1Department of Pathology,Changzheng Hospital, Navy Medical University (Second Military Medical University), Shanghai 200003, China 2Department of Pathology, Medical School of Nantong University, Nantong 226001, Jiangsu, China

Acyl-CoA synthetase long-chain family member 1 (ACSL1) serves as an intermediate of lipid metabolism. However, its function in hepatic stellate cell (HSC) activation has not been thoroughly investigated. Herein, we discovered that the development of hepatic fibrosis in the human liver, which is linked to HSC activation, was accompanied by a decrease in ACSL1 expression. Further intervention of ACSL1 confirmed that ACSL1 inhibited HSC proliferation and activation while promoting HSC apoptosis.

ACSL1, Liver fibrosis, HSC, Lipid, NF-κB, Fatty acids

Liver fibrosis is caused by a variety of chronic liver injuries, such as various viral hepatitis, cholestasis-related liver disease, excessive alcohol consumption, metabolic dysfunction associated liver disease (MASLD), and metabolic dysfunction associated steatohepatitis (MASH), resulting in chronic inflammation of the liver and dysregulation of the healing process of liver with the potential for progression to cirrhosis or even liver cancer in the absence of timely intervention[1-3]. HSC activation and proliferation are the main links in hepatic fibrosis progression[4], and they are the most important steps in triggering the aggravation of extracellular matrix (ECM) deposition [5]. Hepatic stellate cells (HSCs), also known as fat storage cells that package rich lipid droplets (LDs), are in a quiescent state in the normal liver[6]. The LDs of HSC mainly contain a variety of vitamin A (VA) substances, as well as varying amounts of triglyceride (TG), phospholipid (PL), cholesterol, and free fatty acid (FFA)[7]. Quiescent HSCs release VA after liver damage, triggering lipids to transform into myofibroblasts (MF) that migrate, proliferate, and ultimately lead to liver fibrosis, expressing fibrosis genes and proteins, such as type І collagen alpha 1 (Col1a1), type Ⅲ collagen alpha 1 (Col3a1), smooth muscle actin (α-SMA), etc. [8-10]. Recent preclinical research showed that liver damage causes liver fibrosis, immune cell infiltration, hepatocyte death, and reactive oxygen species (ROS)-induced proinflammatory cytokines increase, suggesting lipid metabolism and related redox or stress-related signaling pathways are important mechanisms in HSC activation and transformation. Acyl-CoA synthetase long-chain (ACSL) on the endoplasmic reticulum and mitochondrial outer membrane forms acyl-coenzyme A (acyl-CoA) from 12–20-carbon-atom long-chain fatty acids (LCFA) [11]. After LCFA is esterified by ACSL, it enters a variety of metabolic processes, including β-oxidation of cellular FFA (catabolism and anabolism) to synthesize a variety of esters[12]. In liver tissue, ACSL1 is the most highly expressed isoform and is an important regulator of lipid metabolism in liver tissue, accounting for 50% of the total ACSL activity [13, 14]. Recent evidence suggests that maternal obesity may play a role in modulating the cumulative susceptibility of offspring to hepatocellular carcinoma (HCC) over multiple generations through the micro-27a-3p-Acsl1 axis [15] and that miR-205 disrupts liver cancer by targeting ACSL1 lipid metabolism in cells[16]. Furthermore, augmented interactions between mitochondrial fatty acid translocase (FAT)/CD36 and ACSL1 lead to increased transport of LCFA to ACSL1, thereby increasing hepatocyte mitochondrial fatty acid oxidation (FAO) and mitigating MASLD[17]. Our prior research has explored the role of the microRNA genome high-throughput chip in the liver fibrosis animal model of Sprague Dawley rats and found that micro-34a/c targets the inhibition of ACSL1 to regulate the activation of HSC and participates in the process of liver fibrosis [18], and the LDs in rat tissues and rat HSC-T6 cells treated with micro-34c mimics were significantly reduced [19]. Collectively, changes in these mechanisms may compromise metabolic flexibility, impact lipid peroxidation, and potentially increase the susceptibility to liver fibrosis. In this study, we report the novel discovery that ACSL1 functions to prevent HSC activation and proliferation as well as liver fibrosis. In initial investigations of human liver tissue and in vitro experiments, ACSL1 expression decreased as hepatic fibrosis advanced, also indicating a possible correlation with the development of HCC. By constructing ACSL1 interference and overexpression lentivirus, we further proved the role of ACSL1 in promoting HSC apoptosis and suppressing HSC growth and activation. Next, we found that NF-κB p65 directly binds to the ACSL1 promoter area, suppressing ACSL1 expression, and may be regulated via the TGF-β1/NF-κB p65 axis. Finally, targeted lipidomics showed that ACSL1 altered the HSC lipid profile and prevented fibrogenesis via altering lipid peroxidation. Thus, ACSL1 serves as a key target to direct the fate of fatty acids in HSC and controls the activity of a major regulator of liver fibrosis.

