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. 2023 Nov 18;9(11):e22464. doi: 10.1016/j.heliyon.2023.e22464

TDO2 deficiency attenuates the hepatic lipid deposition and liver fibrosis in mice with diet-induced non-alcoholic fatty liver disease

Zhi Qin a, Min Zhou b,∗
PMCID: PMC10700617  PMID: 38074859

Abstract

Purpose

Non-alcoholic fatty liver disease (NAFLD) represents an increasingly prevalent set of liver diseases. Tryptophan 2,3-dioxygenase 2 (TDO2) is the major enzyme of tryptophan catabolism and is abnormally expressed in liver cancer, but the function of TDO2 in NAFLD remains unclear. The current study was designed to probe into the effect and mechanism of TDO2 on NAFLD.

Methods

C57BL/6 mice and TDO2-knockout (KO) mice were fed with a high-fat diet for 16 weeks to construct the NAFLD model in vivo; primary hepatocytes isolated from TDO2-KO mice were exposed to palmitate (PA) to establish the NAFLD model in vitro. The expression of TDO2 was determined using Western blot. The function and mechanism of TDO2 were evaluated by enzyme-linked immunosorbent assay, hematoxylin-eosin staining, Oil Red O staining, immunohistochemical assay, and Western blot.

Results

The expression of TDO2 in the liver tissue of NAFLD mice was more than three times that in the control group. Functionally, TDO2 knockout reduced hepatic lipid deposition and liver fibrosis in NAFLD mice in vivo and primary hepatocytes induced by 200 μM PA in vitro. Mechanistically, the loss of TDO2 restrained hepatic lipid deposition and expression levels of fibrosis-related markers in PA-treated primary hepatocytes, and these trends were partially reversed by 10 ng/ml receptor activator of the nuclear factor kappa-B ligand (RANKL, an activator of the NF-κB pathway).

Conclusion

Knocking out TDO2 repressed hepatic lipid deposition and liver fibrosis in mice with NAFLD, and reduced hepatic lipid deposition and expressions of fibrosis-related markers in PA-treated primary hepatocytes by inactivating the NF-κB pathway.

Keywords: TDO2, NF-κB, Hepatic lipid deposition, Liver fibrosis, NAFLD

1. Introduction

Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by a series of liver pathological changes such as simple steatosis, non-alcoholic steatohepatitis, and cirrhosis [1,2]. At present, the incidence of NAFLD is on the rise globally, which severely affects the national health and economic systems [3,4]. Except for lifestyle interventions, the treatments for NAFLD include medication and surgical approaches, but these have limited specificity and multiple side effects [5,6]. Hence, there is an urgent need to better understand the pathogenesis of NAFLD and develop novel treatments for NAFLD.

It is generally believed that hepatic lipid deposition induces simple fatty liver and increases the susceptibility of the liver to various other injury factors, and eventually leads to the occurrence of NAFLD [7,8]. Meanwhile, liver fibrosis is another momentous predictor of liver-related complications in NAFLD [9]. Persistent lipid overload in NAFLD facilitates liver fibrosis, which in turn accelerates the NAFLD progression [10]. Thus, elucidating the underlying mechanism of hepatic lipid deposition and liver fibrosis helps alleviate NAFLD.

Tryptophan is mainly catalyzed to kynurenine by indoleamine 2, 3-dioxygenase 1 (IDO1) and tryptophan 2, 3-dioxygenase 2 (TDO2) [11], and kynurenine mediates cancer immune escape [12]. TDO2 is a heme-containing enzyme and exerts regulatory functions in various human tumors by mediating tumor cell growth [13,14]. Meanwhile, TDO2 overexpression is a key mechanism that helps balance the kynurenine axis and inflammation in the liver [15]. TDO2 is highly expressed in the liver and mediates energy homeostasis [16]. As has been reported, TDO2 expressions are increased in liver cancer tissues, and TDO2 overexpression is intimately interrelated to the poor prognosis of patients with liver cancer [14]; the expressions of TDO2 lead to the development of liver cancer through up-regulating tryptophan metabolism [17], implying that TDO2 is a potential therapeutic target for liver cancer. However, the function of TDO2 in NAFLD has not been reported. In the current study, we attempted to investigate whether TDO2 mediated the NAFLD progression and preliminarily confirmed that TDO2 was highly expressed in NAFLD. On this basis, this study further authenticated that the loss of TDO2 relieved hepatic lipid deposition and liver fibrosis in mice with NAFLD, which suggested that TDO2 was a promising therapeutic target molecule for NAFLD.

