LOX-1基因敲除改善小鼠代谢相关脂肪性肝炎（英文）

LOX-1 gene knockout improves metabolic dysfunction-associated steatohepatitis in mice

摘要

目的 代谢相关脂肪性肝炎(metabolic dysfunction-associated steatohepatitis，MASH)作为代谢相关脂肪性肝病(metabolic dysfunction-associated steatotic liver disease，MASLD)的进行性亚型，主要特征是肝脂肪变性、小叶内炎症和气球样变性，可进展至肝纤维化和肝硬化。凝集素样氧化型低密度脂蛋白受体1(lectin-like oxidized low- density lipoprotein receptor-1，LOX-1)是清道夫受体家族中的一员，能够识别和结合氧化型低密度脂蛋白。本研究旨在探究LOX-1在MASH进展中的作用。 方法 通过基因表达综合(Gene Expression Omnibus，GEO)数据库分析检测MASLD小鼠肝LOX-1的表达。免疫荧光染色检测肝纤维化患者肝组织LOX-1和α-平滑肌肌动蛋白(alpha-smooth muscle actin，α-SMA)表达水平。采用CRISPR/胱天蛋白酶9(caspase-9，Cas9)技术构建LOX-1基因敲除(Lox-1^-/-)小鼠，取鼠尾组织进行基因型鉴定和分析。将野生型(wild type，WT)和Lox-1^-/-小鼠随机分为对照组和模型组，构建西方饮食模型，收集小鼠血清和肝组织，进行如下检测：采用生化检测试剂盒检测血清谷草转氨酶(aspartate aminotransferase，AST)和谷丙转氨酶(alanine aminotransferase，ALT)水平；苏木精-伊红(hematoxylin and eosin，HE)染色检测肝组织结构变化；Masson三色染色检测肝胶原纤维形成；油红O染色检测肝脂质沉积；实时荧光定量PCR(real-time quantitative PCR，RT-qPCR)检测肝纤维化标志基因表达。以人肝星状细胞系LX-2为模型，通过siRNA转染敲低LOX-1表达，采用RT-qPCR检测肝纤维化相关标志基因的表达水平，并利用免疫荧光染色观察LOX-1与α-SMA的共定位情况。通过网络药理学预测LOX-1调控MASH的关键靶点，利用检索基因/蛋白质相互作用的搜索工具(search tool for the retrieval of interacting genes/proteins，STRING)数据库构建蛋白质-蛋白质相互作用(protein-protein interaction，PPI)网络，并结合基因本体论(Gene Ontology，GO)富集分析筛选下游通路。 结果 GEO数据库中的GSE30552、GSE40041数据集结果显示，高脂饮食(high fat diet，HFD)模型小鼠和胆管结扎(bile duct ligation，BDL)模型小鼠肝的Lox-1 mRNA水平上调(均P<0.001)。肝纤维化患者肝组织LOX-1和α-SMA表达水平升高。Sanger测序和PCR鉴定结果显示LOX-1基因敲除小鼠构建成功。生化检测结果显示西方饮食模型WT小鼠的血清AST和ALT水平显著升高(均P<0.001)，LOX-1基因敲除后血清AST和ALT水平降低(均P<0.05)。HE染色结果显示，西方饮食模型喂养的WT小鼠肝可见明显的肝细胞气球样变性、脂肪变性、炎症细胞浸润及汇管区纤维增生，LOX-1基因敲除明显改善由西方饮食诱导的肝结构病变。Masson染色结果显示，与对照饲料喂养的小鼠肝组织相比，西方饮食饲料喂养的WT小鼠肝蓝色胶原纤维增多，LOX-1基因敲除抑制胶原纤维形成(均P<0.05)。RT-qPCR结果显示，西方饮食模型小鼠肝的Acta2、Col1a1和Timp1 mRNA水平显著升高，LOX-1基因敲除可降低肝纤维化标志基因表达水平。油红O染色结果显示西方饮食喂养的WT小鼠肝细胞明显增大，呈现大泡性脂肪变性，可见弥漫分布的红色脂滴，LOX-1基因敲除可改善肝脂质沉积(均P<0.001)。RT-qPCR结果显示敲低LOX-1可降低LX-2细胞Acta2、Col1a1和Timp1 mRNA水平(均P<0.05)。免疫荧光结果显示LX-2细胞LOX-1与α-SMA存在共定位，沉默LOX-1可抑制α-SMA表达。网络药理学结果提示LOX-1可能通过调节脂质代谢和胆固醇代谢网络促进MASH进展。 结论 LOX-1基因敲除可改善西方饮食诱导的小鼠MASH，有望成为抗MASH治疗的新靶点。