Patients with liver disease with postoperative pathological findings of hepatic fibrosis/cirrhosis and HCC were selected. In addition, the 6 liver fibrosis tissues (distal adjacent tissues of HCC patients) and 3 normal liver tissues (the distal normal liver tissues of hepatic hemangioma patients) were selected for RNA sequencing (RNA-Seq). Patient inclusion criteria: (1) The initial postoperative pathological assessment of hepatic fibrosis/ cirrhosis and HCC; (2) No preoperative diagnosis of other tumors or history of other tumors; (3) No other anti-tumor treatment such as radiation or chemotherapy before surgery. Pathological results were independently diagnosed and verified by two senior pathologists

Sprague–Dawley rats, aged 8–10 weeks were obtained from the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Liver fibrosis was induced in rats (n = 15) by intraperitoneal injection of dimethylnitrosamine (DMN) (Sigma, St Louis, MO, USA) for three consecutive days per week. Primary rat HSC were isolated from males by in situ liver collagenase perfusion[20]. The HSC (T6 and LX-2 cell lines) were purchased from the China Biomedical Technology Service Center, and verified by STR identification.

Hematoxylin-eosin(HE): Paraffin-embedded tissues were serially sectioned, deparaffinized with xylene, and stained with hematoxylin staining solution for 10 to 15 min. The cells were differentiated in 1% alcohol hydrochloride for about 30s and then stained with 1% eosin. The slices were differentiated for 30s in 95% alcohol and placed in xylene for 1-2min before sealing with neutral gum. Immunohistochemistry (IH): The slides rinsed with PBS to repair the antigen, then added 3% H2O2 to soak for 10 min, rinsed with water and placed in a citric acid buffer, boiled and cooled to room temperature; added primary antibody for 1h at 37°C or refrigerated overnight at 4°C; added secondary antibody for 1h at 37°C, DAB color development for 10 min; hematoxylin staining for 3 min; hydrochloric acid alcohol for 2s, and dehydrated transparent neutral gum to seal the slides. Immunofluorescence (IF): The slides were fixed in formaldehyde solution for 10 min and treated with 0.5% Triton X-100, prepared in PBS, for 20 min at room temperature. Goat-blocking serum was treated for 30 min at room temperature. Then the primary antibody (α-SMA, 1:100) was added and the slides were placed in a wet box and incubated overnight at 4 ° C. The next day, the cells were washed 3 times with PBS-Tween (PBS-T) for 3 min each time. Finally, fluorescent secondary antibodies were added, and the cells were incubated for 1 h at 20 to 37 ° C in a wet box and counterstained with DAPI for 5 min.

Follow the instructions of the SteadyPure Universal RNA Extraction Kit. The mixture of reverse transcription reaction system was gently mixed and centrifuged instantaneously at 1000rpm for 1min and then put into the PCR amplifier. The reaction system was 20µl. The primer list is provided in the table1.

A total of 20ul of protein was loaded and proteins were separated by gel electrophoresis and transferred onto a PVDF membrane. The protein bands were then incubated in a 4°C shaker overnight in antiACSL1(D2H5) rabbit monoclonal antibody (CST9189), anti-Col1α rabbit polyclonal antibody (GB11022), anti-Col3α rabbit polyclonal antibody (GB111323), anti-α SMA (ab5694), anti- NF-κB p65 rabbit polyclonal antibody (ab16502). Anti-GAPDH rabbit monoclonal antibody (ab9485) was used as an internal control.

The ACSL1 gene fragment was amplified by the specified primers and linked to the linearized vector GV358 by ligase. Primer information is in Table S1. Based on the sequence information of ACSL1, three interference target sequences were designed (For detailed sequence design information, see Table S2), and the target with the best kinetic parameters was selected for subsequent experiments through verification

Follow the instructions of the EDU Kit (C10310 Ribotest); add 100μl of fluorescent dye for incubating, then add 100μl of 1×Hoechst 33342 reaction solution to each well, incubate for 30 min avoiding light.