In the current research, in vivo and in vitro experiments were conducted to evaluate the function of TDO2 in NAFLD pathogenesis. The research data preliminarily confirmed that TDO2 was overexpressed in NAFLD. Furthermore, the loss of TDO2 decreased hepatic lipid deposition in NAFLD mice and repressed liver fibrosis in mice with NAFLD. Based on these findings, this study further elucidated the molecular mechanism of TDO2 acting on NAFLD, aiming to provide a theoretical basis and novel biomarkers for the clinical treatment of NAFLD.

2. Materials and methods

2.1. Experimental animals

C57BL/6 mice (20 ± 2 g, 6–8 weeks old, wild type (WT) mice, male), and TDO2-knockout (KO) mice (23 ± 1.5 g, 8 weeks old, male) were provided from Gempharmatech (Nantong, China, https://www.gempharmatech.com/shop/list.html?key=encryption_VERPMg==). Six mice were randomly assigned to each group. All mice were subjected to a controlled environment (25 ± 2 °C, 50 ± 5 % humidity) with a 12 h light/dark cycle. All mice received adaptive feeding for a week [18].

For the mice in WT-high-fat diet (HFD) or TDO2KO-HFD, WT mice or TDO2-KO mice were fed with an HFD (fat, 61.6 %; carbohydrates, 20.3 %, Research Diets, Shanghai, China) for 16 weeks; for the mice in WT-normal control diet (NCD) or TDO2KO-NCD, WT mice or TDO2-KO mice were fed with an NCD (fat, 10.2 %; carbohydrates, 71.5 %, Research Diets) [19]. All mice were weighed every two weeks. After 16 weeks, all mice were sacrificed by intraperitoneal injection of Phenobarbital (250 mg/kg) [20]. Blood samples were collected from the retro-orbital plexus of the mice and centrifuged at 3000 rpm for 10–15 min to obtain mouse serum samples for further analysis; liver tissues were also gathered for the next research. All animal experimental procedures were approved by Wuhan Marine College and were carried out given ARRIVE guidelines (https://arriveguidelines.org).

2.2. Cell isolation, culture, and treatment

Primary hepatocytes were isolated following the previously reported method [21]. After the liver tissues were isolated from mice, the tissues were transferred to Petri dishes (JSHXRT, Taizhou, China). Then, 10 ml buffer B (korea red ginseng (KRG) buffer with 2 mM CaCl2 and 0.5 mg/ml of Collagenase IV) was added to the Petri dish. Subsequently, the liver fascia was torn using forceps and the buffer was filtered with a cell filter (70 μm, Absin, Shanghai, China). The cells were further washed with 10 % Dulbecco's Modified Eagle Medium (DMEM, Solarbio, Beijing, China), and centrifuged at 50×g for 3–5 min.

Primary hepatocytes were maintained in DMEM with 10 % fetal bovine serum (FBS, Solarbio) and 1 % penicillin-streptomycin, and cultured at 37 °C with 5 % CO2. The above isolated primary hepatocytes were incubated with 200 μM palmitate (PA, Sigma-Aldrich, Missouri, USA) and/or 10 ng/ml receptor activator of the nuclear factor kappa-B ligand (RANKL, MedChemExpress, New Jersey, USA) for 48 h [22,23].