Abstract

Objective Metabolic dysfunction-associated steatohepatitis (MASH), a progressive subtype of metabolic dysfunction-associated steatotic liver disease (MASLD), is characterized by hepatic steatosis, lobular inflammation, and hepatocyte ballooning, and may further progress to liver fibrosis and cirrhosis. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a member of the scavenger receptor family, recognizes and binds oxidized low-density lipoprotein. This study aims to investigate the role of LOX-1 in MASH progression. Methods LOX-1 expression in MASLD mouse liver was analyzed using Gene Expression Omnibus (GEO) datasets. Immunofluorescence staining was performed to detect LOX-1 and alpha-smooth muscle actin (α-SMA) levels and co-localization in fibrotic liver tissues and LX-2 cells. LOX-1 knockout (Lox-1^-/-) mice were generated using CRISPR/caspase-9 (Cas9) and genotyped by PCR and Sanger sequencing. Wild-type (WT) and Lox-1^-/- mice were randomized into control and Western diet model groups. Serum and liver samples were collected for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measurement by biochemical kits, liver structure evaluation by hematoxylin and eosin (HE) staining, collagen deposition by Masson staining, lipid accumulation by Oil Red O staining, and fibrotic marker gene expression by real-time quantitative PCR (RT-qPCR). Network pharmacology and search tool for the retrieval of interacting genes/proteins (STRING)-based protein-protein interaction (PPI) with Gene Ontology (GO) enrichment were used to predict downstream targets and pathways. Results The results from the GEO datasets GSE30552 and GSE40041 indicated LOX-1 mRNA was upregulated in high fat diet (HFD) and bile duct ligation (BDL) mouse models (both P<0.001). LOX-1 and α-SMA levels were elevated in fibrotic liver tissues. Lox-1^-/- mice were successfully established. Biochemical tests showed that serum AST and ALT levels were significantly elevated in WT mice fed a Western diet (both P<0.001), and these levels decreased after LOX-1 knockout (both P<0.05). HE staining revealed that WT mice on the Western diet exhibited marked hepatocellular ballooning degeneration, steatosis, inflammatory cell infiltration, and periportal fibroplasia, which were significantly ameliorated by LOX-1 knockout. Masson staining demonstrated increased blue-stained collagen fibers in the liver tissues of WT mice fed the Western diet compared with control-diet mice, and LOX-1 knockout inhibited collagen fiber deposition (all P<0.05). RT‑qPCR results showed that hepatic mRNA levels of Acta2, Col1a1, and Timp1 were significantly increased in Western diet-fed mice, and LOX-1 knockout reduced the expression of these fibrogenic marker genes. Oil Red O staining indicated that hepatocytes in WT mice fed the Western diet were notably enlarged, displayed macrovesicular steatosis, and exhibited diffusely distributed red lipid droplets, whereas LOX-1 knockout alleviated hepatic lipid accumulation (both P<0.001). RT‑qPCR results further demonstrated that knockdown of LOX-1 reduced Acta2, Col1a1, and Timp1 mRNA levels in LX‑2 cells (all P<0.05). Immunofluorescence analysis revealed co‑localization of LOX-1 and α‑SMA in LX‑2 cells, and LOX-1 silencing suppressed α‑SMA expression. Network pharmacology suggested LOX-1 may promote MASH via lipid and cholesterol metabolism networks. Conclusion LOX-1 gene knockout ameliorates Western diet-induced MASH in mice and may serve as a potential therapeutic target.