Collect the the sediment and 10µl of Annexin V-APC was added and stained for 10~15min at room temperature and protected from light; 400µl of 1×binding buffer was added to each tube and detected by flow cytometry.

The ratio of the integrated peak areas of all detected samples was substituted into the linear equation of the standard curve, and further substituted into the calculation formula to obtain the specific value of the absolute content of the substance in the actual sample; the formula for calculating the intracellular lipid content is X=c*V*V1/V2/m, X indicates the content of lipid in the sample, c indicates the ratio of the integrated peak areas in the sample substituted into the standard curve the value of the concentration obtained (nmol/ml), V denotes the compound solution (µl), V 1 denotes the sample extract (µl), V 2 denotes the collection supernatant (µl), and m denotes the number of samples obtained.

ChIP assays were performed using the Simple ChIP Enzymatic Chromatin IP kit (Cell Signaling Technology) following the manufacturer's guidelines. To ensure that the DNA should be enzymatically cut into fragments of approximately 150-900 bp in length. Prepare 2% sample input control, ChIP DNA was eluted, cross-linking was reversed, and protein-free DNA was purified before PCR amplification with site-specific primers covering the ACSL1 promoter region. Primer sequences for the ChIP assay are listed in Table 1.

pGL3-ACSL1-PROMOTER-wildtype plasmids (pGL3-ACSL1-WT), pGL3-ACSL1-PROMOTER- mutant plasmids (pGL3-ACSL1-MUT), pGL3-REPORT luciferase empty plasmids (pGL3), plasmid containing overexpression of NF-κB p65 (pcDNA NF-κB), and control pcDNA (purchased from Shanghai Lanyao Gene Chem Co., Ltd.) were cotransfected into HEK293, and the empty vector PRL-TK was used as control. The fluorescence of Firefly and Renilla luciferase was detected using a microplate reader.

Based on the micro malondialdehyde assay kit (Solarbio). Measure the absorbance of each sample at 450nm, 532nm, and 600nm. After colorimetry, the content of lipid peroxide in the sample can be estimated.

According to the arachidonic acid (AA) ELISA Kit (Sangon Biotech), 100 μl of HRP-labeled streptavidin working solution was added to each reaction well. Add 50 μl stop solution to each well and measure the OD value (within 5 minutes) with a microplate reader at a wavelength of 450 nm.

The study was approved by Changzheng Hospital of Shanghai with the patient’s consent and signed informed consent.The animal experiment received approval from the ethics committee of the Animal Ethics Committee of Navy Medical Universityand enforced uniform standards for animal management.

The Cancer Genome Atlas (TCGA) data difference analysis and lipidomics analysis were used in R studio. SPSS 24.0 statistical software was used to analyze the results. Homogeneity of variance using one-way ANOVA (one-way ANOVA) was tested between groups (using LSD-T test). Kruskal-Wallis test was used when variance was not homogeneous; meanwhile values of each group were compared with each other by Nemenyi test. Statistical significance was considered when p < 0.05. GraphPad Prism 9.0 was used to draw charts.

Transcriptome mRNA sequencing of liver tissue samples from patients undergoing intraoperative hepatectomy showed relatively low ACSL1 expression in liver fibrosis (Figure. 1A). Besides, bioinformatics analysis showed that 24.32% of the significantly down-regulated genes in the liver tissue samples of liver fibrosis were related to metabolic pathways, of which 5.41% were related to lipid metabolism (Figure. 1B). To further verify the expression of ACSL1 in the liver, liver specimens were obtained from individuals who had received postoperative pathological diagnoses of hepatic fibrosis/cirrhosis and HCC. IF staining in liver tissues showed increased expression of fibrous indicator α-SMA, while ACSL1 expression tended to be negative in areas of fibrosis (Figure. 1C). Additionally, IH results showed that ACSL1 was positively expressed in HSC in normal liver tissue, and the degree of positivity was close to that of peripheral hepatocytes; in fibrosis samples, the expression of ACSL1 in HSC was reduced, although ACSL1 remained positive in peripheral hepatocytes. Interestingly, we found that ACSL1 showed faint positive expression in cancer cells, whereas the expression of ACSL1 was further weakened or even negative in HSC transformed into MF in liver fibrosis/ cirrhosis stroma (Figure. 1D). To further verify this finding, the differential expression analysis map was drawn according to the data sets of all liver cancer in the TCGA database. It showed that compared with normal tissue, the expression of ACSL1 in liver cancer samples and paired data decreased significantly (Figure. 1E, F). These findings suggested that ACSL1 may play a role in liver fibrogenesis and a potential association with the development of HCC.

