stress (a microenvironment unsuitable for the survival of liver cells) and adaptation, rather than injury, are the major environmental features faced by NAFL hepatocytes. With lipid deposition, cells enlarge, and the sinus is compressed remarkably. At least three kinds of stress applied to hepatocytes can be deduced during NAFL progression: hypoxic, mechanical, and high-lipid. During chronic hypoxia, a series of transcription factors (TFs) are activated, such as hypoxia-inducible factor (HIF), HIF-1α–like factor (HLF), activator protein 1 (AP-1), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). These TFs regulate expression of target genes to facilitate cell survival in low-oxygen environments [ 22 , 23 ]. In mammals, HIF is the main TF in hypoxic stress and targets a wide range of genes, including those related to hypoxic adaptation, development of inflammation, and tumor growth. HIF is a “molecular switch” that regulates the expression of low-oxygen target genes in the body and is one of the most important pathways mediating decreases in cellular-oxygen concentration [ 24 ]. Recent studies have shown that HIF-1 and HIF-2 can regulate the expression of interleukin 1α (IL-1α) and produce different effects in different cell lines [ 25 ]. This finding establishes a possible link between hypoxic and inflammatory factors. We speculate that absent inflammatory-cell involvement, inflammatory factors might be produced by parenchymal/stromal cells induced by hypoxic stress. These inflammatory factors have two important functions: they can (1) respond to hypoxic signals and (2) activate HSCs to produce fibrotic effects. IL-1 is likely such an inflammatory factor. IL-1β is an early-discovered inflammatory factor that promotes collagen synthesis in osteoblasts and HSCs, enhances the synthesis and release of TGF-β, induces the expression of adhesion factors and their receptors, promotes production of tissue inhibitor of metalloproteinases (TIMP), and inhibits ECM degradation. Recently, IL-1β has been found to play an important role in the progression of NAFL to NASH and fibrosis [ 26 – 29 ], but its source and specific mechanism of action have not yet been discovered. Yes-associated protein (YAP) is a TF co-stimulatory molecule and also an effector molecule in multiple signaling pathways. Highly expressed in activated fibroblasts, YAP promotes interstitial-cell activation by regulating the expression of various activation-related genes, including growth factors [ 30 , 31 ]. Research has found that YAP undergoes nuclear translocation in the early stages of liver injury [ 32 ], and we speculate that it may be involved in the process of NAFL fibrosis. In this article, we report that stress and adaptation induced NAFL hepatocytic fibrosis, which was mediated by HIF-1α and YAP regulation and IL-1β autocrine regulation. Materials and methods Animals We purchased male adult C57BL/6J mice (4 weeks old) from the Experimental Animal Center of Lanzhou University (Lanzhou, China; No. SCXK 2018-0002) and housed them individually in a specific pathogen–free (SPF) environment (12-h light/dark cycle, 22°C, and 65–70% humidity) with free access to food and water. After 1 week of adaptive feeding with a chow diet (CD; 3.16 kcal/g, 11.4% kcal from fat, 64% from carbohydrates, 25% from protein), we fed the animals either CD (n = 9) or a mixture of CD and high-fat feed (n = 15; 5.58 kcal/g, 66.5% kcal from fat, 22% from carbohydrates, 11% from protein) for 1, 2, or 3 months. The HFD was prepared as previously reported [ 33 ]; briefly, lard, egg yolk powder (Jinjianli, Beijing, China), CD powder (KeAo Corp., Beijing, China), and sucrose were mixed at a ratio of 1:1:1.5:0.4. The mice’s diets were changed weekly. All animals were sacrificed in compliance with the Guideline for the Ethical Review of Laboratory Animal Welfare (No. GB/T35892-2018). This study was approved by the Medical Ethics Committee of Lanzhou University (Lanzhou, China; No. jcyxy20190302) and conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Sample collection We anesthetized mice with pentobarbital sodium (5 mg/100 g body mass), followed by three skin sterilizations using 75% ethanol and iodophor. After the abdominal cavity was opened, animals were sacrificed via inferior vena cava blood drainage. We removed the livers, washed them in pre-cooled phosphate-buffered saline (PBS; #P1010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and dried them using filter paper. The left lateral lobe was sampled for free fatty acids (FFAs) and low-density lipoprotein cholesterol (LDL-C) testing, the middle lobe was fixed in 50 mL of 4% neutral formaldehyde for histological testing (including HE, oil red O, and IHC staining), and the rest of the liver was stored at − 80°C after a quick freeze in liquid nitrogen. Free fatty acid and low-density lipoprotein cholesterol determinations We detected FFA concentrations in liver tissues using a non-esterified fatty acid (NEFA) reagent kit (#KG7571; KeyGEN Biotech, Nanjing, China) per manufacturer’s instructions. Briefly, liver tissues were homogenated in 9× volume ethanol on ice for 2 min and centrifuged at 2500 rpm and 4°C for 10 min, after which the supernatant was collected and reacted with copper reagent to acquire a fatty acid copper–salt solution. After adding the color reagent, we measured the absorbance (optical density [OD]) of the solution at 440 nm using an ultraviolet visible (UV/Vis) spectrophotometer (SPECORD 50 Plus; Analytik Jena GmbH + Co. KG, Jena, Germany). Distilled water and 1000 µM palmitic acid (PA; #SLCC6727; Sigma-Aldrich, Burlington, MA, USA) solution were used as blank and standard controls, respectively, and chloroform was used for zero adjustment. We calculated FFA concentration as follows: [(OD sample − OD blank )/(OD standard − OD blank )] × standard concentration (mM) × volume (L)/weight (g) We detected LDL-C concentrations in liver tissues using a LDL-C reagent kit (#A113-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The tissue homogenate supernatant was incubated with reagents at 37°C for 5 min, and OD values were measured on an enzyme-linked immunosorbent assay (ELISA) reader (Synergy NEO2; Agilent Technologies, Inc., Santa Clara, CA, USA). LDL-C concentration was calculated as follows: [(ΔOD sample − ΔOD blank )/(ΔOD standard − ΔOD blank )] × standard concentration (mM) × volume (L)/weight (g) Cell culturing and high lipid–microenvironment in vitro model We purchased Hep G2 (#CL-0103) and Hepa 1–6 (#CL-0105) cells from Pricella Life Science and Technology Co., Ltd. (Wuhan, China). Cells were maintained in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; #SH30022.01; HyClone [Cytiva, Marlborough, MA, USA]) with 10% fetal bovine serum (FBS; #FBSST-01033; OriCell; Cyagen Biosciences, Jiangsu, China) and 1% penicillin–streptomycin mixture (100×; #P1400; Solarbio) at 37°C with 5% CO 2 . To establish a high-lipid microenvironment (HLME) in vitro model, we cultured cells in starvation medium (DMEM with 1% FBS) for 12 h after they reached 60% confluence, and then subjected them to 2 ml PA/bovine serum albumin (BSA; #WXBD1900V; Sigma-Aldrich, USA) medium for 24 h, with or without HIF-1α inhibitor PX-478 (#HY-10231, MedChemExpress, USA). Hep G2 cells were cultured in 0 µM (0.2% BSA), 200 µM, 400 µM, and 600 µM PA; Hepa 1–6 cells were cultured in 0 µM (0.1% BSA), 100 µM, and 200 µM PA. We produced PA/BSA medium by mixing 10 mM PA/BSA solution with DMEM at different concentrations; PA/BSA solution was produced by mixing 20% BSA solution (1.20 g BSA dissolved in 6 mL DMEM) with 20 mM PA solution (0.037 g PA in 6 mL of 0.1 sodium hydroxide) and stirring at 50°C for 30 min. Hematoxylin and eosin staining We fixed liver tissues in 4% paraformaldehyde (PFA; #G1102; Solarbio, China) and embedded them in paraffin. The sections were then deparaffinized with xylene and ethanol and stained with Harris hematoxylin and eosin (#G1150; Solarbio, China) using conventional methods. The slices were then observed under a microscope. Oil Red O staining We fixed liver tissues in 4% paraformaldehyde (PFA; #G1102; Solarbio) and embedded them in Tissue-Tek O.C.T. Compound (#4583; Sakura Finetek, Tokyo, Japan), followed by frozen sectioning on a freezing microtome (#CM1950; Leica, Wetzlar, Germany). Sections were treated with 60% isopropanol and stained with Oil Red O (ORO; #1320-06-5; Shandong Xiya Chemical Technology Co., Ltd., Linyi, China) for 15 min, after which nuclei were counterstained with hematoxylin (#S2100; Solarbio). We observed sections under a Nikon-ECLIPSE 80i/DS-Ri2/NIS-Elements D microscope (Nikon, Tokyo, Japan) and analyzed images using ImageJ software v6.0 (National Institutes of Health [NIH], Bethesda, MD, USA). PA-treated cell slides were fixed at room temperature (RT) for 15 min with 4% PFA and subjected to the same staining process as tissue sections. Immunohistochemical staining We then dewaxed the paraffined sections using xylene and gradient alcohol. Antigens were retrieved using sodium citrate (pH 6.0; #C1010; Solarbio), endogenous peroxidase was inhibited with H 2 O 2 (#SP-9001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China), and nonspecific background antigens were blocked with goat serum (#SP-9001; Beijing Zhongshan Jinqiao) at 37°C for 30 min. We then incubated the slides with primary antibodies (Abs) overnight at 4°C and secondary Abs for 15 min at RT. The reaction was visualized using diaminobenzidine (DAB; 1:40; #ZLI-9018; Beijing Zhongshan Jinqiao), and nuclei were counterstained using hematoxylin (#S920; Solarbio). Primary Abs were collagen III (COLIII) rabbit polyclonal (1:600; #22734-1-AP) and interleukin 1 receptor type I (IL1R1) rabbit polyclonal (1:500; #27348-1-AP; both Proteintech, Wuhan, China). The secondary Ab was goat anti-rabbit (#SP-9001; Beijing Zhongshan Jinqiao). Slides were observed under the Nikon ECLIPSE microscope and analyzed using ImageJ and GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). Cellular immunofluorescence Cells slides were fixed with 4% neutral formaldehyde for 15 min, permeated with 0.5% Triton X-100 (#T8200; Solarbio) for 15 min, and then pretreated with goat serum (#SP-9001; Beijing Zhongshan Jinqiao) for 30 min at RT. We then incubated the slides with primary Abs overnight at 4°C and fluorescent-labeled secondary Abs for 30 min at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; #C0065; Solarbio) at RT for 5 min and observed under the Nikon ECLIPSE microscope at UV-2A (EX330-380, BA410) for DAPI, B2A (EX450-490, BA520) for YAP, and G2A (EX510-560, BA590) for HIF-1α. We analyzed fluorescence intensity using ImageJ. Primary Abs were YAP (1:400) mouse monoclonal (#66900-1-Ig), HIF-1α (1:200) rabbit polyclonal (#20960-1-AP), both from Proteintech, and IL1R1 (1:200) rabbit polyclonal (#ab106278; Abcam, Cambridge, UK). Secondary Abs were Alexa Fluor 488 goat anti-mouse (1:800; #SA0006-1) and Alexa Fluor 594 goat anti-rabbit (1:800; #SA0006-4; both Proteintech). Real-time polymerase chain reaction We extracted total ribonucleic acid (RNA) via the phenol chloroform method. Briefly, liver tissues or cells were homogenated in RNAiso Plus (#9108/9109; TaKaRa, Shiga, Japan) lysis buffer (30 mg:100 mL), and total nucleic acid was extracted using 200 µL chloroform. After centrifugation at 12,000 g and 4°C for 15 min, we extracted RNA and precipitated the solution using isopropanol. RNA concentration was detected using a Thermo Scientific NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A reverse transcription kit (#RR047A; TaKaRa) was used per manufacturer’s instructions to remove genomic deoxyribonucleic acid (gDNA) and acquire efficient complementary DNA (cDNA). We performed real-time quantitative polymerase chain reaction (RT-qPCR) on a QuantStudio 3 real-time fluorescence quantitative PCR instrument with QuantStudio Design & Analysis Software (Thermo Fisher, Waltham, MA, USA), using a TB Green Premix Ex TaqII kit (#RR820A; TaKaRa). The three-step procedure was followed: hold stage: 95°C for 30 s; PCR stage: 95°C for 5 s, 60°C for 30 s; melt curve stage: 95 ° C for 15 s, 60 ° C for 1 min, 95 ° C for 1 s, 25 ° C for 5 min). We calculated gene expression levels using the 2 −ΔΔ Ct method: Δ Ct = Ct target gene – MEAN Ct internal reference , ΔΔCt = Δ Ct – MEAN Δ Ct control target gene ) Primers were as shown in Table 1 . Table 1 Primers for real-time polymerase chain reaction detection Mouse Forward Reverse Acta2 5′-AACACGGCATCATCACCAAC-3′ 5′-CACAGCCTGAATAGCCACATAC-3′ Actin 5′-GGCTGTATTCCCCTCCATCG-3′ 5′-CCAGTTGGTAACAATGCCATGT-3′ Col1a1 