Zhou MY, Fang RF, Tang H, Xia XX, Xie Q, Yao DF, Sai WL, Yao M. Activated cGAS-STING signaling promotes malignancy in metabolic dysfunction-associated fatty liver disease via mitochondrial DNA and immune cell dysfunction. World J Hepatol 2026; 18(7): 121423 [DOI: 10.4254/wjh.121423]
Corresponding Author of This Article
Min Yao, PhD, Postdoc, Professor, Vice Director, Department of Immunology, Medical School of Nantong University, Nantong University, No. 19 Qixiu Road, Nantong 226001, Jiangsu Province, China. erbei@ntu.edu.cn
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Zhou MY, Fang RF, Tang H, Xia XX, Xie Q, Yao DF, Sai WL, Yao M. Activated cGAS-STING signaling promotes malignancy in metabolic dysfunction-associated fatty liver disease via mitochondrial DNA and immune cell dysfunction. World J Hepatol 2026; 18(7): 121423 [DOI: 10.4254/wjh.121423]
World J Hepatol. Jul 27, 2026; 18(7): 121423 Published online Jul 27, 2026. doi: 10.4254/wjh.121423
Activated cGAS-STING signaling promotes malignancy in metabolic dysfunction-associated fatty liver disease via mitochondrial DNA and immune cell dysfunction
Meng-Ya Zhou, Hao Tang, Min Yao, Department of Immunology, Medical School of Nantong University, Nantong University, Nantong 226001, Jiangsu Province, China
Rong-Fei Fang, Department of Gastroenterology, The Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Xiao-Xiao Xia, Qun Xie, Department of Infectious Diseases, Haian People’s Hospital, Haian 226600, Jiangsu Province, China
Deng-Fu Yao, Wen-Li Sai, Research Center of Clinical Medicine, The Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Author contributions: Zhou MY and Fang RF contributed equally to this article, they are the co-first authors of this manuscript; Zhou MY, Fang RF, and Tang H collected blood specimens for analysis; Zhou MY, Fang RF, and Yao DF conceptualized and designed the research; Xia XX, Sai WL, and Yao M acquired the funding and wrote the manuscript; Zhou MY, Xie Q, and Yao M were instrumental and responsible for data reanalysis and reinterpretation, figure plotting, comprehensive literature search, and the preparation and submission of the current version of the manuscript, with a new focus on immunological functions for the potential underlying mechanisms of metabolic dysfunction-associated fatty liver disease; Yao DF, Sai WL, and Yao M are crucial for the publication of this manuscript; Sai WL and Yao M contributed equally to this article, they are the co-corresponding authors of this manuscript; and all authors thoroughly reviewed and endorsed the final manuscript.
Supported by National Natural Science Foundation, No. 32470985; Nantong Science and Technology Programs, No. MS2024051; Nantong Control of Infectious Diseases, No. NTCRB2025016; and Nantong Health Commission of China, No. QN2025064.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Nantong University, China, approval No. P20230327-001.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: The authors declare that they have no conflict of interest.
Corresponding author: Min Yao, PhD, Postdoc, Professor, Vice Director, Department of Immunology, Medical School of Nantong University, Nantong University, No. 19 Qixiu Road, Nantong 226001, Jiangsu Province, China. erbei@ntu.edu.cn
Received: March 24, 2026 Revised: April 14, 2026 Accepted: June 1, 2026 Published online: July 27, 2026 Processing time: 122 Days and 6.3 Hours
Abstract
BACKGROUND
Activated cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) is associated with hepatocellular carcinoma (HCC) progression. However, its mechanisms in metabolic dysfunction-associated fatty liver disease (MAFLD) progression remain to be identified.
AIM
To investigate the dynamic alterations in cGAS-STING activation and hepatic immune cells during MAFLD malignancy.
METHODS
Approved by ethics committees, dynamic models of MAFLD were generated in Sprague-Dawley rats fed a high-fat diet supplemented with 2-fluorene acetylamino acid. Livers were grouped into MAFLD, metabolic dysfunction-associated steatohepatitis, liver cirrhosis and HCC groups on the basis of hematoxylin and eosin staining, with healthy rats used as controls. The mitochondrial ultrastructures were observed by electron microscopy. Hepatic immune cells were analyzed by single-cell sequencing. Carnitine palmitoyl transferase II (CPT-II or CPT2) and cGAS-STING were detected by enzyme-linked immunosorbent assay, real time quantitative polymerase chain reaction, immunohistochemistry, and multicolor immunofluorescence.