The isolation of hepatic stellate cells were performed following the reported method [24]. The details were: The liver of mice was successively infused with 35 ml calcium-free HePes-phosphate buffer A through the superior vena cava, then with 30 ml pronase (0.5 mg/ml), and finally with 30 ml collagenase D (0.5 mg/ml). Followed by the cut liver tissues were digested in a DNase mixture with collagenase D and pronase for 25 min, cell suspension was centrifuged at 50×g for 5 min to remove the remaining liver parenchymal cells. The supernatant was collected, centrifuged at 900×g for 10 min, and non-parenchymal cells were collected. The collected non-parenchymal cells were re-suspended in 15 % OptiPrep, thoroughly mixed and transferred to a 15 ml centrifuge tube. Subsequently, 5 ml 11.5 % OptiPrep and 2 ml Gey's balanced salt solution (Sigma-Aldrich) were separately layered onto cell suspensions. Tubes were centrifuged at 2000×g for 30 min and cell layer between 11.5 % OptiPrep and GBSS was collected. The resulting cells are purified hepatic stellate cells. The cells were washed twice and inoculated with medium (10 % FBS, 1 % glucose, 2 % penicillin/streptomycin) at 37 °C. Hepatic stellate cells were cultured for three days in a conditioned medium (CM) of different groups of liver cells (WT-ctrl-CM, WT-PA-CM, TDO2KO-ctrl-CM, TDO2KO-PA-CM, TDO2KO-PA + RANKL-CM) [19].

2.3. Western blot

Liver tissues and primary hepatocytes were lysed in Radio Immunoprecipitation Assay (Solarbio). Then, the total proteins were determined using the Bicinchoninic acid (BCA) Protein Assay Kit (Sbjbio, Nanjing, China). The proteins (50 μg) were further run on 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Sbjbio) and transferred to polyvinylidene fluoride membranes (Millipore, Massachusetts, USA). After the membranes were blocked using 5 % bovine serum albumin (BSA) blocking solution (Solarbio) for 1 h, the membranes were incubated with antibodies against TDO2 (ab259359, 1:1000, Abcam, Cambridge, UK), SREBF1 (ab28481, 1:500, Abcam), PPARγ (ab178860, 1:1000, Abcam), FABP1 (ab171739, 1:2000, Abcam), CPT1α (ab220789, 1:1000, Abcam), alpha-SMA (α-SMA, ab7817, 0.341 μg/ml, Abcam), collagen I (ab138492, 1:2000, Abcam), p–NF–κB (phospho S536, ab239882, 1:1000, Abcam), NF-κB (ab288751, 1:1000, Abcam), p-IκBα (AF5851, 1:1000, Beyotime, Shanghai, China), IκBα (AF5204, 1:500, Beyotime), and GAPDH (ab8245, 1:2000, Abcam) overnight at 4 °C. Afterward, the membranes were further incubated with secondary antibodies (Abcam). Protein bands were visualized using enhanced chemiluminescence reagent (Absin) and Image J software (National Institutes of Health, USA).

2.4. Enzyme-linked immunosorbent assay

The contents of total cholesterol (TCH), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum samples were determined at optical delnsity (OD) 450 nm using different ELISA Kits (Mlbio, Shanghai, China) and was following the standard curve [25].

2.5. Detection of cellular TG levels

Cellular TG levels in primary hepatocytes were quantified following the reported methods [21]. Primary hepatocytes were digested with 0.25 % trypsin (Sigma-Aldrich) and the cells were collected to determine protein concentration. Followed by the organic phase was evaporated using nitrogen, the lipid was dissolved with Triton X-100 solution (3 % V/V, Sigma-Aldrich). Cellular TG levels were tested given the manufacturer's instructions and normalized by protein concentrations.

2.6. Hematoxylin-eosin staining

Harvested mouse liver tissues were immobilized with 10 % formalin (MKbio, Shanghai, China) and embedded in paraffin. After liver tissues were made into 5 μm paraffin sections, the sections were assessed by conventional hematoxylin-eosin (HE) staining (Beyotime). NAFLD Activity Score is assessed by pathologists based on the degree of steatosis, hepatocyte swelling, and liver fibrosis to evaluate NAFLD progression [26].

2.7. Oil red O staining and masson staining

Lipid accumulation in mouse liver tissues and primary hepatocytes was assessed by Oil Red O staining. For the lipid accumulation in mouse liver tissues, the liver tissues were first made into 5 μm sections. Then, the sections were washed with PBS, and the sections were incubated in oil red O reagent (Abcam) for 30 min and were further incubated with hematoxylin (MedChemExpress) for 2 min to stain the nuclei. For the lipid accumulation in primary hepatocytes, primary hepatocytes were fixed with 10 % formalin for 8 min, followed by a brief flush of the cells in isopropyl alcohol. Soon after, the cells were incubated with oil red O reagent for 25 min, and the nuclei were stained with hematoxylin for 2 min. Ultimately, images were observed using a fluorescence microscope (Olympus, Japan).