Graphical abstract

关键词

凝集素样氧化型低密度脂蛋白受体1 / 西方饮食模型 / 代谢相关脂肪性肝炎 / 代谢相关脂肪性肝病 / 基因表达综合数据库

Key words

lectin-like oxidized low-density lipoprotein receptor-1 / Western diet model / metabolic dysfunction-associated steatohepatitis / metabolic dysfunction-associated steatotic liver disease / Gene Expression Omnibus dataset

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has emerged as a leading cause of advanced chronic liver disease, affecting 20%-25% of the global population^[1-2]. Among its progressive subtypes, metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive lipid accumulation and chronic hepatic inflammation, which can lead to advanced fibrosis, cirrhosis, and eventually hepatocellular carcinoma^[3-4]. The underlying pathogenesis of MASH involves lipid dysregulation, oxidative stress, and the activation of fibrogenic pathways, particularly those mediated by hepatic stellate cells (HSCs)^[5]. The approval of Rezdiffra^TM represents a significant advancement in the field of MASH treatment, but its limited indication reflects the current boundaries of clinical evidence, with limited applicability to patients with mild hepatic fibrosis (F0-F1) and severe cirrhosis^[6]. Thus, there is an urgent need to identify new therapeutic targets.

One critical pathogenic mechanism in MASLD/MASH is the disruption of hepatic lipid and cholesterol homeostasis^[7-8]. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by Olr1 gene, is a scavenger receptor responsible for binding, internalizing, and degrading oxidized low-density lipoprotein (ox-LDL)^[9-10]. It is primarily expressed in endothelial cells, platelets, macrophages, smooth muscle cells, and cardiomyocytes, where it mediates pro-inflammatory and pro-fibrotic responses upon ox-LDL engagement^[11-12]. While LOX-1 has been extensively studied in cardiovascular diseases such as atherosclerosis and myocardial ischemia^[11-13], its potential role in the development and progression of MASLD/MASH remains poorly understood.

The Western diet is one of the classical models for the study of MASLD and MASH, with similar phenotypic and histological characteristics of human MASLD/MASH^[14]. In this study, we aim to investigate the function of LOX-1 in MASH by using LOX-1 knockout mice subjected to a Western diet to induce hepatic steatosis and fibrosis and further explore the underlying molecular mechanisms through in vitro validation and network pharmacology analysis.

1 Materials and methods

1.1 Ethics statement

The establishment and use of these commercial tissues was approved by the Ethics Committee of Xiangya Hospital, Central South University (approval No.: 202203718). Animal studies were approved by Institutional and Local Committee on the Care and Use of Animals (approval No.: CSU-2023-0363). All animal procedures were performed following the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH). All procedures involving human samples were conducted in accordance with ethical standards, with informed consent obtained from all donors.

1.2 Tissue microarrays

De-identified tissue microarrays (TMAs) from normal and fibrotic liver tissues were purchased from Bioaitech Co. Ltd (Xi’an, China).

1.3 Animal experiments

Male wild-type (WT) and Lox-1^-/- mice generated by Cyagen Biosciences (Suzhou, China) were used for the study. The mice were housed in a controlled temperature (25±1) ℃ with a 12-hour light/dark cycle and allowed free access to sterilized food and water. We complied with the principles of the 3Rs (replacement, reduction, and refinement) and respected the highest ethical and animal welfare standards in conducting the animal experiments.

WT and Lox-1^-/- mice (6-8 weeks old) were fed chow diet or Western diet for 16 weeks (5 mice in each group). Chow diet (TP26352) and Western diet (TP26304, caloric composition: 42% fat, 14% protein, 44% carbohydrate, and 0.2% cholesterol) were purchased from Trophic Animal Feed High-tech Co. Ltd (Nantong, China).

1.4 PCR

The DNA was extracted from the mouse tail and amplified by polymerase chain reaction (PCR). The PCR reaction system included DNA, primers, 2×Taq premix and ddH₂O, and the amplified products were subjected to agarose gel electrophoresis for visualization and image acquisition. Primer sequences for PCR were listed in Supplement Table 1 (https://doi.org/10.57760/sciencedb.xbyxb.00142).

1.5 Liver histological staining

Liver specimens were preserved in 4% paraformaldehyde and then dehydrated. One part of liver tissues was embedded in paraffin blocks and cut into 4 μm sections. Hematoxylin and eosin (HE) and Masson’s trichrome staining were performed. One part of liver tissues was fixed in cold propylene glycol and then incubated in Oil Red O solution for staining. Images were visualized and scanned.

1.6 Biochemical analysis

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were enzymatically measured as recommended by manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

1.7 Cell culture and transfection

LX-2 cells were purchased from the Cell Bank of Chinese Academy of Sciences. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were transfected with control or LOX-1 siRNA using lipofectamine RNAiMax transfection reagent. The sequence for LOX-1 siRNA: CTCGGAAGCTGAATGAGAA.