RESULTS
Dynamic models of MAFLD malignancy were successfully established, and hepatocytes with diffuse macrovascular steatosis, unequal nuclear sizes, disordered arrangements and damaged mitochondrial ultrastructures were identified. The activated hepatic STING expressions at the mRNA or protein levels were significantly greater (P < 0.001) in the liver cirrhosis and HCC groups. CPT2 was markedly downregulated (P < 0.001). Serum alpha-fetoprotein and Wnt3a levels were significantly increased (P < 0.001). The expression levels of vimentin-1, interferon-I, nuclear factor kappa-B, transforming growth factor-β1 and tumor necrosis factor-α progressively increased (P < 0.001). Mechanistically, accumulated lipids damage mitochondria, resulting in the downregulation of CPT2 or mitochondrial DNA expressions and the activation of the cGAS-STING pathway. Alterations in immune cells with a weak inflammatory microenvironment promoted MAFLD transformation and malignancy.
CONCLUSION
Activation of cGAS-STING in MAFLD with mitochondrial damage promoted hepatocarcinogenesis via an immune escape mechanism.
Core Tip: Hepatocyte cyclic guanosine monophosphate-adenosine monophosphate synthase, as a DNA sensor, recognizes damaged mitochondrial DNA with aberrant lipid metabolism to activate stimulator of interferon genes to trigger the expressions of inflammatory factors, such as type-I interferon, which might be closely related to metabolic dysfunction-associated fatty liver disease (MAFLD) progression. However, the molecular mechanisms underlying MAFLD malignancy are still unclear. In this study, the activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes signaling pathway was systematically evaluated in a lipid accumulation model, and the pathogenesis of MAFLD was clarified by evaluating the interactions among hepatocyte injury, inflammatory factors, and immune cells in hepatocarcinogenesis.
Citation: Zhou MY, Fang RF, Tang H, Xia XX, Xie Q, Yao DF, Sai WL, Yao M. Activated cGAS-STING signaling promotes malignancy in metabolic dysfunction-associated fatty liver disease via mitochondrial DNA and immune cell dysfunction. World J Hepatol 2026; 18(7): 121423
The incidence of liver diseases caused by hepatitis B virus or hepatitis C virus infection is declining[1,2], while metabolic dysfunction-associated fatty liver disease (MAFLD) is becoming the most common chronic liver disease[3,4]. The pathogenesis of MAFLD is complex, and abnormal fat accumulations in the liver lead to hepatocyte inflammation with mitochondrial DNA (mtDNA) damage[5,6]. MAFLD is not only related to metabolic diseases, such as obesity, drug resistance, and cardiovascular diseases[7,8], but is also accompanied by the activation of multiple signaling pathways. Hepatocyte malignancy can progress to liver cirrhosis (LC) or hepatocellular carcinoma (HCC) via the activation of multiple signaling pathways[9,10]. Recently, the activated cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in MAFLD has attracted the attention of basic and clinical researchers and has become a hotspot in molecular oncology.
Hepatocyte cGAS acts as a DNA sensor and can recognize damaged mtDNA fragments from aberrant lipid metabolism, activate STING to trigger the expressions of inflammatory factors such as type-I interferon (IFN-I)[11], mediate lipid metabolism and the inflammatory response of hepatocytes, and regulate apoptosis pathways, which may be closely related to MAFLD progression[12,13]. The association between the increased incidence of MAFLD in the general population and HCC is still not fully understood, especially the intrinsic connection between the activation of cGAS-STING signaling and malignant hepatocyte transformation[14]. However, the alterations or molecular mechanisms of cGAS-STING signaling during MAFLD malignancy are still unclear. The objectives of this study were to investigate the dynamic changes in GAS-STING signaling and the interactions among hepatocyte injury, inflammatory factors and immune cells in the process of progression from a healthy liver to hepatocarcinogenesis; clarify the pathogenesis of MAFLD; and discuss the application prospects of its key molecules.
MATERIALS AND METHODS
MAFLD models and pathological grouping
MAFLD models were approved (No. P20230327-001) by the Animal Care and Use Committee of Nantong University, China. Male Sprague-Dawley rats (SD, n = 66, 4-week-old, body weight 105-110 g) were fed a high-fat diet (HFD) and then fed a HFD supplemented with 0.05% 2-fluorenyl acetamide (2-FAA, Sigma, United States) to induce hepatocarcinogenesis. All procedures performed on the animals were conducted in accordance with previously described methods[15]. The rats were sacrificed every two weeks, and the livers were divided into MAFLD, metabolic dysfunction-associated steatohepatitis (MASH), LC, and HCC groups on the basis of liver pathological examinations with hematoxylin and eosin staining; normal rat livers were used as controls.