Primary HSCs were deemed quiescent during the first 3 days and activated after 10 days[21]. Initially, we obtained primary rat HSCs and performed IF staining, and α-SMA revealed resting and activated HSC phenotypes at days 2 and 14 successfully (Figure. 2A). Then ACSL1 expression was measured on days 2, 7, and 14 days with a gradual downregulation in response to HSC activation (Figure. 2B, C). Furthermore, we staged liver fibrosis according to the METAVIR staging standard, which is the diagnostic standard for liver fibrosis. The HE staining revealed a progressive deterioration in liver fibrosis from stages F0 to F4, while IH analysis indicated a decrease in ACSL1 expression as the degree of fibrosis increased (Figure. 2D). To validate this trend in vitro, the cytokine TGF-β1 was employed to expedite the process of fibrogenesis, which plays an important role in promoting HSC activation[22]. At a concentration of 10ng/ml, both Col1a1 and α-SMA exhibited varied degrees of growth. However, ACSL1 expression decreased (Figure. 2E). The protein analysis further revealed that TGF-β1 led to a decrease in ACSL1 expression in human HSCs in a concentration-dependent manner, and ACSL1 gradually decreased after a concentration gradient of 5ng/ml (Figure. 2F). Nevertheless, we didn’t catch this trend in rat HSC-t6 (Figure. 2G). Together these findings indicated a reduction in ACSL1 expression during the progression of liver fibrosis and primary HSC activation, displaying a negative correlation with the fibrotic stage.

Fluorescence microscopy confirmed that LX-2 cell lines successfully infected with GFP-labeled lentiviral constructs LV-ACSL1 KD1, KD2, KD3, and LV-NC (Figure. 3A). The KD1 group exhibited the highest reduction of ACSL1 by qRT-PCR (Figure. 3B). Following lentiviral interference of ACSL1 expression, a substantial upregulation in the expression of Col1a1 and Col3a1 was observed, while the expression of α-SMA remained relatively stable (Figure. 3C, D). The EdU staining assay, followed by microscopic observation and quantification of the labeled cells showed that the EdU positive rate of LV-ACSL1 KD was (54.62±0.22) % and the EdU positive rate was (51.96±0.22) % in the control group (Figure. 3E, F). Furthermore, the lentiviral-mediated stable overexpression of ACSL1 with an abundance of ACSL1 gene was 307.288 times higher (Figure. S1A). In the apoptosis peak chart, minimal cell distribution was observed in the lower right quadrant, while a small number of apoptotic cells were present in the upper right quadrant. According to the peak chart analysis of the proportion of apoptotic cells, the proportion of dead cells increased by 5.04 times after overexpression of ACSL1 (Figure. 3G, H). These observations indicate that ACSL1 may have a role in inhibiting both HSC activation and proliferation, and promoting the apoptosis of activated HSC, potentially resulting in additive effects.

To further explore the regulatory mechanisms by which ACSL1 affects HSC activation, potential transcription factors capable of binding to the ACSL1 promoter sequence were predicted by the PROMO website (https://alggen.lsi.upc.es/cgi-bin/) and identified an inflammatory pathway-related transcription factor NF-κB among them (Figure. S1B). Whereas various inflammatory cells and inflammatory signaling pathways are the main regulators of MF proliferation in the liver[23]. Preliminary verification of the combination of the two was performed in hTFtarget online tool (Figure. S1C). Four primer pairs were designed to span the ACSL1 promoter (about 2000bp), each covering about 500 bp units based on the promoter sequence (named P1-P4). A robust binding signal was detected between NF-κB p65 and the promoter region amplified by the second pair of ACSL1 primers. There was no discernible specific signal when employing a negative control antibody (Figure. 4A). Next, to further determine the binding site and the regulation of ACSL1 by NF-κB p65, NF-κB p65 overexpression and ACSL1 wild-type and mutant plasmids were constructed. The result of dual-luciferase reporter gene experiments showed that overexpression of NF-κBp65 significantly impacted ACSL1 promoter activity, resulting in the inhibition of ACSL1 expression (Figure. 4B). The protein analysis results also exhibit concordance (Figure. 4C, D). Altogether these data demonstrated that NF-κB p65 was found to bind directly to the ACSL1 promoter region, leading to the suppression of ACSL1 expression.