5′-TTGGTCCCGCTGGCAAGAATG-3′ 5′-CTGTCACCTTGTTCGCCTGTCTC-3′ Col3a1 5′-CCTCCCAGAACATTACATACC-3′ 5′-TTCGCCTTCATTTGATCCC-3′ Hif1α 5′-GTCCCAGCTACGAAGTTACAGC-3′ 5′-CAGTGCAGGATACACAAGGTTT-3′ Il1β 5′-TGGTGTGTGACGTTCCCATT-3′ 5′-CAGCACGAGGCTTTTTTGTTG-3′ Yap 5′-ACCCTCGTTTTGCCATGAAC-3′ 5′-TGTGCTGGGATTGATATTCCGTA-3′ Il1r1 5′-CTGCTGTCGCTGGAGATTGAC-3′ 5′-TTGGCAGGTACAAACCAAAGAT-3′ Timp 5′-TGGCATCTGGCATCCTCTTG-3′ 5′-CGCTGGTATAAGGTGGTCTCG-3′ Human ACTA2 5′-CCGGGACTAAGACGGGAATC-3′ 5′-TTGTCACACACCAAGGCAGT-3′ ACTIN 5′-CCTGGCACCCAGCACAAT-3′ 5′-GGGCCGGACTCGTCATAC-3′ COL1A1 5′-TGTTCAGCTTTGTGGACCTCCG-3′ 5′-CCGTTCTGTACGCAGGTGATTG-3′ COL3A1 5′-GAAGATGTCCTTGATGTGC-3′ 5′-AGCCTTGCGTGTTCGATAT-3′ HIF1α 5′-ACGAGAGGTTCCCTAATTTCCA-3′ 5′-ATGCCACCAGTACATTGGGAT-3′ IL1β 5′-AGCCATGGCAGAAGTACCTG-3′ 5′-CCTGGAAGGAGCACTTCATCT-3′ YAP 5′-GAACAATGACGACCAATAGCTC-3′ 5′-TAGTCCACTGTCTGTACTCTCA-3′ IL-1R1 5′-GGCTGAAAAGCATAGAGGGAAC-3′ 5′-CTGGGCTCACAATCACAGG-3′ TIMP1 5′-CACTGTTGGCTGTGAGGAA-3′ 5′-AAGGTGACGGGACTGGAA-3′ Rat Col1a1 5′-CTGGCGCTTCAGGTCCAAT-3′ 5′-GGCACCATCCAAACCACTGA-3′ Col3a1 5′-CGAGGTAACAGAGGTGAAAGA-3′ 5′-AACCCAGTATTCTCCGCTCTT-3′ Western blot We extracted total protein using radioimmunoprecipitation assay (RIPA)/phenylmethylsulfonyl fluoride (PMSF) cell lysate (100:1; respectively #R0010 and #P0100; Solarbio). Total protein concentration was measured using bicinchoninic acid (BCA; #PC0020; Solarbio). We separated protein samples via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% separating gel), transferred them onto an Immobilion polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich), and probed them with the above-indicated primary Abs at 4°C overnight, followed by the appropriate secondary horseradish peroxide (HRP)–conjugated immunoglobulin G (IgG) Ab at RT for 1 h. The following Abs were used: primary: rabbit polyclonal COLI (1:800; #14695-1-AP), COLIII (1:800; #22734-1-AP), HIF-1α (1:1000; #20960-1-AP), YAP (1:1000; #13584-1-AP), actin alpha 2 smooth muscle (ACTA2; 1:6000; #14395-1-AP), MMP-2 (1:1000; #10373-2-AP), β-actin (1:10000; #66009-1-Ig), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:6000; #10494-1-AP; all from Proteintech, Chicago, IL, USA), phosphorylated YAP (p-YAP; 1:1000; #13008S), p-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) (1:1000; #4511T), p–NF-κB p65 (Ser536) (93H1) (1:1000; 3033T; all three from Cell Signaling Technology [CST], Danvers, MA, USA); mouse monoclonal HIF-1α (#ab8366; Abcam, Cambridge, UK), YAP (1:400; #66900-1-Ig; Proteintech); rabbit polyclonal COLIII (1:1000; #ab184993), rabbit polyclonal COLI (1:1000; #ab260043), and rabbit polyclonal IL1R1 (1:500; #ab106278; all three from Abcam); secondary: HRP-labeled goat anti-rabbit (1:5000; #RS0002; ImmunoWay Biotechnology Co., Plano, TX, USA). Protein bands were visualized using an electrochemiluminescence (ECL) kit (Super ECL Detection Reagent; #3608ES60; Yeasen Biotechnology Co., Ltd., Shanghai, China) and a chemiluminescence imaging system (FUSION Solo 6S Edge; Vilber, Marne-la-Vallée, France). We used ImageJ to analyze grayscale band values. Immunoprecipitation We added HIF-1α (0.2–2.0 µg; #ab8366; Abcam), YAP (0.2 − 2.0 µg; #66900-1-Ig; Proteintech; Input group) or mouse IgG (1–2 µg; #sc-2025; Santa Cruz Biotechnology, Dallas, TX, USA; IgG group) to total protein solution (100–500 µg in 1 mL lysate) and incubated the mixture at 4°C on a shaker for 12 h. The Ab–antigen complex was then pulled down by adding 20 µL of Protein A/G PLUS Agarose (#sc-2003; Santa Cruz) to the mixture and incubating it on a shaker at 4°C for 6 h. After centrifugation at 4°C and 2500 rpm for 5 min, we collected the precipitate as a magnetic-bead Ab–antigen complex and detected the target proteins via western blot (WB). Transient transfection of small interfering RNA Hep G2 cells were transfected with short interfering RNA (siRNA) when cell density reached 60% confluence. We mixed GP Transfection Mate transfection reagent (5.5 µL; GenePharma, Shanghai, China) in 200 µL DMEM and 20 µM siRNA oligo (7.5 µL) in 200 µL DMEM at RT for 15 min to produce a complex transfection solution, which was then added to 1.6 mL preheated culture medium. Cells were transfected at 37°C and 5% CO 2 for 6 h. After another 60 h of cultivation, we used WB to detect protein expression. Sequences of HIF-1α, GAPDH, and negative control (NC) are described in Table 2 . The experimental designation was as follows: positive control (siGAPDH), NC, transfection reagent (GP Transfer Mate), treatment control (0 µM PA), experimental (200 µM PA), treatment control + siHIF-1α, and experimental (200 µM PA) + siHIF-1α. Table 2 Sequences of human-gene siRNA Human Sense Antisense HIF-1α 5'-CAGGCCACAUUCACGUAUATT-3' 5'-UAUACGUGAAUGUGGCCUGTT-3' GAPDH 5'-UGACCUCAACUACAUGGUUTT-3' 5'-AACCAUGUAGUUGAGGUCATT-3' NC 5'-UUCUCCGAACGUGUCACGUTT-3' 5'-ACGUGACACGUUCGGAGAATT-3' HIF-1α, hypoxia-inducible factor 1α; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC, negative control. Bioinformatic analysis We searched the genomic information for HIF-1α and the IL1β using the National Center for Biotechnology Information [NCBI] database (Bethesda, MD, USA) and predicted the binding relationship between HIF-1α and IL1β using AlphaFold 3, the University of California at Santa Cruz (UCSC) Genome Database, the Gene Transcription Regulation Database (GTRD), Gene Expression Profiling Interactive Analysis (GEPIA), and the Jackson-based Sequence Analysis Parameter (JASPAR) database. Statistical analysis All data were expressed as mean ± standard deviation (SD). We conducted all statistical analyses using SPSS v25.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8. Comparisons between two groups were conducted using Student’s t test, and those between multiple groups were conducted via one-way analysis of variance (ANOVA). We analyzed OD values of immunofluorescence (IF) images using ImageJ software. All experiments were repeated at least three times, with three technical replicates set each time. Differences were considered statistically significant at P < 0.05. Results 1. A high -lipid microenvironment induced the NAFL hepatocytic fibrotic phenotype Compared with CD mice, the body and liver weights of HFD mice significantly increased at all three time points and showed a time-dependent pattern ( P < 0.05; Fig. 1A). Morphologically, lipid was deposited in hepatocytes to form droplets in various numbers and sizes (from micro- to macrovesicular), the severity of which was time dependent ( P < 0.05; Figs. 1B–C). We did not observe hepatocytic necrosis or inflammatory-cell infiltration, which suggested establishment of NAFL (Fig. 1B). Compared with CD mice, FFA and LDL-C levels in HFD mouse livers significantly increased ( P < 0.05; Table 3, Fig. 1D); in HFD1 mice in particular, liver LDL-C levels were significantly elevated ( P < 0.05), suggesting that the HFD induced a high-fat microenvironment and abnormal lipid metabolism in liver cells. Hepatocytes were enlarged and hepatic sinusoids were compressed to different degrees, accompanied by gray-yellow liver color (Fig. 1B), indicating decreased blood perfusion of the hepatic sinusoids and a relatively hypoxic microenvironment. HFD mice also showed obvious fibrosis. Compared with CD mice, levels of Col1a1 and Col3a1 messenger RNA (mRNA) significantly increased in HFD mice ( P < 0.05; Fig. 2A), and the level of Timp1 in HFD3 mice significantly increased ( P 0.05). In addition, MMP-2 protein levels significantly increased in HFD mice ( P ˂ 0.01; Fig. 2B). Immunohistochemical (IHC) staining showed that COL3A1 was deposited around both sinusoids and hepatocytes, presenting a typical grid-like appearance (Fig. 2C). Table 3. FFA and LDL-C levels in C57BL/6J mouse liver tissue Group FFA (mmol/g) LDL-C (mmol/g) CD1 15.68 ± 0.40 0.84 ± 0.11 HFD1 18.31 ± 1.48 * 2.29 ± 0.30 *** CD2 14.42 ± 3.77 1.20 ± 0.09 HFD2 22.16 ± 3.06 * 2.89 ± 0.44 ** CD3 14.86 ± 0.96 1.01 ± 0.02 HFD3 17.39 ± 0.67 * 2.98 ± 0.58 * Note: Compared with CD mice at the same time points. * P < 0.05; ** P < 0.01; *** P < 0.001. 2. Nonalcoholic fatty-liver fibrosis originated from activation of hepatocytes, not of hepatic stellate cells HSCs are the main source of liver fibrosis. However, HSC activation is always secondary to hepatocytic injury and inflammation such as NASH; such lesions are absent in NAFL. Therefore, we next investigated the source of fibrosis in NAFL. We detected expression of α-smooth muscle actin (α-SMA), the marker of HSC activation, in liver tissues. The results showed that compared with CD mice, mRNA and protein levels of Acta2 in the livers of HFD mice at each time point were not statistically significant ( P > 0.05; Figs. 2A–B), which suggested absence of HSC activation during NAFL fibrosis. We cultured Hep G2 and Hepa 1-6 cells with different concentrations of PA for 24 h and then detected lipid deposition and fibrotic phenotype. The results showed that PA treatment significantly increased lipid droplet deposition in cytoplasm, which increased with PA concentration ( P < 0.05; Fig. 3A). PA treatment promoted the expression of COL1A1 , COL3A1 , and TIMP1 in Hep G2 cells and Col3a1 , Timp1 and Acta2 in Hepa 1-6 cells at different concentrations ( P 0.05; Fig. 3B), suggesting that fibrosis originated from hepatocytes in NAFL. 3. IL-1β autocrine mechanisms in hepatocytic fibrosis in a high-lipid microenvironment IL-1β is the most potent pro-fibrotic factor in NASH. Therefore, we further assessed whether hepatocytic fibrosis during NAFL was associated with IL-1β. We showed that, compared with CD mice, mRNA levels of Il1β were significantly upregulated in HFD2 and HFD3 mice ( P ˂ 0.05; Fig. 4A). In our in vitro experiment, mRNA expression of IL-1β was significantly higher in PA groups than in the control group ( P < 0.05; Fig. 4B). We detected the effect of IL-1β on hepatocytes. The results showed that the IL-1β receptor IL1R1 was distributed in hepatocytes and was also upregulated during PA treatment (Figs. 4C–D). Recombined IL-1β significantly upregulated protein expression of COLI and COLIII in Hep G2 cells ( P < 0.05; Fig. 5E). These results indicated that hepatocytic fibrosis in NAFL was related to IL-1β autocrine mechanisms. 4. The high -lipid microenvironment induced HIF-1α/YAP upregulation in hepatocytes With lipid deposition, the hepatocytes enlarged and the sinuses were compressed, meaning that relative hypoxic and mechanical stress might have been induced in the liver. Since HIF-1α and YAP are the respective biomarkers of hypoxia and mechanical stress and play key roles in lipid metabolism and OS in NAFLD, we detected the HIF-1α/YAP signal in the liver. Expression of Hif1α significantly increased in HFD1 and HFD2 mice but decreased in HFD3 mice ( P ˂ 0.05), while that of Yap was also upregulated in HFD mice ( P ˂ 0.01; Fig. 5A). These results suggested the possibility of hypoxic and mechanical stress in the livers of NAFL mice. We further cultured Hep G2 and Hepa 1-6 cells in a high-lipid environment (HLME). The results showed that (a) expression of HIF1α (human) in Hep G2 cells and Hif1α (mouse) in Hepa 1-6 cells was significantly higher than in the control group, and (b) YAP (human) in Hep G2 cells or Yap (mouse) in Hepa 1-6 cells showed significantly higher gene expression in the PA groups than in the control group (both P < 0.05; Fig. 5B). In Hep G2 cells, protein levels of HIF-1α significantly increased in each PA treatment group, with statistical significance in the 600-μM group ( P < 0.05; Fig. 5B). There was no significant change in YAP protein level in the PA treatment groups (Fig. 5B); however, the protein level of p-YAP (a marker of YAP degradation) decreased significantly ( P < 0.05), indicating activation of YAP (Fig. 5B). In Hepa 1-6 cells, expression of HIF-1α and YAP protein in cells treated with different concentrations of PA significantly increased ( P < 0.05). These results suggested that PA was an independent factor producing hypoxic and mechanical stress in hepatocytes. We then detected HIF-1α/YAP interaction. IF results showed that PA induced an increase in the expression of HIF-1α and YAP proteins in a dose-dependent manner, and HIF-1α co-localized with YAP in the nucleus (Fig. 6A). The Co-IP experiment suggested an interaction between HIF-1α and YAP (Fig. 6B), and expression of YAP protein was reduced in the siHIF-1α group (Fig. 6C). These results indicated that YAP expression was regulated by the TF HIF-1α. 5. HIF-1α targeted the Il1 β gene in hepatocytes Both HIF-1α signaling and IL-1β secretion were upregulated in the HLME during NAFL development. Therefore, we