The degree of liver fibrosis in mice was evaluated using Masson's Trichrome Stain Kit (Solarbio) following the reagent manufacturer's standard procedures. Photographs were captured under a microscope (Olympus) at a scale bar of 100 μm.

2.8. Immunohistochemical assay

The harvested 5 μm sections were incubated with 3 % H2O2 methanol for 8 min to block endogenous peroxidase activity. Afterward, the sections were incubated with anti-α-SMA (Abcam) and anti-p–NF–κB (Abcam) overnight at 4 °C. After washing with PBS, the sections were incubated with secondary antibodies (Abcam) at room temperature for 1 h. The sections were further incubated with DAB substrate (Abcam) for visualization. Followed by washing and dehydration, the sections were sealed with cover glass and imaged with a fluorescence microscope (Olympus).

2.9. Statistical analysis

All analyses were conducted by GraphPad Prism software (version 8.0) and presented as mean ± standard deviation (SD). All imaging quantifications (including NAS scores) were performed by blind observers. The percentage of positive area was analyzed by Image J software (National Institutes of Health). Student's t-test was applied to compare differences between two groups, and one-way ANOVA followed by Tukey's post-test was conducted to compare differences among multiple groups. P < 0.05 was considered a statistical significance.

3. Results

3.1. Knockout of TDO2 alleviates hepatic lipid deposition of NAFLD mice

Based on the high expression of TDO2 in high-fat animals [27], this study attempted to investigate the expression pattern of TDO2 in NAFLD. As expected, the Western blot assay clarified that TDO2 was increased in the liver tissues of NAFLD mice (Fig. 1A). Meanwhile, no expressions of TDO2 were examined in TDO2KO-NCD and TDO2KO-HFD groups, hinting that TDO2 was knocked out in mice (Fig. 1A). Further analysis expounded that the body weight of mice was elevated in WT-HFD, and TDO2 knockout caused a reduction in the body weight of mice by nearly a quarter (Fig. 1B–C). A similar conclusion was confirmed in the liver weight analysis (Fig. 1D).

Fig. 1.

Fig. 1

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Influence of TDO2 on hepatic lipid deposition in NAFLD mice. Mice were grouped into WT-NCD, WT-HFD, TDO2KO-NCD, and TDO2KO-HFD. Each group was randomly assigned 6 mice. (A) The protein levels of TDO2 were detected using Western blot. (B–C) Analysis of the body weight of mice. (D) Detection of the liver weight of mice. (E–K) The liver to body weight ratio, and the levels of TCH, TG, LDL-C, HDL-C, ALT, and AST were measured by different commercial kits. (L) Comparison of the SREBF1, PPARγ, FABP1, and CPT1α protein levels using Western blot. (M) The pathological changes in liver tissues in different groups of mice were analyzed by HE staining (scale bar: 50 μm). (N) The lipid accumulation in the mice liver tissues was assessed using Oil Red O staining (scale bar: 100 μm). *P < 0.05 vs. WT-HFD. **P < 0.01 vs. WT-NCD. WT-NCD: wild type-normal control diet; WT-HFD: wild type-high-fat diet; TDO2KO-NCD: TDO2 knockout-normal control diet; TDO2KO-HFD: TDO2 knockout-high-fat diet. Data are shown as mean ± SD.

As displayed in Fig. 1E–K, the liver/body weight, TCH, TG, LDL-C, ALT, AST contents were increased, and HDL-C was decreased in the serum samples from NAFLD mice, while these trends were partially reversed after TDO2 knockout. SREBF1 and PPARγ are genes associated with lipogenesis [28]; FABP1 and CPT1α are fatty acid metabolism-related genes [29]. Western blot corroborated that the protein levels of SREBF1, PPARγ, FABP1 were up-regulated in WT-HFD, and CPT1α was down-regulated, and TDO2 knockout reversed these effects (Fig. 1L). Meanwhile, the NAFLD activity score was increased in WT-HFD, and the NAFLD activity score was lower in TDO2KO-HFD than in WT-HFD, prompting that TDO2 knockout effectively restrained NAFLD progression in mice (Fig. 1M). Oil Red O staining analysis of lipid accumulation in mice liver tissues presented similar trends (Fig. 1N). Taken together, these data demonstrated that TDO2 knockout reduced hepatic lipid deposition in NAFLD mice.