1.8 RT-qPCR

Total RNA was extracted from the liver tissues of mice or LX-2 human hepatic stellate cells with RNAquick purification kit (Shanghai Yishan Biotechnology, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg of DNase I-treated RNA using the HiScript II Q Select RT SuperMix for qPCR (Vazyme, Nanjing, China). Quantitative PCR was performed in three technical replicates with the SYBR Green Master Mix (Vazyme, Nanjing, China). Transcript levels were normalized to β-actin (for mouse samples) or GAPDH (for LX-2 cells). Primer sequences for real time-quantitative PCR (RT-qPCR) were listed in Supplement Table 2 (https://doi.org/10.57760/sciencedb.xbyxb.00142).

1.9 Immunofluorescence

For immunofluorescence, LX-2 cells were transfected with control or LOX-1 siRNA. Paraffin-embedded liver sections were deparaffinized, rehydrated, and microwave heated for 15 min in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval and then permeabilized in 0.4% Triton X-100 for 10 min at room temperature. Paraffin sections and cell culture slides were washed with PBS and incubated by 5% BSA at room temperature. Then the slides were incubated with anti-alpha-smooth muscle actin(α-SMA) mouse (ab7817, Abcam, USA) and anti-LOX-1 rabbit (PA595750, Invitrogen, USA) antibodies overnight at 4 ℃. After washing, the slides were incubated for 2 hours with Alexa Fluor 647 Donkey anti-mouse immunoglobulin (Ig) G (A-31571, Invitrogen, USA) and Alexa Fluor 488 Donkey anti-rabbit IgG (A-21206, Invitrogen, USA). DAPI were used for nuclei staining. Immunofluorescent staining images were obtained by Zeiss LSM800.

1.10 PPI network construction and key targets enrichment analysis

The intersection of related targets of LOX-1 and MASH was assessed, and the intersection targets were loaded into the search tool for the retrieval of interacting genes/proteins (STRING) platform to generate a network of protein-protein interaction (PPI). The “LOX-1-related” gene set was generated using GeneMANIA, with “Olr1” as the seed gene. The top 100 genes ranked by composite association score were retained. The “MASH-related” gene set was retrieved using GeneCards-GeneAnalytics with the search terms: “metabolic dysfunction-associated steatohepatitis” OR “MASH” OR “NASH” OR “nonalcoholic steatohepatitis”. A relevance score >50 yielded 253 high-confidence genes. Gene Ontology (GO) enrichment analysis was performed on core network genes.

1.11 Statistical analysis

All analyses were performed using GraphPad Prism 10 software. Statistical analyses were performed using a two-tailed Student’s t-test or one-way ANOVA with Student-Newman-Keuls post hoc analysis. Error bars in figures represent the standard deviation. P<0.05 was considered as statistical significance.

2 Results

2.1 Upregulated LOX-1 in MASLD mice and human fibrotic livers

In both the GSE30552 and GSE40041 datasets, Lox-1 mRNA levels were significantly upregulated in MASLD mouse models induced by a high fat diet (HFD) or bile duct ligation (BDL) (both P<0.001, Figure 1A). Moreover, immunofluorescence staining and quantification revealed higher LOX-1 protein levels in liver tissues from patients with fibrosis compared to healthy controls (Figure 1B). To define the contribution of LOX-1 to MASLD progression, we generated LOX-1 knockout (Lox-1^-/-) mice using CRISPR/caspase-9 (Cas9)-mediated gene editing. The murine LOX-1 gene (Olr1), located on chromosome 6 and composed of 8 exons, was targeted by deleting exons 5 and 6, which resulted in a frameshift mutation and functional knockout of the gene (Figure 1C). Successful genotyping and deletion were confirmed by PCR and Sanger sequencing (Figure 1D).

2.2 Attenuated hepatic injury in MASH mice by LOX-1 deletion

To explore the role of LOX-1 in MASH, WT and Lox-1^-/- mice were fed Western diet for 16 weeks. Serum biochemical analysis showed that Western diet feeding significantly elevated serum ALT and AST levels in WT mice (both P<0.001), whereas LOX-1 knockout effectively reduced these levels (both P<0.05, Figure 2A). Histological examination by HE staining and NAFLD activity score (NAS) showed extensive hepatocellular ballooning, steatosis and inflammatory cell infiltration in the livers of Western diet-fed WT mice. In contrast, these pathological changes were markedly alleviated in Lox-1^-/- mice (Figure 2B-2C).