further assessed HIF-1α’s regulation of Il1 β gene expression. First, we conducted a bioinformatic analysis of the regulatory relationship. Human Il1 β is located on chromosome 2 at positions 112829751–112836779. AlphaFold 3 predicted the spatial structure of HIF-1α binding with the promoter of human Il1 β. The binding relationship indicated a high probability, as indicated by the UCSC Genome Database (score = 265) and the GEPIA database ( r = 0.43, P < 0.05). The JASPAR database predicted 12 potential binding sites within the 2000-bp upstream and 100-bp downstream intervals of Il1 β , with a relative profile score threshold of 80% (MA0259.2), which suggested that HIF-1α could bind to the IL1β promoter to stimulate IL-1β expression (Fig. 7A). Our in vitro experiment suggested that the HIF-1α inhibitor PX-478 significantly inhibited expression of IL-1β (Fig. 7B) and activation of the downstream pathway and collagen expression during PA treatment (Fig. 7C). Discussion NAFLD is a multifactorial, multistage, and slowly progressing disorder. Due to its strong insidious onset and few disease-related clinical symptoms in the NAFL stage, lipid metabolism and the fibrotic mechanism in NAFLD are still not fully understood. NASH and HSCs are traditionally considered the sources of fibrosis [ 34 ]. This study aimed to investigate hepatocytes in the NAFL stage as such a source. In this article, we reported the parenchymal-cell origin of fibrosis during NAFL. To our knowledge, this is the first report of fibrosis occurring in the NAFL stage. Compared with injury- and inflammation-activated fibrosis (repair fibrosis), this fibrosis, which derived from parenchymal-cell adaptation (adaptive fibrosis), was mild. This parenchymal fibrosis was initiated by microenvironmental stress and the inflammatory mediator autocrine. Inflammatory cells always infiltrate secondary to cell or tissue injury or necrosis, such as hepatitisvirus, alcohol-induced injury, or carbon tetrachloride–induced injury. However, in many subclinical situations ( e.g. , microenvironmental change), parenchymal cells are in a state of adaptation rather than necrosis or injury. Inflammation is considered absent at this stage, and therefore many related phenomena cannot be well interpreted through the lens of inflammation. Our results suggested a state of “molecular inflammation” in which inflammatory mediators were active but leukocytic infiltration was absent. Inflammatory molecules were secreted by parenchymal cells during stress adaptation and epigenetic changes. To distinguish from injury-induced inflammation dependent on leukocytic infiltration, this molecular inflammation dependent on parenchymal-cell adaptation could be termed “adaptive inflammation.” According to our results, adaptive inflammation triggered the same signaling pathway to help parenchymal cells self-regulate and thus adapt to changes in the environment. In this study, the HLME induced hepatocytes to autosecrete IL-1β, resulting in collagen secretion. Many studies have reported that parenchymal cells, including various tumor cells [ 35 , 36 ], secrete inflammatory mediators under physiological conditions [ 37 – 39 ] or during adaptation to stress [ 40 , 41 ]. Therefore, molecular inflammation should be considered a common pathological phenomenon that is often ignored. This concept could help introduce the idea of inflammatory mechanisms to various pathological adaptive processes. On the other hand, inflammatory mediators mainly activate other immunocytes to initiate inflammation cascade reactions or activate interstitial cells to initiate the repair process through paracrine or endocrine mechanisms. In some cases, inflammatory mediators can also regulate themselves by positive or negative feedback. Herein, we showed that the inflammatory mediator of parenchymal cells acted on itself, mainly through autocrine regulation, because HSCs did not appear to be activated by the high level of IL-1β during NAFL development. This study took microenvironmental change as the breakthrough point of the NAFL fibrotic mechanism. The stress of continuous lipid deposition could cause development of a microenvironment unsuited for cell survival. Enlarged hepatocytes can compress the sinus and block influx of red blood cells. In this study, we detected FFA concentrations and HIF-1α and YAP expression in liver tissues. The results supported the existence of hyperlipid, hypoxic, and mechanical-compression stress. Interestingly, the results of the in vitro experiments indicated that PA (high lipid) was an independent factor inducing high expression of HIF-1α and YAP in hepatocytes. HIF is a key oxygen-regulated transcription activator, and HIF-1α is an important functional component that is extremely sensitive to oxygen concentration [ 42 ]. The latter plays an important role in regulating cellular responses to hypoxia. Experiments have shown that HIF-1α is also induced in NAFLD hepatocytes by different causes [ 43 ]; for example, expression of HIF-1α increases in hepatocytes from obese patients with NAFLD [ 44 ]. Studies have also found that HIF-1α in hepatocytes mediates the development of NAFLD fibrosis in NAFLD mouse models [ 45 ], such as by promoting NAFLD fibrosis via activation of the phosphatase and tensin homolog (PTEN)/p65 signaling pathway [ 46 ]. Multiple downstream target genes of HIF-1α are involved in processes of disease regulation, including glycolysis, erythropoiesis, angiogenesis, and vascular remodeling. Previous studies have shown that IL-1β is a direct target of HIF-1α [ 47 , 48 ] and both human and mouse IL-1β promoter regions carry multiple HIF-1α–binding sites [ 49 ]. Additionally, the HIF-1α/IL-1β signaling pathway enhances liver cancer epithelial–mesenchymal transition (EMT) [ 50 ]. In this article, we showed that HIF-1α activated IL-1β expression and the downstream inflammatory pathway in hepatocytes. YAP, as a transcription regulatory cofactor, plays a regulatory role in the Hippo signaling pathway [ 51 ]. Mechanical signaling is the second important factor in regulation of YAP function [ 52 ]. YAP exerts regulatory effects by nuclear localization and binding to members of the transcriptional enhancer factor domain family (TEAD) [ 53 , 54 ]. We showed that HIF-1α’s activation of IL-1β expression was associated with YAP binding and activity. IL-1β is a key inflammatory factor in the NASH stage and is closely related to NAFLD fibrosis [ 55 ]. During the NASH stage, pro-inflammatory cytokines and chemokines are produced by hepatocytes and Kupffer cells. No inflammatory response is believed to occur in the liver during this stage. Our results demonstrated that in the HFD-induced NAFL stage, HIF-1α/YAP might also induce hepatocytic expression of IL-1β and promote development of fibrosis. This suggested that a HLME promoted expression of HIF-1α and YAP in hepatocytes, activated the secretion of the pro-fibrotic inflammatory factor IL-1β by hepatocytes, and then acted on itself, leading to occurrence of NAFL fibrosis. HIF-1α interacted with YAP to jointly activate transcription of Il1β (Fig. 8 ). However, some key points need further clarification. For example, can HIF-1α directly activate the expression of collagen genes without the intervention of IL-1β? Furthermore, is YAP necessary for HIF-1α to function? In conclusion, this study reported an important fibrotic process during the adaptation stage of parenchymal cells that included the inflammatory factor autocrine. These two phenomena suggested a new concept in the pathological process of inflammation; i.e., molecular inflammation occurring in parenchymal cells independent of leukocytes. Some problems need to be investigated further: first, how the regulatory mechanisms of high lipid (FFA, PA), hypoxia (HIF-1α), and mechanical microenvironment (YAP) induced high expression of IL-1β; second, the epigenetic mechanism of collagen expression in hepatocytes. In summary, HIF-1α, YAP, and IL-1β could be valuable targets to inhibit hepatocytic fibrosis induced by a HLME. Abbreviations Ab: antibody; AP-1: activating protein-1; BSA: bovine serum albumin; CD: chow diet; cDNA: complementary deoxyribonucleic acid; COL1A1: collagen type 1 α1 chain; COL3A1: collagen type 3 α1 chain; DAB: diaminobenzidine; DAPI: 4′,6-diamidino-2-phenylindole; DMEM: Dulbecco’s Modified Eagle’s Medium; ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; gDNA: genomic deoxyribonucleic acid; HFD: high-fat diet; HIF: hypoxia-inducible factor; HLF: HIF-1α–like factor; HLME: high-lipid microenvironment; HRP: horseradish peroxide; HSCs: hepatic stellate cells; IF: immunofluorescence; IgG: immunoglobulin G; IHC: immunohistochemistry; IL-1β: interleukin-1β; LDL-C: low-density lipoprotein cholesterol; MAFLD: metabolic-dysfunction–associated fatty-liver disease; MMP-2: matrix metalloproteinase 2; MOD: mean optical density; mRNA: messenger ribonucleic acid; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty-liver disease; NASH: nonalcoholic steatohepatitis; NF-κB: nuclear factor κ-light-chain-enhancer of activated B cells; OD: optical density; PA: palmitic acid; PFA: paraformaldehyde; PVDF: polyvinylidene difluoride; PMSF: phenylmethylsulfonyl fluoride; RIPA: radioimmunoprecipitation assay; RNA: ribonucleic acid; RT: room temperature; RT-PCR: real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; siRNA: short interfering ribonucleic acid; SPF: specific-pathogen–free; TEAD: transcriptional enhancer factor domain family; TFs: transcription factors; TGF: transforming growth factor; TIMP: tissue inhibitor of metalloproteinases; WB: western blot; YAP: yes-associated protein. Declarations Conflict of interest The authors declare that there are no conflicts of interest regarding the publication of this article. Funding statement This work was supported by the National Natural Science Foundation of China [Grant Nos. 81670776 and 81970734] to JGZ. Authors’ contributions JGZ contributed to the study concept and design. XWS and JGZ contributed to the analysis and interpretation of data and drafted the manuscript. HZ began the investigation of hepatocytic fibrosis and completed animal acquisition. TTN completed the study on autocrine regulation mechanisms and also performed the image editing. All authors contributed to the acquisition of data and critical revision of the manuscript. All authors approved the final manuscript prior to submission. HZ and TTN contributed equally to this work; JGZ, XWS and HA are equal co-corresponding authors. Acknowledgments We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Author details Hua Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Ting-Ting Niu, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Wen-Jiao Lin, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Xiao Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Xuan Xuan Kou, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Jing-Xin Deng, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Li-Li Yang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Peng Fei Xin, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Xue Gao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Cai Yun Zhou, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Wen Min Gao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Lan Xie, Department of Sports Medicine, Third Hospital, Peking University, Peking, 100085, China. E-mail: [email protected] . Yu-Meng Hao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Zhao-Yang Li, pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Jian-Gang Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. Tel: 86 15095387695; E-mail: [email protected] . Xiao Wei Sun, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected] . Hua AI, Department of Sports Medicine, Third Hospital, Peking University, Peking, 100085, China. E-mail: [email protected] . References Maurice J, Manousou P. Non-alcoholic fatty liver disease. Clin Med (Lond). 2018, 18(3):245-250. Anstee QM, Targher G, Day CP. Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nat Rev Gastroenterol Hepatol. 2013, 10(6):330-344. Gofton C, Upendran Y, Zheng MH, George J. MAFLD: How is it different from NAFLD? Clin Mol Hepatol. 2023, 29(Suppl):S17-S31. Boccatonda A, Andreetto L, D'Ardes D, Cocco G, Rossi I, Vicari S, Schiavone C, Cipollone F, Guagnano MT. 