HCC tissues
With the use of data from the TCGA database (https://www.cancer.gov/tcga), carnitine palmitoyl transferase II (CPT-II/CPT2) mRNA (CPT2) data from human liver cancer (liver hepatocellular carcinoma, n = 269) or normal liver (n = 50) samples were analyzed. With the approval of the Nantong University Ethics Committee (No. 2021-43), 12 cases of postoperative HCC and paired adjacent noncancerous tissues were collected from patients with MAFLD-related HCC on the basis of pathological diagnoses. Electron microscopy was used to examine mitochondrial damage and mtDNA function, and the transcriptional status of CPT2 on the inner mitochondrial membrane (IMM) was evaluated. The expressions of CPT-II and STING-related inflammatory mediators [e.g., IFN-I, nuclear factor kappa-B (NF-κB), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α)] were analyzed by immunohistochemistry or enzyme-linked immunosorbent assay (ELISA).
Western blot analysis
Rat livers (100 mg) were homogenized in radioimmunoprecipitation assay lysis buffer and phenylmethanesulfonylfluoride (Multi Sciences Biotech Co., China). Manual steps for protein extraction were performed, and the proteins were centrifuged at 12000 rpm for 15 minutes. The proteins were subsequently quantified using a Bicinchoninic acid kit (Epizyme Biomed. Technol., Shanghai, China), mixed with 5 × loading buffer (Solarbio, Beijing, China) at a ratio of 4:1 in radioimmunoprecipitation assay buffer, boiled for 1 min, and loaded onto a 4%-15% sodium dodecyl sulfate polyacrylamide gel for electrophoresis. The proteins were subsequently transferred onto a 0.22-μm nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, United States), blocked with 5% skim milk at 25 °C for 2 hours, and then incubated first with antibodies overnight at 4 °C. The next day, the membrane was washed 3 times in Tris-buffered saline Tween-20 solution, incubated at 25 °C for 2 hours with horseradish peroxidase (GE Healthcare, United States)-labeled secondary antibodies, and imaged using a gel imaging system (BD Bioscience, United States), after which the proteins were quantified using ImageJ software. The antibodies used included STING (Abbkine, Wuhan, China); CPT-II (Abcam, ab181114, United States); Wnt3a (Huamei Biotech, China); and vimentin-1 (VIM-1) (Fisher, China).
Total RNA purification
Total RNA from 100 mg of liver tissue was purified in RNAlater (Ambion, TX, United States) according to the instructions for TRIzol reagent (Gibco BRL, United States) and dissolved in 30 μL of diethyl pyrocarbonate water. The quantification and purity of the RNAs were determined by a NanoDrop ND1000 spectrophotometer and an Agilent RNA 6000 NANO KIT.
Total RNA was reverse transcribed into complementary DNA. CPT-II and STING genes were amplified using SYBR Green reagent (TaKaRa, Japan) on an ABI Pri00 Real-Time polymerase chain reaction System (Applied Biosystems, United States), with β-actin as the internal reference. The primer sequences used were as follows: CPT-II gene[16] amplification primers (forward: 5’-TGGTCGATGAAAAGCCTCCA-3’ and reverse: 5’-TTGGGATTCCGCTCACAC-3’); STING gene amplification primers (forward: 5’-CCTGGACCTTCAGAGCTTGG-3’ and reverse: 5’-CTGCAGTCCTGGCAAGATCA-3’)[17]; and β-actin gene primers (forward: 5’-GAAGACTCTGGCATGCTA-3’ and reverse: 5’-CACGCTGAGCCAGTCAGTGTA-3’). The polymerase chain reaction conditions were as follows: Denaturation at 94 °C for 3 minutes, followed by annealing at 62 °C for 30 seconds, and extension at 72 °C for 30 seconds. This was repeated for 40 cycles. Each sample was tested in triplicate, and the mRNA expression levels of the respective genes were calculated using the 2-ΔΔCt method.
Immunofluorescence analysis
Multiplex immunofluorescence staining was performed using a PerkinElOPAL IHC Kit (ABSIN, Shanghai, China). After blocking with antibody diluent, primary antibodies against STING (1:1000; Abcam, United Kingdom); Wnt3a (Huamei Biotech, China); and VIM-1 (1:1000; Abcam, United Kingdom) were added and incubated for 1 hour; the negative control group was treated with antibodies. Detection was performed using OPAL Polymer horseradish peroxidase Antibody (Waltham, MA, United States), and signals were visualized using OPAL Tyramide Signal Amplification. The sections were placed in ethylenediaminetetraacetic acid buffer (pH 8.0) and heated in a microwave. After staining, the samples were washed and mounted with glycerin in the dark at 25 °C. Two pathologists independently and blindly evaluated the samples and calculated scores on the basis of the intensity and quantity of positive cells using Image-Pro Plus 6.0 software (Rockville, MD, United States).