Since NF-κB can be activated by TGF-β and mediates transcriptional activation of TGF-β target genes in multiple cell types[24, 25], we stimulated HSC with different concentrations of TGF-β1 and found a graded elevation in the expression of NF-κB p65. Notably, significant increases were observed at TGF-β1 concentrations of 20 ng/ml, 30 ng/ ml, and 40 ng/ml compared to the control group, 5 ng/ml, and 10 ng/ml, with the highest expression noted at 20 ng/ml (Figure. 4D). In order to clarify the regulatory function, we found that TGF-β1 pathway inhibitor SB-431542 significantly inhibited the protein expression of NF-κB p65 in the rescue assay; however, the addition of TGF-β1 stimulation resulted in a significant increase in the expression of NF-κB p65 (Figure. 4E). In addition, SB-431542 also significantly inhibited the protein expression of Col1a1, which was restored by TGF-β1. The protein expression of ACSL1 exhibited an inverse pattern, experiencing a significant increase following SB-431542 treatment (Figure. 4F). Notably, the promotional effect was restored upon the addition of TGF-β1, providing evidence that the TGF-β1 pathway promotes NF-κB p65 and inhibits ACSL1. However, antagonism of Smad2/3 alone cannot completely prevent fibrosis and may lead to increased inflammation, suggesting that other pathways downstream of TGF-β1 may also play an important role in fibrogenesis[26]. Our investigation also found that BAY 11-7821 inhibits fibrosis formation, decreases Col1a1 and α-SMA expression, and significantly increases ACSL1 expression (Figure. 4G). These further indicate that NF-κB pathway promotes liver fibrogenesis and has an inhibitory effect on ACSL1, with the potential of regulating by TGF-β1.

To further clarify the mechanism by which ACSL1 activates HSC, the engineered strain and its corresponding controls were subjected to LC-MS/MS-based targeted quantitative lipidomics analysis. Principal component analysis (PCA) showed that PCA in two virus groups and the control group exhibited a clear separation between the groups (Figure. S2A-D). Comparisons between PCA after ACSL1 overexpression and PCA from the control virus also showed segregation (except for the CON2 group, which had large outliers and was subjected to orthogonal partial least squares discriminant analysis later) (2D: Figure.5A; 3D: Figure.5B). While the PCA may lack sensitivity to variables with minimal correlations. Within partial least squares Discriminant Analysis (PLS-DA) modeling, the X-matrix information is partitioned into two distinct categories: one representing information directly related to Y (the primary component for prediction), and the other representing information unrelated to Y (the orthogonal principal component). Following correction, it is evident that the group disparities between the control virus group and the ACSL1 overexpression group exhibited a significant separation, with the orthogonal component score registering a substantial value of 38.1% (Figure. 5C). In addition, S-plot plots of OPLS-DA showed significant aggregation of differential metabolites upon ACSL1 overexpression (Figure. 5D). Lipid composition analysis identified lipids including glycerides, phospholipids, sheath lipids, and cholesterol ester, etc. (Figure. 5E), meanwhile revealed the presence of 27 distinct lipid subclasses and TG exhibiting the highest abundance across all groups, comprising a total of 261 distinct species in all samples (Figure. S2E). The maximal level of 0.25% was observed in the proportion of lipid components, indicating that there was a tiny variation in the changes of lipid subclasses with lentivirus transfection (Figure. S2F). ACSL1 significantly increased the abundance of sphingomyelin (SM) metabolites while significantly inhibited carnitine (CAR) metabolites (Fig. 5F) in all classes. The total FFA concentration increases in the early stage of primary HSC activation, while it decreases in the later stage [27]. Interestingly, among the lipid subclasses, the greatest abundance was observed within the category of oleic acid (18:1), and palmitic acid (16:0), succeeded by eicosapentaenoic acid (20:1) and arachidonic acid (AA) (20:4). Intriguingly, the abundance of all lipid species exhibited an increase, except for class AA (20:4), which experienced a reduction after ACSL1 overexpression (Figure. 5G). Conversely, all members of the TG subclass demonstrated increased abundance following ACSL1 overexpression (Figure. 5H). Subsequently, lipid species meeting the criteria of a fold change of ≥20%, along with a P-value < 0.05, and variable importance in projection (VIP) value >1, as determined by Welch's test, were categorized as statistically significant. This screening process identified a total of 24 differential metabolites, comprising 18 up-regulated and 6 down-regulated lipid species (Figure. 5I). Interestingly, the largest multiplicative difference in up-regulated metabolites was TG (48:4)14:1. Nine up-regulated metabolites belonged to TG and three belonged to diglycerides (DG). The interplay between differential metabolites revealed that TG (48:4) 14:1 exhibited a striking negative correlation with CAR (18:0) and 10 differential metabolites showed the greatest negative correlation with CAR (18:0) (Figure. 5J). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differential metabolites identified enrichments in regulating metabolic pathways and glycerolipid metabolism pathways (Figure. 5K). The above results underscore a conspicuous modification in the cellular lipid profile after the enhancement of ACSL1 expression, shedding light on the impact of ACSL1 on these metabolites in HSC.