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Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYDACCYYEhg8FYKYB8VoYZxiQqIWBmYckLfKzGx6/tjGoS2xgb94mwVBzh7AWxjkH0qxzDNgSG3iOlUkwHHtGWAuzREKacY4BT2KDRI6ZBGPDYcJa2EBaLAwkEhvk3xCphUciIfkxg4EB0BYeIrVIAG1h7DFIMG7jSSu2SDhGhBb5GTnJH35U1Mn2sx/eeONDDRFagE5LkwBRbCAigRgNDAzshz8Qp3AUjIJRMApGLAAAv7QyqBO5DswAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-4143-9461","institution":"Pathology Institute, School of Basic Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jian-Gang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-27 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08:35:15","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173238,"visible":true,"origin":"","legend":"","description":"","filename":"rs65383402structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/12d12b47c949bc80e3a0622a.xml"},{"id":92838649,"identity":"3e65cda5-d573-4078-bcf5-6de9c07a09cf","added_by":"auto","created_at":"2025-10-06 08:27:14","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186112,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/e9e89b0aa5aec18ff332d327.html"},{"id":92838643,"identity":"81c50ebe-f33b-4b51-a803-9fb39a236283","added_by":"auto","created_at":"2025-10-06 08:27:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19241990,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-fat diet–induced nonalcoholic fatty-liver C57BL/6J mouse model and microenvironmental stress. (A) Body and liver weights of mice with NAFL. (B) Histology and gross appearance of mouse livers at different time points. Hematoxylin and eosin (H\u0026amp;E) staining; bar = 50 μM. (C) Lipid deposition analysis in hepatocytes. ORO staining and mean optical density (MOD) were compared; bar = 50 μm. (D) FFA and LDL-C concentrations in liver tissue. Mice were fed CDs or and HFDs for 1 (CD1/HFD1), 2 (CD2/HFD2), or 3 (CD3/HFD3) months. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/9c000058d746c2e890ee247b.png"},{"id":92837872,"identity":"e74a513f-2272-4e74-a757-8a3868a5a842","added_by":"auto","created_at":"2025-10-06 08:19:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15145252,"visible":true,"origin":"","legend":"\u003cp\u003eNonalcoholic fatty-liver fibrosis induced by a high-fat diet in C57BL/6 mice. (A) Messenger RNA levels of \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eCol3a1\u003c/em\u003e, \u003cem\u003eTimp1\u003c/em\u003e, and \u003cem\u003eActa2\u003c/em\u003e in livers of NAFL mice. (B) Expression of COL1A1, COL3A1, α-smooth muscle actin (α-SMA), and MMP-2 proteins in mice. (C) Expression pattern of COL3A1 in livers of HFD3 mice; IHC staining (bar = 50 μm). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/1e82400718144e2b2cca654b.png"},{"id":92837865,"identity":"fc3debcf-37a6-4553-8cce-e2d36f900665","added_by":"auto","created_at":"2025-10-06 08:19:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10764501,"visible":true,"origin":"","legend":"\u003cp\u003eA high-lipid microenvironment induced hepatocytic fibrosis without hepatic stellate-cell activation.\u003c/p\u003e\n\u003cp\u003e(A) Lipid deposition in Hep G2 and Hepa 1-6 cells cultured in PA solution for 24 h. Hep G2: 0 μM, 200 μM, 400 μM, and 600 μM PA; Hepa 1-6: 0 μM, 100 μM, and 200 μM PA. ORO staining; bar = 50 μm. (B) Expression of fibrosis-related genes. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/44a2d449a608fc97ac4918e8.png"},{"id":92837867,"identity":"2c0cc59c-984e-4cea-82f2-396962b37f71","added_by":"auto","created_at":"2025-10-06 08:19:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9477405,"visible":true,"origin":"","legend":"\u003cp\u003eInterleukin-1β autocrine mechanisms in nonalcoholic fatty liver. (A) \u003cem\u003eIL1β\u003c/em\u003e expression was upregulated in the livers of HFD C57BL/6J mice. (B) Expression of \u003cem\u003eIL1β\u003c/em\u003e in Hep G2 and Hepa 1-6 cells exposed to different concentrations of PA for 24 h. (C) Expression of IL-1R1 in mouse livers,bar = 100 μm. (D) Expression of IL1R1 in Hep G2 cells, bar = 20 μm. (E) Expression of COLI and COLIII in Hep G2 cells stimulated with recombinant human IL-1β (10 ng/mL). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/11b3b73a83a5911d552078b9.png"},{"id":92837871,"identity":"c9aa17c0-c89e-4bc2-aed6-68c53ae5b8e2","added_by":"auto","created_at":"2025-10-06 08:19:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3544758,"visible":true,"origin":"","legend":"\u003cp\u003eThe high-lipid microenvironment induced hypoxic and mechanical stress. (A) HIF-1α/YAP expression was upregulated in HFD mice. (B) PA upregulated HIF-1α/YAP expression in Hep G2 and Hepa 1-6 cells. Cells were cultured with different concentrations of PA for 24 h.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/f75d928fc154527c65ba2435.png"},{"id":92837870,"identity":"e0b2455e-f65a-4e0e-b196-37845afb9837","added_by":"auto","created_at":"2025-10-06 08:19:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5280633,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between HIF-1α and YAP. Hep G2 cells were cultured with different concentrations of PA for 24 h. (A) HIF-1α co-localized in the nucleus with YAP during PA treatment. IF staining; bar = 50 μm. (B) Interaction between HIF-1α and YAP. Hep G2 cells were cultured with 200 μM PA for 24 h. With HIF-1α Ab was used as immunoprecipitation (IP), the Input group showed YAP protein expression; when YAP Ab was used as IP, the Input group showed HIF-1α protein expression. (C) YAP expression was dependent on HIF-1α regulation. Hep G2 cells were cultured with 200 μM PA for 24 h and transfected with siRNA for 15 min.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/0645bd91ea36c193322b23ec.png"},{"id":92837878,"identity":"87c98862-f590-4d55-b0da-4adff9c4bddd","added_by":"auto","created_at":"2025-10-06 08:19:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5318383,"visible":true,"origin":"","legend":"\u003cp\u003eHIF-1α regulated \u003cem\u003eIl1β\u003c/em\u003e gene expression in hepatocytes. (A) Bioinformatic analysis showed HIF-1α binding with the promoter of \u003cem\u003eIl1β\u003c/em\u003e. (B) Effect of HIF-1α inhibitor PX-478 on \u003cem\u003eIl1β\u003c/em\u003e gene expression. (C) Effect of HIF-1α inhibitor PX-478 on IL-1βsignaling and collagen expression.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/f79b024f8b0dd6989cd48bcf.png"},{"id":92838644,"identity":"391fd964-4410-4e82-8942-29fcb39d7ccc","added_by":"auto","created_at":"2025-10-06 08:27:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":550615,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of adaptive-fibrosis mechanism in NAFL. FFAs promoted expression of hypoxia-sensitive factor HIF-1α and pressure-sensitive factor YAP in hepatocytes. Both factors entered the nucleus and activated the expression and secretion of the pro-fibrotic inflammatory factor IL-1β, which then acted on itself via regulation of autocrine mechanisms, leading to NAFL fibrosis. HIF-1α interacted with YAP to jointly activate the transcription of \u003cem\u003eIl1β\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/abc45009d97200ba2d006d26.png"},{"id":92839904,"identity":"98a12628-58f7-4fdf-a656-5e821680f5fd","added_by":"auto","created_at":"2025-10-06 08:43:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":65690865,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6538340/v1/0b7ab12b-315a-4100-9feb-8af7343f410e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"HIF-1α/YAP induces interleukin 1β autocrine in hepatocytes, leading to hepatocytic fibrosis: adaptive fibrosis in nonalcoholic fatty liver","fulltext":[{"header":"Key Points","content":"\u003cp\u003eFibrosis occurred at the NAFL stage and originated from hepatocytes rather than hepatic stellate cells.\u003c/p\u003e\n\u003cp\u003eHigh-lipid, hypoxic, and mechanical stress was present, with lipid deposition in hepatocytes. High lipid levels were an independent factor.\u003c/p\u003e\n\u003cp\u003eA high-fat diet or palmitic acid induced high expression of COL1A1, COL3A1, MMP-2, HIF-1\u0026alpha;, YAP, and IL-1\u0026beta; in hepatocytes.\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha;/YAP regulated expression of IL-1\u0026beta;, the receptor of which was highly expressed in hepatocytes; IL-1\u0026beta; in turn regulated hepatocytic fibrosis via autocrine regulation.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eEconomic development and lifestyle changes have contributed to nonalcoholic fatty-liver disease (NAFLD), increasingly becoming a major global-health problem [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NAFLD generally refers to liver damage with histological changes similar to those seen in alcoholic fatty liver, but its cause is related to metabolic dysfunction; therefore, it is also referred as to metabolic dysfunction\u0026ndash;associated fatty liver disease (MAFLD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. High-fat diets (HFDs), obesity, and insulin resistance (metabolic syndrome [MetS]) are the main causes of NAFLD [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePathologically, NAFLD progresses from nonalcoholic fatty liver (NAFL; also called steatosis) to nonalcoholic steatohepatitis (NASH), and then to fibrosis, which occurs secondary to NASH [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Lipid deposition causes oxidative stress (OS) and peroxidation in mitochondria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], leading to hepatocytic damage. This endogenous injury further activates Kupffer cells to release inflammatory factors such as transforming growth factor-β (TGF-β), inducing inflammatory response (NASH) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and further activating hepatic stellate cells (HSCs) as myofibroblasts to secrete extracellular matrix (ECM), leading to fibrosis [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. HSCs are considered the main source of ECM and play a crucial role in the formation of NASH fibrosis [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The TGF-β/Smad signaling pathway is a key mechanism in HSC activation [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, little is known about fibrosis during the NAFL stage, which is absent in oxidative injury, inflammatory-cell reactions, and HSC activation.\u003c/p\u003e\u003cp\u003eStress (a microenvironment unsuitable for the survival of liver cells) and adaptation, rather than injury, are the major environmental features faced by NAFL hepatocytes. With lipid deposition, cells enlarge, and the sinus is compressed remarkably. At least three kinds of stress applied to hepatocytes can be deduced during NAFL progression: hypoxic, mechanical, and high-lipid. During chronic hypoxia, a series of transcription factors (TFs) are activated, such as hypoxia-inducible factor (HIF), HIF-1α\u0026ndash;like factor (HLF), activator protein 1 (AP-1), and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). These TFs regulate expression of target genes to facilitate cell survival in low-oxygen environments [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn mammals, HIF is the main TF in hypoxic stress and targets a wide range of genes, including those related to hypoxic adaptation, development of inflammation, and tumor growth. HIF is a \u0026ldquo;molecular switch\u0026rdquo; that regulates the expression of low-oxygen target genes in the body and is one of the most important pathways mediating decreases in cellular-oxygen concentration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recent studies have shown that HIF-1 and HIF-2 can regulate the expression of interleukin 1α (IL-1α) and produce different effects in different cell lines [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This finding establishes a possible link between hypoxic and inflammatory factors. We speculate that absent inflammatory-cell involvement, inflammatory factors might be produced by parenchymal/stromal cells induced by hypoxic stress. These inflammatory factors have two important functions: they can (1) respond to hypoxic signals and (2) activate HSCs to produce fibrotic effects. IL-1 is likely such an inflammatory factor. IL-1β is an early-discovered inflammatory factor that promotes collagen synthesis in osteoblasts and HSCs, enhances the synthesis and release of TGF-β, induces the expression of adhesion factors and their receptors, promotes production of tissue inhibitor of metalloproteinases (TIMP), and inhibits ECM degradation. Recently, IL-1β has been found to play an important role in the progression of NAFL to NASH and fibrosis [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but its source and specific mechanism of action have not yet been discovered. Yes-associated protein (YAP) is a TF co-stimulatory molecule and also an effector molecule in multiple signaling pathways. Highly expressed in activated fibroblasts, YAP promotes interstitial-cell activation by regulating the expression of various activation-related genes, including growth factors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Research has found that YAP undergoes nuclear translocation in the early stages of liver injury [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and we speculate that it may be involved in the process of NAFL fibrosis. In this article, we report that stress and adaptation induced NAFL hepatocytic fibrosis, which was mediated by HIF-1α and YAP regulation and IL-1β autocrine regulation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eWe purchased male adult C57BL/6J mice (4 weeks old) from the Experimental Animal Center of Lanzhou University (Lanzhou, China; No. SCXK 2018-0002) and housed them individually in a specific pathogen\u0026ndash;free (SPF) environment (12-h light/dark cycle, 22\u0026deg;C, and 65\u0026ndash;70% humidity) with free access to food and water. After 1 week of adaptive feeding with a chow diet (CD; 3.16 kcal/g, 11.4% kcal from fat, 64% from carbohydrates, 25% from protein), we fed the animals either CD (n\u0026thinsp;=\u0026thinsp;9) or a mixture of CD and high-fat feed (n\u0026thinsp;=\u0026thinsp;15; 5.58 kcal/g, 66.5% kcal from fat, 22% from carbohydrates, 11% from protein) for 1, 2, or 3 months. The HFD was prepared as previously reported [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; briefly, lard, egg yolk powder (Jinjianli, Beijing, China), CD powder (KeAo Corp., Beijing, China), and sucrose were mixed at a ratio of 1:1:1.5:0.4. The mice\u0026rsquo;s diets were changed weekly.