Single-cell RNA sequencing
Single-cell suspensions were prepared from five liver tissue samples from MAFLD, MASH, LC, HCC and normal rats. The cell activities were determined, and the samples were subsequently centrifuged at 4 °C and 300 × g for 5 minutes after they were passed through a cell strainer. After the supernatant was discarded, 1000 μL of cell protective solution was added, the prepared single-cell samples were resuspended, and single-cell RNA sequencing was performed via the 10 × genomics method on Gene Denovo. The numbers and ratios of hepatocytes at the single-cell level were determined via R language (4.1.3), cellranger (6.1.0), urat (4.1.0), and CellChat (V2.1.0).
Immunohistochemistry
Liver immunohistochemistry (IHC) was performed using a nonbiotin detection system. Paraffin sections were deparaffinized, rehydrated, and washed with water, after which antigen retrieval was performed. Liver sections were incubated with peroxidase blocking reagent at 25 °C, followed by overnight incubation with primary rabbit anti-rat CPT-II (Abcam, ab181114, China). Liver sections were then incubated with enhancer solution and enzyme-labeled anti-rabbit IgG polymer, developed with fresh diaminobenzidine tetrachloride solution, counterstained with hematoxylin, blued, dehydrated, cleared in xylene, and mounted with neutral gum. The sections were observed and photographed using an OLYMPUS microscope. The integrated optical density values and areas of the observed fields were measured using Image-Pro Plus 6.0 software to calculate the relative levels of positive staining and image analysis.
Mitochondrial ultrastructure
Livers were cut into small pieces, fixed with glutaraldehyde/polyform-aldehyde, washed in phosphate buffer saline (pH 7.4) for 30 minutes, and fixed in osmium tetroxide (OsO4) at 4 °C. The samples were immersed in low and high concentrations of dimethyl sulfoxide, frozen in liquid nitrogen, and thawed in dimethyl sulfoxide. They were then fixed in OsO4, stained with tannic acid, dehydrated, frozen in tert-butanol (ES2030; Hitachi, Japan), dried with silver paint, and ion-sputtered with a platinum-palladium coating (E1010; Hitachi). Cellular ultramicrostructures were evaluated using field emission scanning electron microscopy (S4100; Hitachi, Japan). Livers were cut into small pieces, immersed in glutaraldehyde/polyformaldehyde at 4 °C, fixed in OsO4 in phosphate buffer saline, dehydrated, embedded in epoxy resin (Epon 812), sectioned, and observed under an HT7700 transmission electron microscope (Japan) to examine changes in mitochondrial ultrastructure.
ELISA
Serum concentrations of STING, Wnt3a, IFN-I, NF-κB, and TGF-β1 in rats were quantitatively determined by ELISA according to the instructions of the STING (Wuhan Fine Biotech, China); Wnt3a (ADL, United States); IFN-I (Cusabio, United States); NF-κB (Cusabio, United States); and TGF-β1 (Cusabio, United States) kits. Their standard curves were generated, and the respective concentrations or tissue ratios were calculated.
Statistical analysis
Quantitative data are expressed as the means ± SDs, while qualitative data are summarized as counts (n) and percentages (%). Analysis was performed using SPSS version 23.0 statistical software. The results were analyzed using one-way analysis of variance, Student’s t test and χ2 tests. Image-Pro Plus 6.0, ImageJ, GraphPad Prism 5.0 and Photoshop were used to construct the figures. A P value < 0.05 was considered to indicate statistical significance.
RESULTS
MAFLD models and histopathological grouping
Liver histopathologies and dynamic alterations under lipid accumulation during MAFLD malignancy are shown in Figure 1. Obvious changes in the gross liver (Figure 1A) progressed from a bright red liver overall (Figure 1A1) to nodules on the liver surface following excessive fat intake (Figure 1A2-5). After hematoxylin and eosin staining (Figure 1B), the livers were divided into the normal control (NC) (Figure 1B1), MAFLD, MASH, LC, and HCC groups. The granular degeneration of hepatocytes at the early stage was observed with occasional atypical nuclei due to massive amounts of lipids (Figure 1B2). The number of cell layers in the hepatic plates at the middle stage significantly increased, the focal areas exceeded three layers (Figure 1B3), the nuclei were enlarged or had a marked state of fibrosis or cirrhosis, and a very slight increase in the nuclear/cytoplasmic ratio was occasionally detected (Figure 1B4). The liver structure was lost at the late stage, the cells were arranged in nests or thick cords, and the nuclei were coarsened or the nuclear/cytoplasmic ratio increased (Figure 1B5). Lipids were stained with oil red O, which revealed numerous vacuoles (Figure 1C). Compared with those in the NC group (Figure 1C1), the lipid levels in the MAFLD (t = 14.158; P < 0.001; Figure 1C2), MASH (t = 8.498; P = 0.004; Figure 1C3), LC (t = 14.133; P = 0.002; Figure 1C4), and HCC groups (t = 9.875; P < 0.001; Figure 1C5) were significantly greater, and massive abnormal fat accumulations were observed in the livers of all the groups except for the NC group. A model was used to analyze the activation and mechanisms of the cGAS-STING pathway.