3.2. TDO2 deficiency reduces liver fibrosis in mice with NAFLD

Considering that liver fibrosis is the pathological factor of NAFLD [30], we further investigated whether TDO2 mediated liver fibrosis in NAFLD. Masson staining verified that the liver fibrosis in WT-HFD mice was more severe than that in WT-NCD mice, and TDO2 knockout alleviated the liver fibrosis in WT-HFD mice (Fig. 2A). Furthermore, the level of fibrotic marker α-SMA was elevated in WT-HFD, and this elevation was reversed by TDO2 knockout (Fig. 2B). As expected, the protein levels of fibrotic markers α-SMA and collagen I in liver tissues presented similar trends (Fig. 2C). Conclusively, TDO2 knockout repressed liver fibrosis in NAFLD mice.

Fig. 2.

Fig. 2

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Regulation of TDO2 on liver fibrosis in NAFLD mice. Mice were grouped into WT-NCD, WT-HFD, TDO2KO-NCD, and TDO2KO-HFD. (A) The degree of liver fibrosis in mice was evaluated using Masson staining (scale bar: 100 μm). (B) The expression of α-SMA was quantified by an IHC assay (scale bar: 100 μm). (C) Detection of the protein levels of α-SMA and collagen I (fibrotic markers) in liver tissues using Western blot. *P < 0.05 vs. WT-HFD. **P < 0.01 vs. WT-NCD. Data are shown as mean ± SD.

3.3. Loss of TDO2 inactivates the NF-κB pathway in NAFLD mice

Previous studies repeatedly confirm that the NF-κB pathway exerts momentous functions in NAFLD pathogenesis [31,32]. Thus, this research tried to determine whether the NF-κB pathway was required for TDO2 to mediate the NAFLD process. Firstly, Western blot analysis authenticated that the protein levels of p–NF–κB and p-IκBα (pivotal targets of the NF-κB pathway) were increased by nearly tripled in WT-HFD, and these increases were reversed after knocking out TDO2 (Fig. 3A). Afterward, the IHC assay further corroborated that the expression of p–NF–κB was higher in WT-HFD than in WT-NCD, and the TDO2 deficiency decreased the p–NF–κB expression (Fig. 3B). All in all, these experimental data suggested that TDO2 knockout reduced the activation of the NF-κB pathway.

Fig. 3.

Fig. 3

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TDO2 mediates the NF-κB pathway in mice with NAFLD. Mice were grouped into WT-NCD, WT-HFD, TDO2KO-NCD, and TDO2KO-HFD. (A) The protein levels of p–NF–κB, NF-κB, p-IκBα, and IκBα were measured by Western blot. (B) Comparison of the p–NF–κB expression using IHC analysis (scale bar: 100 μm). *P < 0.05 vs. WT-HFD. **P < 0.01 vs. WT-NCD. Data are shown as mean ± SD.

3.4. TDO2 knockdown reduces the lipid accumulation and fibrosis of PA-induced primary hepatocytes

Subsequently, this study further clarified the TDO2 function in the NAFLD model in vitro. As reported, wild-type hepatocytes exposed to PA are applied as an in vitro model of NAFLD [22]. Our data further revealed that the percentage of Oil Red O staining in primary hepatocytes was increased nearly six-fold in WT-PA, and knocking out TDO2 reversed this increase (Fig. 4A). The contents of cellular TG in different groups displayed similar trends (Fig. 4B). Also, PA treatment up-regulated the protein levels of SREBF1, PPARγ, FABP1, and down-regulated CPT1α, while these trends were reversed after TDO2 deficiency (Fig. 4C). Moreover, α-SMA and collagen I were increased in WT-PA-CM, and TDO2 knockout reversed these elevations (Fig. 4D). In short, knocking out TDO2 restrained the lipid accumulation and fibrosis of primary hepatocytes induced by PA.

Fig. 4.