\u003c/p\u003e\u003cp\u003e All animals were sacrificed in compliance with the Guideline for the Ethical Review of Laboratory Animal Welfare (No. GB/T35892-2018). This study was approved by the Medical Ethics Committee of Lanzhou University (Lanzhou, China; No. jcyxy20190302) and conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and Replacement, Refinement and Reduction of Animals in Research (NC3Rs).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003e We anesthetized mice with pentobarbital sodium (5 mg/100 g body mass), followed by three skin sterilizations using 75% ethanol and iodophor. After the abdominal cavity was opened, animals were sacrificed via inferior vena cava blood drainage. We removed the livers, washed them in pre-cooled phosphate-buffered saline (PBS; #P1010; Beijing Solarbio Science \u0026amp; Technology Co., Ltd., Beijing, China), and dried them using filter paper. The left lateral lobe was sampled for free fatty acids (FFAs) and low-density lipoprotein cholesterol (LDL-C) testing, the middle lobe was fixed in 50 mL of 4% neutral formaldehyde for histological testing (including HE, oil red O, and IHC staining), and the rest of the liver was stored at \u0026minus;\u0026thinsp;80\u0026deg;C after a quick freeze in liquid nitrogen.\u003c/p\u003e\n\u003ch3\u003eFree fatty acid and low-density lipoprotein cholesterol determinations\u003c/h3\u003e\n\u003cp\u003eWe detected FFA concentrations in liver tissues using a non-esterified fatty acid (NEFA) reagent kit (#KG7571; KeyGEN Biotech, Nanjing, China) per manufacturer\u0026rsquo;s instructions. Briefly, liver tissues were homogenated in 9\u0026times; volume ethanol on ice for 2 min and centrifuged at 2500 rpm and 4\u0026deg;C for 10 min, after which the supernatant was collected and reacted with copper reagent to acquire a fatty acid copper\u0026ndash;salt solution. After adding the color reagent, we measured the absorbance (optical density [OD]) of the solution at 440 nm using an ultraviolet visible (UV/Vis) spectrophotometer (SPECORD 50 Plus; Analytik Jena GmbH\u0026thinsp;+\u0026thinsp;Co. KG, Jena, Germany). Distilled water and 1000 \u0026micro;M palmitic acid (PA; #SLCC6727; Sigma-Aldrich, Burlington, MA, USA) solution were used as blank and standard controls, respectively, and chloroform was used for zero adjustment. We calculated FFA concentration as follows:\u003c/p\u003e\u003cp\u003e[(OD\u003csub\u003esample\u003c/sub\u003e \u0026minus; OD\u003csub\u003eblank\u003c/sub\u003e)/(OD\u003csub\u003estandard\u003c/sub\u003e \u0026minus; OD\u003csub\u003eblank\u003c/sub\u003e)] \u0026times; standard concentration (mM) \u0026times; volume (L)/weight (g)\u003c/p\u003e\u003cp\u003eWe detected LDL-C concentrations in liver tissues using a LDL-C reagent kit (#A113-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The tissue homogenate supernatant was incubated with reagents at 37\u0026deg;C for 5 min, and OD values were measured on an enzyme-linked immunosorbent assay (ELISA) reader (Synergy NEO2; Agilent Technologies, Inc., Santa Clara, CA, USA). LDL-C concentration was calculated as follows:\u003c/p\u003e\u003cp\u003e[(ΔOD\u003csub\u003esample\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ΔOD\u003csub\u003eblank\u003c/sub\u003e)/(ΔOD\u003csub\u003estandard\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;ΔOD\u003csub\u003eblank\u003c/sub\u003e)] \u0026times; standard concentration (mM) \u0026times; volume (L)/weight (g)\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culturing and high lipid\u0026ndash;microenvironment\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003emodel\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe purchased Hep G2 (#CL-0103) and Hepa 1\u0026ndash;6 (#CL-0105) cells from Pricella Life Science and Technology Co., Ltd. (Wuhan, China). Cells were maintained in high-glucose Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; #SH30022.01; HyClone [Cytiva, Marlborough, MA, USA]) with 10% fetal bovine serum (FBS; #FBSST-01033; OriCell; Cyagen Biosciences, Jiangsu, China) and 1% penicillin\u0026ndash;streptomycin mixture (100\u0026times;; #P1400; Solarbio) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. To establish a high-lipid microenvironment (HLME) \u003cem\u003ein vitro\u003c/em\u003e model, we cultured cells in starvation medium (DMEM with 1% FBS) for 12 h after they reached 60% confluence, and then subjected them to 2 ml PA/bovine serum albumin (BSA; #WXBD1900V; Sigma-Aldrich, USA) medium for 24 h, with or without HIF-1α inhibitor PX-478 (#HY-10231, MedChemExpress, USA). Hep G2 cells were cultured in 0 \u0026micro;M (0.2% BSA), 200 \u0026micro;M, 400 \u0026micro;M, and 600 \u0026micro;M PA; Hepa 1\u0026ndash;6 cells were cultured in 0 \u0026micro;M (0.1% BSA), 100 \u0026micro;M, and 200 \u0026micro;M PA. We produced PA/BSA medium by mixing 10 mM PA/BSA solution with DMEM at different concentrations; PA/BSA solution was produced by mixing 20% BSA solution (1.20 g BSA dissolved in 6 mL DMEM) with 20 mM PA solution (0.037 g PA in 6 mL of 0.1 sodium hydroxide) and stirring at 50\u0026deg;C for 30 min.\u003c/p\u003e\n\u003ch3\u003eHematoxylin and eosin staining\u003c/h3\u003e\n\u003cp\u003eWe fixed liver tissues in 4% paraformaldehyde (PFA; #G1102; Solarbio, China) and embedded them in paraffin. The sections were then deparaffinized with xylene and ethanol and stained with Harris hematoxylin and eosin (#G1150; Solarbio, China) using conventional methods. The slices were then observed under a microscope.\u003c/p\u003e\n\u003ch3\u003eOil Red O staining\u003c/h3\u003e\n\u003cp\u003eWe fixed liver tissues in 4% paraformaldehyde (PFA; #G1102; Solarbio) and embedded them in Tissue-Tek O.C.T. Compound (#4583; Sakura Finetek, Tokyo, Japan), followed by frozen sectioning on a freezing microtome (#CM1950; Leica, Wetzlar, Germany). Sections were treated with 60% isopropanol and stained with Oil Red O (ORO; #1320-06-5; Shandong Xiya Chemical Technology Co., Ltd., Linyi, China) for 15 min, after which nuclei were counterstained with hematoxylin (#S2100; Solarbio). We observed sections under a Nikon-ECLIPSE 80i/DS-Ri2/NIS-Elements D microscope (Nikon, Tokyo, Japan) and analyzed images using ImageJ software v6.0 (National Institutes of Health [NIH], Bethesda, MD, USA). PA-treated cell slides were fixed at room temperature (RT) for 15 min with 4% PFA and subjected to the same staining process as tissue sections.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e\u003cp\u003eWe then dewaxed the paraffined sections using xylene and gradient alcohol. Antigens were retrieved using sodium citrate (pH 6.0; #C1010; Solarbio), endogenous peroxidase was inhibited with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (#SP-9001; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China), and nonspecific background antigens were blocked with goat serum (#SP-9001; Beijing Zhongshan Jinqiao) at 37\u0026deg;C for 30 min. We then incubated the slides with primary antibodies (Abs) overnight at 4\u0026deg;C and secondary Abs for 15 min at RT. The reaction was visualized using diaminobenzidine (DAB; 1:40; #ZLI-9018; Beijing Zhongshan Jinqiao), and nuclei were counterstained using hematoxylin (#S920; Solarbio). Primary Abs were collagen III (COLIII) rabbit polyclonal (1:600; #22734-1-AP) and interleukin 1 receptor type I (IL1R1) rabbit polyclonal (1:500; #27348-1-AP; both Proteintech, Wuhan, China). The secondary Ab was goat anti-rabbit (#SP-9001; Beijing Zhongshan Jinqiao). Slides were observed under the Nikon ECLIPSE microscope and analyzed using ImageJ and GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCellular immunofluorescence\u003c/h3\u003e\n\u003cp\u003eCells slides were fixed with 4% neutral formaldehyde for 15 min, permeated with 0.5% Triton X-100 (#T8200; Solarbio) for 15 min, and then pretreated with goat serum (#SP-9001; Beijing Zhongshan Jinqiao) for 30 min at RT. We then incubated the slides with primary Abs overnight at 4\u0026deg;C and fluorescent-labeled secondary Abs for 30 min at RT. Nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; #C0065; Solarbio) at RT for 5 min and observed under the Nikon ECLIPSE microscope at UV-2A (EX330-380, BA410) for DAPI, B2A (EX450-490, BA520) for YAP, and G2A (EX510-560, BA590) for HIF-1α. We analyzed fluorescence intensity using ImageJ. Primary Abs were YAP (1:400) mouse monoclonal (#66900-1-Ig), HIF-1α (1:200) rabbit polyclonal (#20960-1-AP), both from Proteintech, and IL1R1 (1:200) rabbit polyclonal (#ab106278; Abcam, Cambridge, UK). Secondary Abs were Alexa Fluor 488 goat anti-mouse (1:800; #SA0006-1) and Alexa Fluor 594 goat anti-rabbit (1:800; #SA0006-4; both Proteintech).\u003c/p\u003e\n\u003ch3\u003eReal-time polymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eWe extracted total ribonucleic acid (RNA) via the phenol chloroform method. Briefly, liver tissues or cells were homogenated in RNAiso Plus (#9108/9109; TaKaRa, Shiga, Japan) lysis buffer (30 mg:100 mL), and total nucleic acid was extracted using 200 \u0026micro;L chloroform. After centrifugation at 12,000\u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min, we extracted RNA and precipitated the solution using isopropanol. RNA concentration was detected using a Thermo Scientific NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A reverse transcription kit (#RR047A; TaKaRa) was used per manufacturer\u0026rsquo;s instructions to remove genomic deoxyribonucleic acid (gDNA) and acquire efficient complementary DNA (cDNA). We performed real-time quantitative polymerase chain reaction (RT-qPCR) on a QuantStudio 3 real-time fluorescence quantitative PCR instrument with QuantStudio Design \u0026amp; Analysis Software (Thermo Fisher, Waltham, MA, USA), using a TB Green Premix Ex TaqII kit (#RR820A; TaKaRa). The three-step procedure was followed: hold stage: 95\u0026deg;C for 30 s; PCR stage: 95\u0026deg;C for 5 s, 60\u0026deg;C for 30 s; melt curve stage: 95\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 15 s, 60\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 1 min, 95\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 1 s, 25\u003csup\u003e\u0026deg;\u003c/sup\u003eC for 5 min). We calculated gene expression levels using the 2\u003csup\u003e\u0026minus;ΔΔ\u003c/sup\u003e\u003csub\u003eCt\u003c/sub\u003e method:\u003c/p\u003e\u003cp\u003e\u003csup\u003eΔ\u003c/sup\u003e\u003csub\u003eCt\u003c/sub\u003e = Ct\u003csub\u003etarget gene\u003c/sub\u003e \u0026ndash; MEAN Ct\u003csub\u003einternal reference\u003c/sub\u003e, ΔΔCt\u0026thinsp;=\u0026thinsp;\u003csup\u003eΔ\u003c/sup\u003e\u003csub\u003eCt\u003c/sub\u003e \u0026ndash; MEAN Δ Ct\u003csub\u003econtrol target gene\u003c/sub\u003e)\u003c/p\u003e\u003cp\u003ePrimers were as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers for real-time polymerase chain reaction