Figure 1 Rat liver histopathological grouping and lipid accumulations.
A: Livers from rats fed a normal diet (A1), a high-fat diet (HFD, A2), or a high-fat diet plus 2-fluorenyl acetamide at the early (A3), middle (A4) or last stage (A5); B: Histopathological groupings of the normal control (B1, n = 12), metabolic dysfunction-associated fatty liver disease (B2, n = 12), metabolic dysfunction-associated steatohepatitis (B3, n = 17), liver cirrhosis (B4, n = 15) and hepatocellular carcinoma (B5, n = 10) groups; C: Sections corresponding to the above livers were subjected to Oil Red O staining, and lipid accumulations in hepatocytes from metabolic dysfunction-associated steatohepatitis (C2), metabolic dysfunction-associated steatohepatitis (C3), liver cirrhosis (C4) and hepatocellular carcinoma (C5) rats but not from normal control rats (C1) were evaluated. NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma; HE: Hematoxylin and eosin.
Figure 3 Dynamic changes in liver stimulator of interferon genes expressions at the mRNA or protein level.
A: Stimulator of interferon genes (STING) mRNA amplification plot by real time quantitative polymerase chain reaction; B: Melting curve of STING mRNA; C: Amplification curves of STING mRNA; D: Liver STING mRNA expressions among the different groups (n = 5/each); E: Liver STING immunoblot analysis in the normal control: 1, 11, and 13; metabolic dysfunction-associated fatty liver disease: 2, 5; metabolic dysfunction-associated steatohepatitis: 3, 4, 7; liver cirrhosis: 6, 8, 9; and hepatocellular carcinoma: 10, 12, 14 (n = 5/each) groups; F: Analysis of the relative ratios of liver STING to β-actin in the different groups. cP < 0.001 vs the control group. NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma; STING: Stimulator of interferon genes.
Figure 5 Dynamic alteration of cell clustering in the liver.
A: Clustering of liver cells in control rats; the numbers of cells in liver cell subpopulations; B: Dynamic changes in cell subpopulations in different livers (n = 5; that is, normal control, metabolic dysfunction-associated fatty liver disease, metabolic dysfunction-associated steato-hepatitis, liver cirrhosis and hepatocellular carcinoma) during malignant progression. The following cell types were identified: Hepatocytes (0, 20, 23, 24, 27); B cells (2, 25, 28); endothelial cells (3, 1; natural killer cells (6, 10, 13); T cells, monocytes, macrophages (7, 9, 12, 15); dendritic cells (8); neutrophils (11); epithelial cells (19); red blood cells (21); plasma cells (26); hepatocytes (29); and dendritic cells (33). Unidentified cells (1, 4, 5, 14, 16, 18, 22, 30, 31, 32). t-SNE-1: T-distributed stochastic neighbor embedding-1; t-SNE-2: T-distributed stochastic neighbor embedding-2; NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma.
Biochemistry during MAFLD malignancy
The dynamic changes in the levels of liver enzymes, lipids and tumor markers during MAFLD malignancy are shown in Table 1. The morphological changes in rat hepatocytes after 2-FAA induction included MASH, LC and HCC, accompanied by cell injury manifested by the release of alanine aminotransferase and aspartate aminotransferase into the blood, and their activities significantly increased. The concentrations of triglycerides (TG), total cholesterol, and low-density lipoprotein (LDL) in the blood, except for high-density lipoprotein, were also significantly greater than those in the NC group. The differences in the serum levels of alpha-fetoprotein (AFP) and Wnt3a were significant in the LC group (P < 0.05) but were extremely high in the HCC group (P < 0.001). These data indicated that a HFD plus 2-FAA promoted MAFLD malignancy, and the presence of HCC was confirmed by liver histopathology.
Table 1 Serum biochemistry parameters and hepatocellular carcinoma markers during metabolic dysfunction-associated fatty liver disease progression, mean ± SD.