Fig. 4

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TDO2 regulates the lipid accumulation and fibrosis of primary hepatocytes treated by PA. After primary hepatocytes were isolated from WT and TDO2KO mice, the cells were exposed to 200 μM PA for 48 h. (A) Analysis of the lipid accumulation in primary hepatocytes using Oil Red O staining (scale bar: 200 μm). (B) The concentrations of cellular TG were determined by a commercial kit. (C) Comparison of the SREBF1, PPARγ, FABP1, and CPT1α protein levels using Western blot. (D) Hepatic stellate cells were cultured for three days in a conditioned medium (CM) of different groups of liver cells (WT-ctrl-CM, WT-PA-CM, TDO2KO-ctrl-CM, TDO2KO-PA-CM). The protein levels of α-SMA and collagen I were measured by Western blot. *P < 0.05 vs. WT-PA. **P < 0.01 vs. WT-Ctrl. Ctrl: control; PA: palmitate. Data are shown as mean ± SD.

TDO2 influences hepatic lipid deposition and liver fibrosis in PA-induced primary hepatocytes by the NF-κB pathway.

Next, we investigated whether TDO2 mediated hepatic lipid deposition and liver fibrosis in the in vitro model of NAFLD through NF-κB. As presented in Fig. 5A, the protein levels of p–NF–κB and p-IκBα were increased after PA treatment, while TDO2 knockout reversed this trend. RANKL is an activator of the NF-κB pathway [33]. Western blot analysis further affirmed that the protein levels of p–NF–κB and p-IκBα were decreased by nearly two-thirds in TDO2KO-PA, and this decrease was partially reversed after RANKL treatment (Fig. 5B). Also, the percentage of Oil Red O staining in primary hepatocytes was reduced in TDO2KO-PA, while RANKL reversed this reduction (Fig. 5C). Similarly, analysis of the cellular TG in primary hepatocytes presented the same trend (Fig. 5D). Furthermore, the protein levels of α-SMA and collagen I were decreased in TDO2KO-PA-CM, and this effect was reversed by RANKL treatment (Fig. 5E). In summary, TDO2 knockout repressed hepatic lipid deposition and liver fibrosis in PA-treated primary hepatocytes by inactivating the NF-κB pathway.

Fig. 5.

Fig. 5

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TDO2 mediates hepatic lipid deposition and liver fibrosis in PA-induced primary hepatocytes via the NF-κB pathway. (A) Primary hepatocytes were isolated from WT and TDO2KO mice, the cells were then exposed to 200 μM PA for 48 h. The protein levels of p–NF–κB, NF-κB, p-IκBα, and IκBα were tested using Western blot. (B) Primary hepatocytes were isolated from WT and TDO2KO mice, the cells were then exposed to 200 μM PA and/or 10 ng/ml for 48 h. Detection of the p–NF–κB, NF-κB, p-IκBα, and IκBα protein levels by Western blot. (C) Comparison of the lipid accumulation in primary hepatocytes using Oil Red O staining (scale bar: 200 μm). (D) Analysis of the cellular TG by commercial kit. (E) Hepatic stellate cells were cultured for three days in a CM of different groups of liver cells (WT-ctrl-CM, WT-PA-CM, TDO2KO-ctrl-CM, TDO2KO-PA-CM, TDO2KO-PA + RANKL-CM). The protein levels of α-SMA and collagen I were determined using Western blot. *P < 0.05 vs. WT-PA or TDO2KO-PA. **P < 0.01 vs. WT-Ctrl. Data are shown as mean ± SD.

4. Discussion

As a global epidemic disease, NAFLD has brought great pressure on the health system [34]. This study aimed to provide effective target molecules for NAFLD treatment. Here, we found for the first time that TDO2 was highly expressed in liver tissues of NAFLD, and TDO2 knockout restrained hepatic lipid deposition and liver fibrosis in NAFLD models in vivo and in vitro. Furthermore, we confirmed that TDO2 deficiency repressed hepatic lipid deposition and liver fibrosis in PA-treated primary hepatocytes by inactivating the NF-κB axis. Our findings provided a promising target for the clinical treatment of NAFLD.