detection\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eActa2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-AACACGGCATCATCACCAAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CACAGCCTGAATAGCCACATAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eActin\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GGCTGTATTCCCCTCCATCG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCAGTTGGTAACAATGCCATGT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCol1a1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-TTGGTCCCGCTGGCAAGAATG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CTGTCACCTTGTTCGCCTGTCTC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCol3a1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CCTCCCAGAACATTACATACC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TTCGCCTTCATTTGATCCC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHif1α\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GTCCCAGCTACGAAGTTACAGC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CAGTGCAGGATACACAAGGTTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIl1β\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-TGGTGTGTGACGTTCCCATT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CAGCACGAGGCTTTTTTGTTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eYap\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-ACCCTCGTTTTGCCATGAAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TGTGCTGGGATTGATATTCCGTA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIl1r1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CTGCTGTCGCTGGAGATTGAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TTGGCAGGTACAAACCAAAGAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTimp\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-TGGCATCTGGCATCCTCTTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CGCTGGTATAAGGTGGTCTCG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHuman\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eACTA2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CCGGGACTAAGACGGGAATC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TTGTCACACACCAAGGCAGT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eACTIN\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CCTGGCACCCAGCACAAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GGGCCGGACTCGTCATAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOL1A1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-TGTTCAGCTTTGTGGACCTCCG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCGTTCTGTACGCAGGTGATTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOL3A1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GAAGATGTCCTTGATGTGC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AGCCTTGCGTGTTCGATAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHIF1α\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-ACGAGAGGTTCCCTAATTTCCA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-ATGCCACCAGTACATTGGGAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIL1β\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-AGCCATGGCAGAAGTACCTG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CCTGGAAGGAGCACTTCATCT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eYAP\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GAACAATGACGACCAATAGCTC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-TAGTCCACTGTCTGTACTCTCA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eIL-1R1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-GGCTGAAAAGCATAGAGGGAAC-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-CTGGGCTCACAATCACAGG-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTIMP1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CACTGTTGGCTGTGAGGAA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AAGGTGACGGGACTGGAA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCol1a1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CTGGCGCTTCAGGTCCAAT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-GGCACCATCCAAACCACTGA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCol3a1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026prime;-CGAGGTAACAGAGGTGAAAGA-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u0026prime;-AACCCAGTATTCTCCGCTCTT-3\u0026prime;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot\u003c/h2\u003e\u003cp\u003eWe extracted total protein using radioimmunoprecipitation assay (RIPA)/phenylmethylsulfonyl fluoride (PMSF) cell lysate (100:1; respectively #R0010 and #P0100; Solarbio). Total protein concentration was measured using bicinchoninic acid (BCA; #PC0020; Solarbio). We separated protein samples via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% separating gel), transferred them onto an Immobilion polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich), and probed them with the above-indicated primary Abs at 4\u0026deg;C overnight, followed by the appropriate secondary horseradish peroxide (HRP)\u0026ndash;conjugated immunoglobulin G (IgG) Ab at RT for 1 h. The following Abs were used: primary: rabbit polyclonal COLI (1:800; #14695-1-AP), COLIII (1:800; #22734-1-AP), HIF-1α (1:1000; #20960-1-AP), YAP (1:1000; #13584-1-AP), actin alpha 2 smooth muscle (ACTA2; 1:6000; #14395-1-AP), MMP-2 (1:1000; #10373-2-AP), β-actin (1:10000; #66009-1-Ig), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:6000; #10494-1-AP; all from Proteintech, Chicago, IL, USA), phosphorylated YAP (p-YAP; 1:1000; #13008S), p-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) (1:1000; #4511T), p\u0026ndash;NF-κB p65 (Ser536) (93H1) (1:1000; 3033T; all three from Cell Signaling Technology [CST], Danvers, MA, USA); mouse monoclonal HIF-1α (#ab8366; Abcam, Cambridge, UK), YAP (1:400; #66900-1-Ig; Proteintech); rabbit polyclonal COLIII (1:1000; #ab184993), rabbit polyclonal COLI (1:1000; #ab260043), and rabbit polyclonal IL1R1 (1:500; #ab106278; all three from Abcam); secondary: HRP-labeled goat anti-rabbit (1:5000; #RS0002; ImmunoWay Biotechnology Co., Plano, TX, USA). Protein bands were visualized using an electrochemiluminescence (ECL) kit (Super ECL Detection Reagent; #3608ES60; Yeasen Biotechnology Co., Ltd., Shanghai, China) and a chemiluminescence imaging system (FUSION Solo 6S Edge; Vilber, Marne-la-Vall\u0026eacute;e, France). We used ImageJ to analyze grayscale band values.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation\u003c/h2\u003e\u003cp\u003eWe added HIF-1α (0.2\u0026ndash;2.0 \u0026micro;g; #ab8366; Abcam), YAP (0.2\u0026thinsp;\u0026minus;\u0026thinsp;2.0 \u0026micro;g; #66900-1-Ig; Proteintech; Input group) or mouse IgG (1\u0026ndash;2 \u0026micro;g; #sc-2025; Santa Cruz Biotechnology, Dallas, TX, USA; IgG group) to total protein solution (100\u0026ndash;500 \u0026micro;g in 1 mL lysate) and incubated the mixture at 4\u0026deg;C on a shaker for 12 h. The Ab\u0026ndash;antigen complex was then pulled down by adding 20 \u0026micro;L of Protein A/G PLUS Agarose (#sc-2003; Santa Cruz) to the mixture and incubating it on a shaker at 4\u0026deg;C for 6 h. After centrifugation at 4\u0026deg;C and 2500 rpm for 5 min, we collected the precipitate as a magnetic-bead Ab\u0026ndash;antigen complex and detected the target proteins via western blot (WB).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTransient transfection of small interfering RNA\u003c/h2\u003e\u003cp\u003eHep G2 cells were transfected with short interfering RNA (siRNA) when cell density reached 60% confluence. We mixed GP Transfection Mate transfection reagent (5.5 \u0026micro;L; GenePharma, Shanghai, China) in 200 \u0026micro;L DMEM and 20 \u0026micro;M siRNA oligo (7.5 \u0026micro;L) in 200 \u0026micro;L DMEM at RT for 15 min to produce a complex transfection solution, which was then added to 1.6 mL preheated culture medium. Cells were transfected at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 6 h. After another 60 h of cultivation, we used WB to detect protein expression. Sequences of HIF-1α, GAPDH, and negative control (NC) are described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The experimental designation was as follows: positive control (siGAPDH), NC, transfection reagent (GP Transfer Mate), treatment control (0 \u0026micro;M PA), experimental (200 \u0026micro;M PA), treatment control\u0026thinsp;+\u0026thinsp;siHIF-1α, and experimental (200 \u0026micro;M PA)\u0026thinsp;+\u0026thinsp;siHIF-1α.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequences of human-gene siRNA\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHuman\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSense\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAntisense\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHIF-1α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-CAGGCCACAUUCACGUAUATT-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-UAUACGUGAAUGUGGCCUGTT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-UGACCUCAACUACAUGGUUTT-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-AACCAUGUAGUUGAGGUCATT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5'-UUCUCCGAACGUGUCACGUTT-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5'-ACGUGACACGUUCGGAGAATT-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHIF-1α, hypoxia-inducible factor 1α; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC, negative control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatic analysis\u003c/h2\u003e\u003cp\u003eWe searched the genomic information for HIF-1α and the \u003cem\u003eIL1β\u003c/em\u003e using the National Center for Biotechnology Information [NCBI] database (Bethesda, MD, USA) and predicted the binding relationship between HIF-1α and \u003cem\u003eIL1β\u003c/em\u003e using AlphaFold 3, the University of California at Santa Cruz (UCSC) Genome Database, the Gene Transcription Regulation Database (GTRD), Gene Expression Profiling Interactive Analysis (GEPIA), and the Jackson-based Sequence Analysis Parameter (JASPAR) database.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). We conducted all statistical analyses using SPSS v25.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8. Comparisons between two groups were conducted using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test, and those between multiple groups were conducted via one-way analysis of variance (ANOVA). We analyzed OD values of immunofluorescence (IF) images using ImageJ software. All experiments were repeated at least three times, with three technical replicates set each time. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eA high\u003c/strong\u003e\u003cstrong\u003e-lipid microenvironment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einduced the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;NAFL hepatocytic fibrotic phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompared with CD mice, the body and liver weights of HFD mice significantly increased at all three time points and showed a time-dependent pattern (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 1A). Morphologically, lipid was deposited in hepatocytes to form droplets in various numbers and sizes (from micro- to macrovesicular), the severity of which was time dependent (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Figs. 1B\u0026ndash;C). We did not observe hepatocytic necrosis or inflammatory-cell infiltration, which suggested establishment of NAFL (Fig. 1B). Compared with CD mice, FFA and LDL-C levels in HFD mouse livers significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Table 3, Fig. 1D); in HFD1 mice in particular, liver LDL-C levels were significantly elevated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), suggesting that the HFD induced a high-fat microenvironment and abnormal lipid metabolism in liver cells. Hepatocytes were enlarged and hepatic sinusoids were compressed to different degrees, accompanied by gray-yellow liver color (Fig. 1B), indicating decreased blood perfusion of the hepatic sinusoids and a relatively hypoxic microenvironment.