2.3 Ameliorated hepatic fibrosis in MASH mice by LOX-1 knockout

Masson’s trichrome staining showed a significant increase in collagen deposition in the pericentral and periportal regions of livers from Western diet-fed WT mice, which was significantly reduced in Lox-1^-/- mice (all P<0.01, Figure 3A). Consistently, RT-qPCR analysis showed that the mRNA expression levels of pro-fibrotic genes, including Acta2, Col1a1 and Timp1, were significantly upregulated in Western diet-fed WT mice, but were markedly downregulated in the LOX-1 knockout group (all P<0.05, Figure 3B).

2.4 Reduced hepatic lipid accumulation in MASH mice　by LOX-1 knockout

Oil Red O staining was performed to assess hepatic lipid accumulation and showed that there were no obvious lipid droplets in the hepatocytes of mice fed chow diet, whereas Western diet-fed WT mice displayed marked macrovesicular steatosis, characterized by diffuse red-stained lipid droplets and enlarged hepatocytes with obscured cell borders (Figure 4A). Notably, Lox-1^-/- mice exhibited significantly reduced lipid accumulation and preserved hepatic architecture under the same dietary conditions (both P<0.001, Figure 4B).

2.5 HSCs activation inhibited by LOX-1 knockdown

LOX-1 was knocked down in LX-2 cells via siRNA transfection. RT-qPCR results showed that LOX-1 silencing significantly reduced the expression of Acta2, Col1a1 and Timp1 mRNA expression (all P<0.05, Figure 5A). Dual immunofluorescence staining showed colocalization of α-SMA and LOX-1 in LX-2 cells. LOX-1 knockdown led to reduced α-SMA protein expression, suggesting impaired HSCs activation (Figure 5B).

2.6 LOX-1-related pathways in MASH progression　identified by network pharmacology

To further explore the mechanistic role of LOX-1 in MASH, we performed a network pharmacology analysis. Nine common target genes were identified from the intersection of LOX-1- and MASH-related gene sets retrieved from the GeneMANIA and GeneCards databases (Figure 6A, Supplement Table 3,https://doi.org/10.57760/sciencedb.xbyxb.00142). A PPI network was constructed using the STRING database (Figure 6B). GO enrichment analysis of the core network genes revealed significant enrichment in biological processes including cholesterol storage regulation and lipid metabolic processes (Figure 6C).

3 Discussion

MASLD is characterized by excessive hepatic fat accumulation (steatosis) due to underlying metabolic dysfunction^[15]. Owing to its complex and heterogeneous nature, the pathogenesis of MASLD and its progressive subtype-MASH remains incompletely understood. Consequently, identifying novel pathogenic mechanisms and therapeutic targets is essential to address the lack of approved pharmacological treatments^[8]. LOX-1 is a well-characterized scavenger receptor responsible for the uptake and degradation of ox-LDL. It has been implicated in a variety of metabolic disorders, including atherosclerosis, diabetes, and cardiovascular disease^{[12, 16-17]}. Although ox-LDL and free cholesterol accumulation have been associated with MASH progression^[18], the function of LOX-1 in MASH progression remains unclear. In the present study, we provided compelling evidence that LOX-1 contributed to the pathogenesis of MASH, and that its genetic deletion attenuated hepatic steatosis, inflammation, and fibrogenesis in a Western diet-induced mouse model.

We first confirmed that LOX-1 deficiency significantly attenuated hepatic steatosis and lipid deposition in MASH mice, recovered impaired hepatic structure and function, as well as interrupted the further progression of MASH to liver fibrosis. In normal physiological conditions, serum levels of AST and ALT remain at basal levels. Upon hepatocellular injury, increased permeability of the hepatocyte membrane allows cytosolic AST and ALT to translocate into the bloodstream, thereby elevating their serum concentrations^[19]. Utilizing LOX-1 knockout mice, our results demonstrated that LOX-1 deficiency obviously reduced serum ALT and AST levels and improved histological features of MASH. These findings are consistent with the hepatoprotective effects observed in other LOX-1 deficient models of metabolic disease^{[17, 20]}. The Western diet is a well-established model for MASH, as it closely mimics the human dietary pattern and induces key features of the disease, including hepatic lipid accumulation, lobular inflammation, hepatocellular ballooning, and mild fibrosis^[21]. Histological staining showed that the livers of mice fed Western diet exhibited hepatocellular macrovesicular and microvesicular steatosis, as evidenced by distinct lipid droplets and ballooned hepatocytes, as well as inflammation with mild fibrogenesis around portal area, suggesting that the MASH model was successfully constructed.