Mitochondrial damage with respect to CPT2 expression status
The localization of mitochondrial CPT2 transcription and its expression status in liver tissues are shown in Figure 2. CPT2 is localized to the IMM and is a key enzyme for fatty acid entry into mitochondrial oxidation (Figure 2A). Compared with that in the normal liver group in the TCGA database, CPT2 expressions were significantly downregulated in the liver hepatocellular carcinoma group (n = 269; P < 0.001; Figure 2B). Damaged mitochondria were observed in the HCC tissues (Figure 2C upper). Compared with those in the paired non-HCC group (n = 12), CPT2 expressions were significantly lower in the HCC group (n = 12; P < 0.001; Figure 2C lower). CPT-II expressions in MAFLD model livers were analyzed by IHC, and CPT-II expressions were significantly lower in HCC tissues (P < 0.001; Figure 2D). The results of the quantitative analysis of the IHC, liver and serum levels of mitochondrial CPT-II during MAFLD malignancy are shown in Table 2. CPT-II expressions progressively decreased in the NC, MAFLD, MASH, LC and HCC groups. The specific concentrations of liver CPT-II (ng/mg tissue protein) and serum CPT-II activity indicated that decreased CPT-II severely hindered the entry of lipids into mitochondrial β-oxidation and exacerbated lipid accumulations, thereby promoting the malignancy of hepatocytes.
Table 2 Dynamic changes in liver carnitine palmitoyl transferase-II specific concentration and activity, mean ± SD.
The dynamic alterations in liver STING expressions at the mRNA or protein level are shown in Figure 3. At the mRNA level, amplification profiles (Figure 3A), melting curves (Figure 3B) and amplification curves (Figure 3C) were generated for hepatic STING mRNA. The relative ratios of STING mRNA to β-actin mRNA were lower in the NC group and slightly greater in the MAFLD group. However, the ratios in the MASH, LC, and HCC groups significantly increased (P < 0.001), especially in the MASH group (Figure 3D). Compared with β-actin, the protein expressions of STING in the liver significantly increased, as determined by Western blotting (Figure 3E). The relative ratios of STING to β-actin did not significantly differ between the NC and MAFLD groups. Liver STINGs were progressively overexpressed during hepatocyte malignancy (Figure 3F). Their ratios in the MASH, LC and HCC groups were significantly greater than those in the NC or MAFLD groups (P < 0.001), especially in the HCC group.
Hepatic or serum STING and inflammatory mediators
The dynamic fluorescence intensities of STING expressions in hepatocytes from the NC, MAFLD, MASH, LC and HCC groups are summarized in Table 3. The correlations between upregulated STING and hepatic Wnt3a and VIM-1 in neoplastic hepatocytes are shown in Figure 4. In the livers of the MAFLD model rats during HCC development, entire lipid vacuoles, white STING (Figure 4A), red Wnt3a (Figure 4B), and green VIM-1 (Figure 4C) were observed in hepatocytes, whereas no fluorescence staining was observed in the NC livers. The dynamic changes in circulating STING levels and inflammatory mediator levels (mean ± SD) are shown in Table 4. Serum STING, IFN-I, NF-κB, TNF-α, and TGF-β1 expression levels were significantly upregulated in HCC rats compared with those in NC rats. These data indicated that increased STING and inflammatory mediator levels are associated with hepatocyte malignancy.
Table 3 Dynamic stimulator of interferon genes immunofluorescence intensity during metabolic dysfunction-associated fatty liver disease malignancy.
Dynamic alterations in immune cells during MAFLD malignancy
The cell subpopulations isolated from livers at different stages are shown in Figure 5. All subtypes (0-33 clusters) were identified in healthy liver tissue (Figure 5), of which 24 subtypes were identifiable: Hepatocyes (0, 20, 23, 24, and 27); B cells (2, 25, and 28); endothelial cells [3, 1; natural killer (NK) cells (6, 10, 13)]; T cells, monocytes, and macrophages (7, 9, 12, and 15); dendritic cells (8); neutrophils (11); epithelial cells (19); red blood cells (21); plasma cells (26); hepatocytes (29); and dendritic cells (DCs) (33). In addition, 10 were unidentifiable (e.g., 1, 4, 5, 14, 16, 18, 22, 30, 31, and 32).
Among all the subtypes, hepatocytes, B cells, endothelial cells, NK cells, T cells, monocytes, macrophages, and conventional DCs (cDCs) were the most abundant. These data indicated that MAFLD malignancy was associated with hepatic cell subtypes, especially immune cells.
Alterations in immune cells and the mechanism of cGAS-STING activation are shown in Figure 6. The gene transcription levels in liver subtypes were significantly upregulated in hepatocytes, macrophages, T cells and endothelial cells (Figure 6A). From MAFLD to MASH, genes were most active in the subtypes of macrophages, NK cells and cDCs (Figure 6B), whereas genes in T cells and endothelial cells were downregulated, and liver cell subtypes were capable of secreting proinflammatory cytokines and chemokines that participate in the inflammatory response of MAFLD, with related genes promoting the malignancy of hepatocytes under lipid accumulation. On the basis of the dynamic observations of the model and validation in clinical tissue, we proposed a mechanism by which the cGAS-STING signaling pathway is activated during MAFLD malignancy (Figure 6C). In hepatocytes, excessive lipids damage mtDNA, which is characterized by the loss of CPT2 in the IMM, and the mtDNA produced by cGAS activates downstream STING, leading to abnormal expressions of inflammatory factors. Hepatic lipotoxicity and immune dysfunction act synergistically to promote the malignant transformation of MAFLD.