\u003c/p\u003e\n\u003cp\u003eHFD mice also showed obvious fibrosis. Compared with CD mice, levels of \u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eCol3a1\u003c/em\u003e messenger RNA (mRNA) significantly increased in HFD mice (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 2A), and the level of \u003cem\u003eTimp1\u003c/em\u003e in HFD3 mice significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Fig. 2A). Protein expression showed similar elevations of COL1A1 and COL3A1, despite HFD3 and CD3 mice showing the same trend in COL1A1 (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). In addition, MMP-2 protein levels significantly increased in HFD mice (\u003cem\u003eP\u003c/em\u003e ˂ 0.01; Fig. 2B). Immunohistochemical (IHC) staining showed that COL3A1 was deposited around both sinusoids and hepatocytes, presenting a typical grid-like appearance (Fig. 2C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. FFA and LDL-C levels in C57BL/6J mouse liver tissue\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"554\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003eFFA (mmol/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eLDL-C (mmol/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eCD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e15.68 \u0026plusmn; 0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.84 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eHFD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e18.31 \u0026plusmn; 1.48\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e2.29 \u0026plusmn; 0.30\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eCD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e14.42 \u0026plusmn; 3.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eHFD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e22.16 \u0026plusmn; 3.06\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e2.89 \u0026plusmn; 0.44\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eCD3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e14.86 \u0026plusmn; 0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1.01 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 161px;\"\u003e\n \u003cp\u003eHFD3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 201px;\"\u003e\n \u003cp\u003e17.39 \u0026plusmn; 0.67\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e2.98 \u0026plusmn; 0.58\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: Compared with CD mice at the same time points. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Nonalcoholic fatty-liver fibrosis originated from activation of hepatocytes, not of hepatic stellate cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHSCs are the main source of liver fibrosis. However, HSC activation is always secondary to hepatocytic injury and inflammation such as NASH; such lesions are absent in NAFL. Therefore, we next investigated the source of fibrosis in NAFL. We detected expression of \u0026alpha;-smooth muscle actin (\u0026alpha;-SMA), the marker of HSC activation, in liver tissues. The results showed that compared with CD mice, mRNA and protein levels of \u003cem\u003eActa2\u003c/em\u003e in the livers of HFD mice at each time point were not statistically significant (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; Figs. 2A\u0026ndash;B), which suggested absence of HSC activation during NAFL fibrosis.\u003c/p\u003e\n\u003cp\u003eWe cultured Hep G2 and Hepa 1-6 cells with different concentrations of PA for 24 h and then detected lipid deposition and fibrotic phenotype. The results showed that PA treatment significantly increased lipid droplet deposition in cytoplasm, which increased with PA concentration (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 3A). PA treatment promoted the expression of \u003cem\u003eCOL1A1\u003c/em\u003e, \u003cem\u003eCOL3A1\u003c/em\u003e, and \u003cem\u003eTIMP1\u003c/em\u003e in Hep G2 cells and \u003cem\u003eCol3a1\u003c/em\u003e, \u003cem\u003eTimp1\u003c/em\u003e and \u003cem\u003eActa2\u003c/em\u003e in Hepa 1-6 cells at different concentrations (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and there was no elevation of \u0026alpha;-SMA (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05; Fig. 3B), suggesting that fibrosis originated from hepatocytes in NAFL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. IL-1\u0026beta; autocrine mechanisms in hepatocytic fibrosis in a high-lipid microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta; is the most potent pro-fibrotic factor in NASH. Therefore, we further assessed whether hepatocytic fibrosis during NAFL was associated with IL-1\u0026beta;. We showed that, compared with CD mice, mRNA levels of \u003cem\u003eIl1\u0026beta;\u0026nbsp;\u003c/em\u003ewere significantly upregulated in HFD2 and HFD3 mice (\u003cem\u003eP\u003c/em\u003e ˂ 0.05; Fig. 4A). In our \u003cem\u003ein vitro\u003c/em\u003e experiment, mRNA expression of IL-1\u0026beta; was significantly higher in PA groups than in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 4B).\u003c/p\u003e\n\u003cp\u003eWe detected the effect of IL-1\u0026beta; on hepatocytes. The results showed that the IL-1\u0026beta; receptor IL1R1 was distributed in hepatocytes and was also upregulated during PA treatment (Figs. 4C\u0026ndash;D). Recombined IL-1\u0026beta; significantly upregulated protein expression of COLI and COLIII in Hep G2 cells (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 5E). These results indicated that hepatocytic fibrosis in NAFL was related to IL-1\u0026beta; autocrine mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe high\u003c/strong\u003e\u003cstrong\u003e-lipid microenvironment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einduced\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;HIF-1\u0026alpha;/YAP upregulation in hepatocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWith lipid deposition, the hepatocytes enlarged and the sinuses were compressed, meaning that relative hypoxic and mechanical stress might have been induced in the liver. Since HIF-1\u0026alpha; and YAP are the respective biomarkers of hypoxia and mechanical stress and play key roles in lipid metabolism and OS in NAFLD, we detected the HIF-1\u0026alpha;/YAP signal in the liver. Expression of \u003cem\u003eHif1\u0026alpha;\u0026nbsp;\u003c/em\u003esignificantly increased in HFD1 and HFD2 mice but decreased in HFD3 mice (\u003cem\u003eP\u003c/em\u003e ˂ 0.05), while that of \u003cem\u003eYap\u003c/em\u003e was also upregulated in HFD mice (\u003cem\u003eP\u003c/em\u003e ˂ 0.01; Fig. 5A). These results suggested the possibility of hypoxic and mechanical stress in the livers of NAFL mice.\u003c/p\u003e\n\u003cp\u003eWe further cultured Hep G2 and Hepa 1-6 cells in a high-lipid environment (HLME). The results showed that (a) expression of \u003cem\u003eHIF1\u0026alpha;\u0026nbsp;\u003c/em\u003e(human) in Hep G2 cells and \u003cem\u003eHif1\u0026alpha;\u0026nbsp;\u003c/em\u003e(mouse) in Hepa 1-6 cells\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas significantly higher than in the control group, and (b) \u003cem\u003eYAP\u0026nbsp;\u003c/em\u003e(human) in Hep G2 cells or \u003cem\u003eYap\u0026nbsp;\u003c/em\u003e(mouse)\u0026nbsp;in\u0026nbsp;Hepa 1-6 cells\u003cem\u003e\u0026nbsp;\u003c/em\u003eshowed significantly higher gene expression in the PA groups than in the control group (both \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 5B). In Hep G2 cells, protein levels of HIF-1\u0026alpha; significantly increased in each PA treatment group, with statistical significance in the 600-\u0026mu;M group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; Fig. 5B). There was no significant change in YAP protein level in the PA treatment groups (Fig. 5B); however, the protein level of p-YAP (a marker of YAP degradation) decreased significantly (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), indicating activation of YAP (Fig. 5B). In Hepa 1-6 cells, expression of HIF-1\u0026alpha; and YAP protein in cells treated with different concentrations of PA significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). These results suggested that PA was an independent factor producing hypoxic and mechanical stress in hepatocytes.\u003c/p\u003e\n\u003cp\u003eWe then detected HIF-1\u0026alpha;/YAP interaction. IF results showed that PA induced an increase in the expression of HIF-1\u0026alpha; and YAP proteins in a dose-dependent manner, and HIF-1\u0026alpha; co-localized with YAP in the nucleus (Fig. 6A). The Co-IP experiment suggested an interaction between HIF-1\u0026alpha; and YAP (Fig. 6B), and expression of YAP protein was reduced in the siHIF-1\u0026alpha; group (Fig. 6C). These results indicated that YAP expression was regulated by the TF HIF-1\u0026alpha;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. HIF-1\u0026alpha; targeted\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe \u003cem\u003eIl1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene in hepatocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth HIF-1\u0026alpha; signaling and IL-1\u0026beta; secretion were upregulated in the HLME during NAFL development. Therefore, we further assessed HIF-1\u0026alpha;\u0026rsquo;s regulation of \u003cem\u003eIl1\u003c/em\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e gene expression. First, we conducted a bioinformatic analysis of the regulatory relationship. Human \u003cem\u003eIl1\u003c/em\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e is located on chromosome 2 at positions 112829751\u0026ndash;112836779. AlphaFold 3 predicted the spatial structure of HIF-1\u0026alpha; binding with the promoter of human \u003cem\u003eIl1\u003c/em\u003e\u003cem\u003e\u0026beta;.\u003c/em\u003e The binding relationship indicated a high probability, as indicated by the UCSC Genome Database (score = 265) and the GEPIA database (\u003cem\u003er\u003c/em\u003e = 0.43,\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt; 0.05). The JASPAR database predicted 12 potential binding sites within the 2000-bp upstream and 100-bp downstream intervals of \u003cem\u003eIl1\u003c/em\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e, with a relative profile score threshold of 80% (MA0259.2), which suggested that HIF-1\u0026alpha; could bind to the \u003cem\u003eIL1\u0026beta;\u003c/em\u003e promoter to stimulate IL-1\u0026beta; expression (Fig. 7A). Our \u003cem\u003ein\u003c/em\u003e\u003cem\u003e\u0026nbsp;vitro\u003c/em\u003e experiment suggested that the HIF-1\u0026alpha; inhibitor PX-478 significantly inhibited expression of IL-1\u0026beta; (Fig. 7B) and activation of the downstream pathway and collagen expression during PA treatment (Fig. 7C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNAFLD is a multifactorial, multistage, and slowly progressing disorder. Due to its strong insidious onset and few disease-related clinical symptoms in the NAFL stage, lipid metabolism and the fibrotic mechanism in NAFLD are still not fully understood. NASH and HSCs are traditionally considered the sources of fibrosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This study aimed to investigate hepatocytes in the NAFL stage as such a source. In this article, we reported the parenchymal-cell origin of fibrosis during NAFL. To our knowledge, this is the first report of fibrosis occurring in the NAFL stage. Compared with injury- and inflammation-activated fibrosis (repair fibrosis), this fibrosis, which derived from parenchymal-cell adaptation (adaptive fibrosis), was mild. This parenchymal fibrosis was initiated by microenvironmental stress and the inflammatory mediator autocrine.\u003c/p\u003e\u003cp\u003eInflammatory cells always infiltrate secondary to cell or tissue injury or necrosis, such as hepatitisvirus, alcohol-induced injury, or carbon tetrachloride\u0026ndash;induced injury. However, in many subclinical situations (\u003cem\u003ee.g.\u003c/em\u003e, microenvironmental change), parenchymal cells are in a state of adaptation rather than necrosis or injury. Inflammation is considered absent at this stage, and therefore many related phenomena cannot be well interpreted through the lens of inflammation. Our results suggested a state of \u0026ldquo;molecular inflammation\u0026rdquo; in which inflammatory mediators were active but leukocytic infiltration was absent. Inflammatory molecules were secreted by parenchymal cells during stress adaptation and epigenetic changes. To distinguish from injury-induced inflammation dependent on leukocytic infiltration, this molecular inflammation dependent on parenchymal-cell adaptation could be termed \u0026ldquo;adaptive inflammation.