Liver functions as a central organ of lipid homeostasis regulation^[22]. In our study, LOX-1 deficiency significantly ameliorated hepatic steatosis and lipid droplet accumulation, suggesting that LOX-1 promotes hepatic lipid dysregulation. Given the established role of LOX-1 in cholesterol uptake and foam cell formation, it is plausible that LOX-1 contributes to excessive lipid deposition in hepatocytes, thereby driving MASLD progression.

During fibrosis progression, extracellular matrix (ECM) proteins accumulate in response to chronic liver injury, and HSCs are a major source of ECM protein production and secretion^[23]. Hepatic steatosis is amplified by increased dietary cholesterol, which enhances ECM accumulation and HSCs activation leading to liver fibrogenesis and cirrhosis^[5]. Importantly, LOX-1 knockout also attenuated hepatic fibrosis, as evidenced by decreased collagen deposition and reduced expression of fibrosis-related genes (Acta2, Col1a1, Timp1). To further validate this effect in vitro, we demonstrated that silencing LOX-1 in LX-2 cells inhibited the activation of HSCs, highlighting a potential direct link between LOX-1 signaling and fibrogenesis. This finding aligned with previous study^[5] showing that free cholesterol accumulation in HSCs promoted their activation and contributes to fibrosis.

Inflammation represents a central pathological hallmark of MASH^[21]. Consistent with established MASH activity scoring criteria, HE staining revealed a significant reduction in lobular inflammatory foci, ballooning degeneration, and inflammatory cell infiltration in Lox-1^-/- mice. In parallel, silencing LOX-1 in LX-2 cells downregulated Acta2, Col1a1, and Timp1 expression, indicating an attenuation of inflammation-driven fibrogenic activation. Given that fibrosis is a downstream consequence of persistent inflammatory injury, the observed amelioration of liver fibrosis further corroborated the alleviation of chronic inflammation. Moreover, network pharmacology implicated LOX-1 involvement in lipid-immune pathways, including cholesterol-associated inflammatory responses. Collectively, these converging findings provide robust evidence that LOX-1 depletion mitigated hepatic inflammation.

Network pharmacology and gene enrichment analyses revealed that LOX-1-associated targets were enriched in pathways related to lipid and cholesterol metabolism. These bioinformatic results supported our experimental findings and suggested that LOX-1 may act as a molecular hub integrating metabolic and fibrotic signaling in MASH. Although further mechanistic studies are warranted, our study laid the foundation for targeting LOX-1 as a novel therapeutic strategy in MASLD/MASH. To further elucidate the specific mechanisms by which LOX-1 contributes to MASH pathology, subsequent studies will employ the palmitic acid-/oleic acid-induced lipotoxicity model in subsequent studies.

In conclusion, our study demonstrated that genetic deletion of LOX-1 effectively attenuates hepatic steatosis, inflammation, and fibrosis in a mouse model of Western diet-induced MASH. These protective effects are likely mediated by the modulation of lipid and cholesterol metabolism and the inhibition of hepatic stellate cell activation. Our findings may provide novel insights into the pathological role of LOX-1 in MASLD/MASH and highlight its potential as a promising therapeutic target for the treatment of this increasingly prevalent disease.

Contributions: HUANG Ruihua Conceptualization, data curation, formal analysis, visualization, original draft writing; YANG Yongyu Resources; ZHOU Shuhan Data curation; ZHU Xiaoyun Conceptualization, writing-review and editing, resources, supervision, project administration, funding acquisition; HU Changping Conceptualization, writing-review and editing, resources, supervision, project administration, funding acquisition. The final version of the manuscript has been read and approved by all authors.

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基金资助

the Natural Science Foundation of Hunan Province, China(211142095031)

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