Due to the correlation between increased unsaturation of FA and molecular lipid peroxidation[28], the species in each class are ordered from fully saturated to highly polyunsaturated. It showed that the content of lipids with unsaturated carbon bond of 0 (saturated fatty acids, SFAs) was the highestproportion, which was significantly higher than unsaturated fatty acids after ACSL1 increased (Figure. 6A). Notably, ACSL1 overexpression had lipid content increases in fatty acyl chains containing 1 double bond, indicative of monounsaturated fatty acids (MUFAs), accompanied by relative decreases in polyunsaturated fatty acids (PUFAs; ≥3 double bonds) species (Figure. 6B). Saturated fatty acids are very stable, while unsaturated acids are more reactive[29]. More specifically, subclasses of lipid metabolites showed a significant rise in SM levels throughout unsaturation degrees 0 and 1, as well as sphingosine metabolites (SPH) at 0 unsaturation, following the overexpression of ACSL1 (Figure. 6C, D). This suggests that the FA is typically in relative stability in HSCs. In addition, cluster analysis of oxidized lipids showed that the overall expression of PUFAs decreased after ACSL1 overexpression compared to the control transfection group (Figure. 6E). The more double bonds in the chain, the more susceptible the molecule is to lipid peroxidation[28]. However, ACSL1 reversed this active trajectory. Additionally, the 3D heatmap of the correlation between carbon chain length and unsaturation showed that the lower the degree of unsaturation, the higher difference of differential metabolites affected by ACSL1(Figure. S2G). Since lipid peroxides can not only induce quiescent HSC to transform into MF but also stimulate MF to secrete more ECM[30], further lipid peroxidation was measured by the level of MDA which is formed during the degradation of polyunsaturated lipids by ROS, and the result showed suppression in the extent of lipid peroxidation within HSC by LV-ACSL1- OE (Figure. 6F). In the presence of ROS, PUFAs including AA undergo lipid peroxidation, leading to ferroptosis[31, 32].The detection of the AA (20:4) expression change in the KEGG ferroptosis metabolic pathway after ACSL1 overexpression also showed that the AA may be involved in the regulation of ACSL1 in HSC (Figure. S2H). Following the knockdown of ACSL1, the content of AA exhibited an increase in comparison to its resting state (Figure. 6G). Further, the induction of AA (20:4) significantly heightened the protein expression levels of fibrosis markers Col1a1 and Col3a1 directly (Figure. 6H). In addition, the ELISA assay showed BAY 11-7821 exhibited significant inhibition of AA expression in HSC (Figure. 6I), an effect that was restored following ACSL1 knockdown (Figure. 6J). The results suggested that ACSL1 induced a reduction in AA content, this inhibitory effect may be regulated by NF-κB pathway. In conclusion, our results demonstrated that ASCL1 has inhibitory effects on the progression of HSC activation, which are influenced by the levels of cell lipid peroxidation.