\u0026rdquo; According to our results, adaptive inflammation triggered the same signaling pathway to help parenchymal cells self-regulate and thus adapt to changes in the environment. In this study, the HLME induced hepatocytes to autosecrete IL-1β, resulting in collagen secretion. Many studies have reported that parenchymal cells, including various tumor cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], secrete inflammatory mediators under physiological conditions [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] or during adaptation to stress [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, molecular inflammation should be considered a common pathological phenomenon that is often ignored. This concept could help introduce the idea of inflammatory mechanisms to various pathological adaptive processes. On the other hand, inflammatory mediators mainly activate other immunocytes to initiate inflammation cascade reactions or activate interstitial cells to initiate the repair process through paracrine or endocrine mechanisms. In some cases, inflammatory mediators can also regulate themselves by positive or negative feedback. Herein, we showed that the inflammatory mediator of parenchymal cells acted on itself, mainly through autocrine regulation, because HSCs did not appear to be activated by the high level of IL-1β during NAFL development.\u003c/p\u003e\u003cp\u003eThis study took microenvironmental change as the breakthrough point of the NAFL fibrotic mechanism. The stress of continuous lipid deposition could cause development of a microenvironment unsuited for cell survival. Enlarged hepatocytes can compress the sinus and block influx of red blood cells. In this study, we detected FFA concentrations and HIF-1α and YAP expression in liver tissues. The results supported the existence of hyperlipid, hypoxic, and mechanical-compression stress. Interestingly, the results of the \u003cem\u003ein vitro\u003c/em\u003e experiments indicated that PA (high lipid) was an independent factor inducing high expression of HIF-1α and YAP in hepatocytes.\u003c/p\u003e\u003cp\u003eHIF is a key oxygen-regulated transcription activator, and HIF-1α is an important functional component that is extremely sensitive to oxygen concentration [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The latter plays an important role in regulating cellular responses to hypoxia. Experiments have shown that HIF-1α is also induced in NAFLD hepatocytes by different causes [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]; for example, expression of HIF-1α increases in hepatocytes from obese patients with NAFLD [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Studies have also found that HIF-1α in hepatocytes mediates the development of NAFLD fibrosis in NAFLD mouse models [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], such as by promoting NAFLD fibrosis via activation of the phosphatase and tensin homolog (PTEN)/p65 signaling pathway [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Multiple downstream target genes of HIF-1α are involved in processes of disease regulation, including glycolysis, erythropoiesis, angiogenesis, and vascular remodeling. Previous studies have shown that IL-1β is a direct target of HIF-1α [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and both human and mouse IL-1β promoter regions carry multiple HIF-1α\u0026ndash;binding sites [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Additionally, the HIF-1α/IL-1β signaling pathway enhances liver cancer epithelial\u0026ndash;mesenchymal transition (EMT) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In this article, we showed that HIF-1α activated IL-1β expression and the downstream inflammatory pathway in hepatocytes. YAP, as a transcription regulatory cofactor, plays a regulatory role in the Hippo signaling pathway [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Mechanical signaling is the second important factor in regulation of YAP function [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. YAP exerts regulatory effects by nuclear localization and binding to members of the transcriptional enhancer factor domain family (TEAD) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. We showed that HIF-1α\u0026rsquo;s activation of IL-1β expression was associated with YAP binding and activity. IL-1β is a key inflammatory factor in the NASH stage and is closely related to NAFLD fibrosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. During the NASH stage, pro-inflammatory cytokines and chemokines are produced by hepatocytes and Kupffer cells. No inflammatory response is believed to occur in the liver during this stage. Our results demonstrated that in the HFD-induced NAFL stage, HIF-1α/YAP might also induce hepatocytic expression of IL-1β and promote development of fibrosis. This suggested that a HLME promoted expression of HIF-1α and YAP in hepatocytes, activated the secretion of the pro-fibrotic inflammatory factor IL-1β by hepatocytes, and then acted on itself, leading to occurrence of NAFL fibrosis. HIF-1α interacted with YAP to jointly activate transcription of \u003cem\u003eIl1β\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). However, some key points need further clarification. For example, can HIF-1α directly activate the expression of collagen genes without the intervention of IL-1β? Furthermore, is YAP necessary for HIF-1α to function?\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, this study reported an important fibrotic process during the adaptation stage of parenchymal cells that included the inflammatory factor autocrine. These two phenomena suggested a new concept in the pathological process of inflammation; i.e., molecular inflammation occurring in parenchymal cells independent of leukocytes. Some problems need to be investigated further: first, how the regulatory mechanisms of high lipid (FFA, PA), hypoxia (HIF-1α), and mechanical microenvironment (YAP) induced high expression of IL-1β; second, the epigenetic mechanism of collagen expression in hepatocytes. In summary, HIF-1α, YAP, and IL-1β could be valuable targets to inhibit hepatocytic fibrosis induced by a HLME.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAb: antibody; AP-1: activating protein-1; BSA: bovine serum albumin; CD: chow diet; cDNA: complementary deoxyribonucleic acid; COL1A1: collagen type 1 \u0026alpha;1 chain; COL3A1: collagen type 3 \u0026alpha;1 chain; DAB: diaminobenzidine; DAPI: 4\u0026prime;,6-diamidino-2-phenylindole; DMEM: Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium; ECM: extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; gDNA: genomic deoxyribonucleic acid; HFD: high-fat diet; HIF: hypoxia-inducible factor; HLF: HIF-1\u0026alpha;\u0026ndash;like factor; HLME: high-lipid microenvironment; HRP: horseradish peroxide; HSCs: hepatic stellate cells; IF: immunofluorescence; IgG: immunoglobulin G; IHC: immunohistochemistry; IL-1\u0026beta;: interleukin-1\u0026beta;; LDL-C: low-density lipoprotein cholesterol; MAFLD: metabolic-dysfunction\u0026ndash;associated fatty-liver disease; MMP-2: matrix metalloproteinase 2; MOD: mean optical density; mRNA: messenger ribonucleic acid; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty-liver disease; NASH: nonalcoholic steatohepatitis; NF-\u0026kappa;B: nuclear factor \u0026kappa;-light-chain-enhancer of activated B cells; OD: optical density; PA: palmitic acid; PFA: paraformaldehyde; PVDF: polyvinylidene difluoride; PMSF: phenylmethylsulfonyl fluoride; RIPA: radioimmunoprecipitation assay; RNA: ribonucleic acid; RT: room temperature; RT-PCR: real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; siRNA: short interfering ribonucleic acid; SPF: specific-pathogen\u0026ndash;free; TEAD: transcriptional enhancer factor domain family; TFs: transcription factors; TGF: transforming growth factor; TIMP: tissue inhibitor of metalloproteinases; WB: western blot; YAP: yes-associated protein.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e \u003cstrong\u003estatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [Grant Nos. 81670776 and 81970734] to JGZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJGZ contributed to the study concept and design. XWS and JGZ contributed to the analysis and interpretation of data and drafted the manuscript. HZ began the investigation of hepatocytic fibrosis and completed animal acquisition. TTN completed the study on autocrine regulation mechanisms and also performed the image editing. All authors contributed to the acquisition of data and critical revision of the manuscript. All authors approved the final manuscript prior to submission. HZ and TTN contributed equally to this work; JGZ, XWS and HA are equal co-corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHua Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eTing-Ting Niu, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eWen-Jiao Lin, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eXiao Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eXuan Xuan Kou, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eJing-Xin Deng, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eLi-Li Yang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003ePeng Fei Xin, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eXue Gao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eCai Yun Zhou, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eWen Min Gao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eLan Xie, Department of Sports Medicine, Third Hospital, Peking University, Peking, 100085, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eYu-Meng Hao, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eZhao-Yang Li, pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eJian-Gang Zhang, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. Tel: 86 15095387695; E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eXiao Wei Sun, Pathology Institute, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China. E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003eHua AI, Department of Sports Medicine, Third Hospital, Peking University, Peking, 100085, China. E-mail: [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaurice J, Manousou P. 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World J Gastroenterol. 2015, 21(45):12787-12799.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nonalcoholic fatty liver, IL-1β, autocrine, hepatocytic fibrosis, HIF-1α/YAP, HLME","lastPublishedDoi":"10.21203/rs.3.rs-6538340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6538340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNonalcoholic fatty-liver disease (NAFLD) has become the most common type of chronic liver disease and a global public-health problem. Fibrosis is always secondary to nonalcoholic steatohepatitis (NASH), an inflammatory fibrotic process in which hepatic stellate cells (HSCs), macrophages, and hepatic sinusoidal endothelial cells (HSECs) may all play roles. However, steatosis-associated fibrosis (\u003cem\u003ei.e.\u003c/em\u003e, NAFL fibrosis) and the key role of hepatocytes in liver fibrosis are not well understood.\u003c/p\u003e\n\u003cp\u003eIn this study, we explored the mechanisms of high-lipid microenvironment (HLME)-induced NAFL hepatocytic fibrosis and interleukin 1β (IL-1β) regulation through the hypoxia-inducible factor 1α (HIF-1α)/yes-associated protein (YAP) signaling pathway. C57BL/6J mice were fed a high-fat diet (HFD) for 1–3 months. Moreover, Hep G2 and Hepa 1-6 cells were treated with palmitic acid (PA) for 24 h to establish a NAFL model. Furthermore, we performed immunohistochemical (IHC) staining, western blot (WB) analysis, real-time quantitative polymerase chain reaction (RT-qPCR), silencing, and co-immunoprecipitation (Co-IP) experiments. The results showed that a HFD or PA induced high expression of collagen type I α1 chain and type III α1 chain (COL1A1, COL3A1), matrix metalloproteinase 2 (MMP-2), HIF-1α, YAP, and IL-1β in hepatocytes compared with the control group. IL-1β receptor was highly expressed in hepatocytes, and HIF-1α/YAP regulated the expression of IL-1β. In conclusion, hepatocytes contributed to NAFL fibrosis by IL-1β autocrine regulation via HIF-1α/YAP signaling in a HLME.\u003c/p\u003e","manuscriptTitle":"HIF-1α/YAP induces interleukin 1β autocrine in hepatocytes, leading to hepatocytic fibrosis: adaptive fibrosis in nonalcoholic fatty liver","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 08:19:08","doi":"10.21203/rs.3.rs-6538340/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11c88601-8af9-4f21-932c-86e2195dfccd","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55694777,"name":"Health sciences/Molecular medicine"},{"id":55694778,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":55694779,"name":"Health sciences/Pathogenesis/Inflammation"}],"tags":[],"updatedAt":"2025-10-06T08:19:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 08:19:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6538340","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6538340","identity":"rs-6538340","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}