The central driving factor of NAFLD is the imbalance of liver lipid metabolism, resulting in lipid deposition in liver cells, which in turn leads to liver oxidation, local inflammation, and cell injury [35]. Critically, accumulated studies show that hepatic lipid deposition accelerates the occurrence of NAFLD. For instance, NAFLD is a series of diseases caused by lipid deposition in liver cells, and the intervention of hepatic lipid deposition can be used as a potential treatment for NAFLD [7]; blocking lipid deposition in NAFLD by stimulating mitochondrial fatty acid oxidation and triacylglycerol efflux attenuates NAFLD [36]. It is noteworthy that our experimental data revealed that TDO2 was increased in liver tissues of NAFLD. Previous studies confirm that SREBF1 and PPARγ (genes related to lipogenesis), FABP1, and CPT1α (genes related to fatty acid metabolism) indicate the degree of lipid deposition [29,37]. Meanwhile, our data further corroborated that TDO2 loss reduced the protein levels of SREBF1, PPARγ, FABP1, and increased CPT1α, implying that TDO2 loss restrained lipid accumulation in NAFLD mice. On this basis, we further demonstrated that the loss of TDO2 decreased the NAFLD activity score, which suggested that TDO2 might be a prospective biomarker for NAFLD treatment.

With the continuous exploration of NAFLD development, liver fibrosis is another critical driver of NAFLD. For example, Powell et al. authenticated that liver fibrosis is a key prognostic marker of total mortality in NAFLD patients [38]; Wandrer et al. verified that liver fibrosis development is associated with a high prevalence of NAFLD, and inhibiting liver fibrosis is a promising approach to alleviate NAFLD [39]. As has been reported, the increased expressions of α-SMA and Collagen I indicate the aggravation of liver fibrosis [40]. Notably, the current research confirmed that TDO2 knockout lessened the degree of fibrosis and decreased the expressions of α-SMA and collagen I in liver tissues, hinting that TDO2 knockout reduced liver fibrosis in NAFLD mice. Therefore, our subsequent research focused on revealing the underlying mechanism of the NAFLD process mediated by TDO2.

Nuclear factor-kappaB (NF-κB) is a homologous or heterodimer complex composed of RelA, NF-κB1, NF-κB2, c-Rel, and RelB [41]. Accumulated evidence affirms that NF-κB and its target genes are vital therapeutic targets for different human diseases [42]. Recent studies have demonstrated that NF-κB has pivotal regulatory functions in NAFLD. Wang et al. expounded that berberine alleviates hepatic steatosis and inflammatory cell infiltration in NAFLD by reversing nuclear NF-κB translocation in primary hepatocytes [43]; Zhao et al. clarified that bicyclol pretreatment reduces inflammation by inactivating the NF-κB axis, thereby alleviating NAFLD [32]. Crucially, TDO2 mediates the NF-KB pathway by promoting the autocrine IL-6 of liver cancer cells [17]. Similar to the above findings, our studies confirmed that the loss of TDO2 reduced the expressions of p–NF–κB and p-IκBα (critical targets for the NF-κB pathway), prompting that TDO2 deficiency reduced the activation of the NF-κB pathway in vivo. Furthermore, we demonstrated that TDO2 had similar regulatory functions on lipid accumulation and fibrosis in PA-treated primary hepatocytes in vitro.

5. Conclusions

This research indicated that TDO2 was increased in NAFLD. Our functional studies further clarified that the loss of TDO2 repressed hepatic lipid deposition and liver fibrosis in NAFLD models in vivo and in vitro, and these beneficial effects were interrelated to the NF-κB pathway. This study suggests that TDO2 might be a novel therapeutic target for NAFLD. Moreover, further detailed studies including gain-of-function TDO2 and other possible signaling pathways are needed to fully elucidate the conclusions of this research. Meanwhile, another limitation of this study was that there were not enough biological samples. We would expand the biological samples in the future to further support the conclusions of this study. Moreover, limitation of this study is the lack of evidence that TDO2 might affect the NF-κB axis. We would carry out further research to enrich the content of this study.

Funding statement

This study was supported by The Scientific Research Program of Hubei Provincial Education Department in 2018 (B2018226).

Data availability statement

Data will be made available on request, and the corresponding author can be contacted if needed.

CRediT authorship contribution statement

Zhi Qin: Writing – original draft, Software, Methodology, Formal analysis, Data curation. Min Zhou: Writing – review & editing, Project administration, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

NA.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e22464.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.pdf (315.2KB, pdf)

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