The purpose of this work was to evaluate the role of ACSL1 in mediating the metabolic section of activation of HSC and liver fibrosis. We observe a histological change of ACSL1 and downregulation in human liver fibrosis and malignancy. We also studied the lipidomic fingerprints of immortalized HSC cell lines that preserve critical quiescent HSC characteristics. By clarifying changes in lipid profiles, differentially expressed metabolites are screened out, and further by intervening on these metabolic substances, their impact on HSC/liver fibrosis is clarified. Collectively, these data highlight the significant impact of ACSL1 alteration on key metabolic processes of HSC activation, which provides a new molecular target for reversing HSC activation/hepatic fibrosis. Sequencing results on liver fibrosis linked ACSL1 to metabolic pathways, supporting early research revealing a large drop in ACSL1 expression in HCC and MASH livers[33]. Further study verified that ACSL1 was reduced during the natural activation of HSC which is associated with the study of Maidina et al[34]. Recent studies have also concluded that liver-specific Y-box binding protein 1 (Ybx1) knockdown significantly ameliorated TGF-βinduced fibrosis in LX2 cells, and the low expression of ACSL1 may be regulated by Ybx1 overexpression[35]. These data suggest that ACSL1 within HSC has a potential negative relationship with the progression of liver fibrosis. Additionally, it is also well acknowledged that HSC activity can be reversed by inducing a quiescent state or promoting apoptosis. HSC can be returned to quiescence by upregulating the anti-apoptotic gene heat shock protein Hspa1a/b following complete activation to MF[36]. Furthermore, HSC has been shown to express death receptors such as fatty acid synthase (FAS, also known as CD95) and tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) receptors and promote HSC apoptosis by binding to their ligands[37], which aligns with our discovery that ACSL1 induces apoptosis in HSCs.

We then investigated the upstream of ACSL1-related regulatory pathways and predicted that the pro-inflammatory factor NF-κB could bind to the ACSL1 promoter, which was consistent with the prediction of Yang et al[38]. Epigallocatechin gallate (EGCG) significantly decreased NF-κB but increased ACSL1 protein expression in rat liver by inhibiting oxidative stress and activating fatty acid transport and oxidation[39]. Although it has been shown that inflammatory stimulation of mouse bone mard-derived macrophages (BMDM) by lipopolysaccharide (LPS) increases ACSL1 mRNA through the transcription factor NF-κB, this regulation appears to be present only under hyperglycemic and inflammatory conditions. In the absence of LPS treatment, p65/RELA was not found to be higher than any binding of IgG to ACSL1[40]. The regulation of ACSL1 by NF-κB p65 may be multifactorial and situation-specific, with different physiological roles depending on the cell of origin, the underlying inflammatory injury, the chronicity of the disease, and the phosphorylation status of NF-κB p65. Moreover, TGF-β or its downstream signaling protein Sma and Mad associated protein 3 (Smad3) also inhibited LPS-stimulated promoter activity in RAW264.7 cells[41]. Next, we used different concentrations of TGF-β1 to stimulate HSC and found that the expression of NF-κB p65 was increased to varying degrees, indicating that TGF-β1 can indeed promote the expression of NF-κB p65. Moreover, the strongest effect was observed when the concentration of TGF-β1 was 20ng/ml, as in other studies, TGF-β1 increased p65 phosphorylation and enhanced NF-κB transcriptional activity[42, 43]. Our study showed that TGF-β1 enhanced ACSL1 promoter activity by promoting NF-κB p65 expression, while NF-κB p65 inhibited ACSL1 expression. Thus, although we tend to favor a model in which NF-κB p65 directly regulates ACSL1 expression, we do not rule out an indirect effect of these factors on ACSL1 regulation, and HSC activation is associated with the secretion of various mediated modulators regulated by NF-κB signaling. Further, ACSL1 expression may also enhance intracellular free fatty acids, which can be utilized to synthesize SM and ceramide (Cer)[44]. Quiescent HSCs are now viewed as plastic cells that regulate liver growth, immunity, inflammation, and energy and nutrient balance[45]. During the activation of primary HSC, the highest concentration of total FFA was palmitate (C16:0), followed by oleate (C18:1) and palmitate (C16:1), followed by a decrease in the content of these substances on days 3 and 7[27]. Some studies have defined that after palmitate supplementation, the expression of adipose differentiation-related protein (ADRP) in HSC is up-regulated, which in turn inhibits HSC activation while down-regulating fibrogenic genes[46]. This suggests that ACSL1 may promote the increase of palmitic acid (16:0) content and inhibit HSC activation. In addition, up-regulated differential metabolites, particularly TG and DG, are primarily influenced by key regulators of adipogenesis, leading to significant changes in lipid metabolites in cells[44]. Moreover, administering L-carnitine can effectively decrease blood TG levels in individuals suffering from liver disease[47].