Figure 6 Alterations in immune cells and a possible mechanism of cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes activation.
A: Clustering of liver immune cells. The cell types identified were hepatocytes, B cells, endothelial cells, natural killer cells, T cells, monocytes, dendritic cells, neutrophils, epithelial cells, red blood cells, plasma cells, hepatocytes, and dendritic cells. Unidentified cells (8, 32, 33); B: Gene transcription of immune cells in metabolic dysfunction-associated fatty liver disease; C: A possible mechanism by which metabolic dysfunction-associated fatty liver disease malignancy is promoted. NK: Natural killer; DC: Dendritic cell; HSC: Hepatic stellate cells; UMAP: Uniform manifold approximation and projection; cGAS: Cyclic guanosine monophosphate-adenosine monophosphate synthase; STING Stimulator of interferon genes; mtDNA: Mitochondrial DNA; LF: Liver fibrosis; AFP: Alpha-fetoprotein; GPC3: Glypican-3; CPT-II: Carnitine palmitoyl transferase II; INF-I: Interferon-I; NF-κB: Nuclear factor kappa-B; TGF-β1: Transforming growth factor β1; TNF-α: Tumor necrosis factor α; MAFLD: Metabolic dysfunction-associated fatty liver disease; LC: Liver cirrhosis.
DISCUSSION
With the effective control of hepatitis B virus and hepatitis C virus infections, the incidence of MAFLD, characterized by abnormal lipid metabolism[18,19], is rapidly increasing worldwide and has become one of the most common chronic liver diseases. The newly discovered activation of the cGAS-STING signaling pathway is closely associated with common chronic liver disease progression. Exogenous or endogenous DNA, such as that involved in hepatitis virus replication and lipid metabolism disorders, is sensed as a cytoplasmic cGAS ligand, which can catalyze cyclic GMP-AMP synthesis to activate STING and release cytokines such as IFN to trigger immune responses[20,21]. The complex pathogenesis of MAFLD includes insulin resistance, cytokine overexpression, mitochondrial damage, lipid peroxidation, lipid metabolism, iron overload, noncoding RNA regulation, genetic or environmental factors, immune changes, and drug effects[22-25]. Although various MAFLD models exist, none can encompass the entire disease spectrum. Therefore, in this study, a dynamic model of MAFLD malignancy was established to explore the activation of the cGAS-STING pathway and analyze its intrinsic links with mitochondrial damage, inflammatory mediators, and immune cells.
Lipid accumulations cause mitochondrial damage in hepatocytes. The CPT system in mitochondria plays a crucial role in the transport of fatty acids across the mitochondrial membrane[26,27]. CPT-II serves as a key enzyme for lipid entry into mitochondrial oxidation, is an important indicator of mitochondrial integrity and is directly associated with the activities of enzymatic reactions that catalyze fatty acid β-oxidation. Additionally, hepatitis virus replication affects CPT-II activity because it is heat sensitive[16,28]. In this study, CPT2 expressions on the IMM were significantly downregulated at the mRNA and protein levels. However, the progressive increase in the levels of lipids (e.g., TG, total cholesterol and LDL) in addition to high-density lipoprotein; liver injury (e.g., alanine aminotransferase and aspartate aminotransferase) and HCC markers (e.g., AFP and Wnt3a) suggests that excessive lipids damage mtDNA, activating the cGAS-STING pathway to exacerbate the inflammatory response and activating Wnt signaling, thereby promoting MAFLD malignancy.
Lipid metabolism disorders can lead to mitochondrial damage in hepatocytes. Activation of the cGAS-STING pathway is associated with damaged mtDNA, lipid antigens, and adipokines[29,30]. STING is expressed and transported from the endoplasmic reticulum to the Golgi complex, where it recruits and phosphorylates TANK-binding kinase 1 and inhibitor of NF-κB kinase, activating interferon regulatory factor 3/7 and NF-κB to trigger the expressions of IFN-I or proinflammatory factors[31]. Additionally, activated inflammatory genes can secrete some mediators, such as TNF-α, NF-κB, and STING[32]. Dynamic modeling first demonstrated the dynamic progressive expressions of TNF-α, NF-κB, STING and Wnt3a in the microenvironment with increasing lipid accumulations across different stages of MAFLD progression. The oxidation of lipids entering mitochondria is impeded, and denaturation is exacerbated, while excessive TG and LDL can activate the Wnt signaling pathway related to hepatocyte transformation[33]. Secreted TGF-β1 activates hepatic stellate cells and accelerates extracellular matrix fibrosis. Sustained inflammation promotes hepatocarcinogenesis[34], suggesting that abnormal activation of cGAS-STING during lipid accumulation could be associated with MAFLD malignancy[5,26].