The biological reactivity of fatty acids is influenced by the double bond position in the carbon chain[28], with lower unsaturation levels suggesting reduced lipid peroxidation susceptibility in HSC. The role of oxidative stress in HSC activation and fibrosis was supported by the stimulation of HSC proliferation and ECM production by prostaglandin F2 class compounds, mediators of lipid peroxidation, in isolated rat HSC[48]. In many cases, ROS material accumulation may be a peroxidation product of the enzymatic generation of arachidonic acyl and adrenal acyl chains. In addition, heightened AA content not only signifies a potential catalyst for HSC activation but also suggests a facilitative role in the progression of hepatic fibrosis formation [27]. On top of that, inflammation also induces the disorder of lipid metabolism and further aggravates the occurrence of liver fibrosis. Palmitic acid induces NF-κB activation to regulate the expression of SUMO-specific protease 2 (SENP2), which in turn promotes palmitic acid-mediated increase in ACSL1 expression[49]. Furthermore, studies have shown that aspirin inhibits abnormal lipid metabolism in HCC cells by disrupting NF-κB-ACSL1 signaling[38], indicating that studies have focused on the regulatory role of NF-κB-ACSL1 signaling axis in liver cancer, and this role is related to abnormal lipid metabolism. We found that ACSL1 inhibits the progression of HSC activation by affecting lipid peroxidation levels in HSC, possibly through the NF-κB p65-ACSL1 signaling axis.

Hepatic fibrosis is a dynamic process triggered by signals like proinflammatory cytokines, hepatocyte apoptosis, growth factor activation, and increased ROS load.A series of paracrine and autocrine loops enhance the cellular response to injury, including fibrogenic signals such as TGF-β1 and connective tissue growth factor (CTGF). Specific lipids act as signaling molecules that may promote tumorigenesis. It is now thought that HSC activation is the same as cancer formation in obtaining energy and that specific lipids may follow the same pathway. As cofactors, lipids can directly bind to transcription factors to regulate the expression of lipid metabolism in a feedback loop, in which ACSL1 may play a mediator role (Figure. 7). In summary, we identified a novel target ACSL1 to regulate HSC activation and liver fibrosis, underscoring the inflammatory pathway with ACSL1-related lipid alteration and provide a new perspective for the prevention and treatment of liver fibrosis. pathways and downstream lipid metabolism by concentrating on ACSL1.

This work was supported by National Natural Science Foundation of China (81370553; 81870418), Medical guidance project of Shanghai Science and Technology Commission(124119a4100).

ACSL1, acyl-CoA synthetase long-chain family member 1;HSC, hepatic stellate cell;ChIP, chromatin immunoprecipitation assay;LC-MS/MS, liquid chromatography and tandem mass spectrometry; NF-κB, nuclear factor kappa B; ; TGF-β1, transforming growth factor beta-1; MASLD, metabolic dysfunction associated liver disease; MASH, metabolic dysfunction associated steatohepatitis; ECM, extracellular matrix; HSCs, hepatic stellate cells; LDs, lipid droplets; VA, vitamin A; TG, triglyceride; PL, phospholipid; FFA, free fatty acid; MF, myofibroblast; Col1a1, type І collagen alpha 1; Col3a1, type Ⅲ collagen alpha 1; α-SMA, smooth muscle actin; ROS, reactive oxygen species; ACSL, acyl-CoA synthetase long-chain; acyl-CoA, acyl-coenzyme A; LCFA, long-chain fatty acid; HCC, hepatocellular carcinoma; FAT, fatty acid translocase; FAO, fatty acid oxidation; DMN, dimethylnitrosamine; HE, hematoxylineosin; IH, immunohistochemistry; IF, immunofluorescence; MDA, malondialdehyde; TBA, thiobarbituric acid; AA, arachidonic acid; TCGA, The Cancer Genome Atlas; PCA, principal component analysis; PLS-DA, Partial least squares Discriminant Analysis; SM, sphingomyelin; CAR, carnitine; VIP, variable importance of projection; DG, diglycerides; KEGG, Kyoto Encyclopedia of Genes and Genomes; SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SPH, sphingosine; PDGF, platelet-derived growth factor; Ybx1, Y-box binding protein 1; FAS, fatty acid synthase; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; EGCG, epigallocatechin gallate; BMDM, mard-derived macrophages; LPS, lipopolysaccharide; Smad3, Sma and Mad associated protein 3; Cer, ceramide; ADRP, adipose differentiation-related protein; SENP2, SUMO-specific protease 2; CTGF, connective tissue growth factor.

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Hongyu Yu. Effect Of ACSL1-Mediated Inhibition Mechanisms On The Progression Of Fibrogenesis And Its Regulation Of Lipid Profile In HSC. International Journal of Gastroenterology and Hepatology 2024.