Dynamic alterations in hepatic cell subpopulations were confirmed during MAFLD progression[35]. Liver immune cells, such as macrophages, Kupffer cells, DCs, neutrophils, NK cells, and NKT cells, coordinate to aid the body’s immune response upon pathogen invasion or hepatitis[36]. In MAFLD, mtDNA damage triggers hepatic immune surveillance, activating the cGAS-STING signaling pathway to regulate immune cells[37], such as NK cells, Kupffer cells (macrophages), T cells, and B cells, thereby enriching and participating in the inflammatory response of MAFLD. In this study, the dynamic alterations in hepatic cell subtypes during MAFLD malignancy were preliminarily investigated. Among the 34 liver cell clusters (10 of which remained to be identified), hepatocytes, B cells, endothelial cells, NK cells, T cells, monocytes, and macrophages were present, and DCs were the most abundant[38].
The genes of some cells were more significantly upregulated, while those of cDCs, macrophages, B cells, NK cells, T cells and endothelial cells were more significantly downregulated. In the MAFLD stage, the genes whose expressions were most significantly upregulated were cDCs, macrophages, B cells, NK cells and T cells, whereas the genes whose expressions were most significantly downregulated were genes in endothelial cells. In the MASH stage, genes in macrophages, NK cells, and cDCs were actively upregulated, whereas genes in T cells and endothelial cells were downregulated. In the HCC stage, genes in macrophages, NK cells, and cDCs were most actively upregulated, intrahepatic cell subpopulations were dynamically enriched, and genes related to the secretion of proinflammatory cytokines and chemokines were either upregulated or downregulated and participate in the inflammatory response[39,40], suggesting that the cGAS-STING signaling pathway is activated in a lipid accumulation microenvironment, promoting the malignancy of hepatocytes[41-43].
Macrophages and Kupffer cells respond to inflammatory stimuli in MAFLD by releasing inflammatory cytokines, triggering interactions with sinusoidal endothelial cells, hepatic stellate cells, neutrophils, monocytes, T cells, and DCs, and thereby participate in the malignant progression of MAFLD[44,45]. A comprehensive analysis of the dynamic model revealed that lipid accumulation is the driving force behind MASH, fibrosis/cirrhosis, and malignant transformation of hepatocytes[46,47]. The balance between hepatic immune tolerance and effective immunity is crucial for liver function and homeostasis. Furthermore, genes in hepatic subpopulations exhibit dynamic changes at the transcriptional level; cGAS detection of mtDNA is closely associated with the activation of the innate immune system and MAFLD malignancy, where inflammatory necrosis-induced DNA damage can chronically activate cGAS-STING to regulate anti-inflammatory and tumor-suppressive effects. Conversely, M2 macrophages, type 2 NKT cells, and regulatory T lymphocytes might be associated with protection against liver injury[48-50].
CONCLUSION
In conclusion, the mechanism underlying the malignant transformation of MAFLD is extremely complex. In lipid-laden hepatocytes, mitochondrial damage results in the release of mtDNA, which directly activates the cGAS-STING pathway. Within the lipid-aggregated microenvironment, this study is the first to preliminarily observe the dynamic alterations in hepatic cell subtypes during MAFLD malignancy. Hepatocyte subtypes undergo dynamic changes, and inflammatory cells are highly active and inhibit the function of immune cells, thereby promoting MAFLD malignancy. Molecules associated with the cGAS-STING pathway, such as CPT2 and STING, serve as molecular targets for early intervention in hepatocyte malignancy. It is therefore inferred that protecting mitochondria, increasing CPT-II activity, reducing reaggregation toxicity and inhibiting inflammatory mediators, as well as specifically blocking STING or using STING as an immune adjuvant to activate immune system activity, could effectively prevent or delay MAFLD malignancy.
ACKNOWLEDGEMENTS
The authors would like to thank the staff of the Research Center of Medical Research, the Affiliated Hospital of Nantong University, China.
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P-Reviewer: Jeong KY, PhD, Assistant Professor, South Korea; Shelat VG, Associate Professor, Singapore; Zheng YY, Associate Research Scientist, Professor, China S-Editor: Bai Y L-Editor: A P-Editor: Wang WB
