Protective effects of dopamine against non-alcoholic steatohepatitis via inhibiting p65 pathways in vivo and in vitro

Abstract

Introduction

Currently, the role and mechanism of dopamine in non-alcoholic steatohepatitis (NASH) remains unclear.

Methods

In vitro experiments utilized FFA and LPS to establish NASH cell models, while a fibrotic cell model was created using TGFβ1 to investigate the impact of dopamine on cellular lipid metabolism, inflammation, and fibrosis. In vivo experiments involved the use of MCD and HFD diets to induce NASH in mouse models for observing the effects of dopamine on NASH disease progression.

Results

Our study showed that dopamine significantly downregulated the expression levels of Caspase 1, IL-1β and IL18 in the HepG2 NASH cell model. In addition, dopamine could inhibit the TGF-β1-induced accumulation of collagen I and α-SMA in LX2 cells. In vivo experiments have shown that dopamine attenuation in mice is associated with MCD diet-induced and HFD-induced steatohepatitis. Mechanically, dopamine inhibits the p65 signaling pathway in NASH.

Conclusion

In conclusion, the present study demonstrates the role of dopamine in ameliorating the symptoms of NASH and provides a direction for future research on the application of the dopaminergic system to liver disease.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a liver metabolic disease that involves fat accumulation in the liver. It is caused by factors such as excessive drinking, long-term use of drugs, hepatitis C, or genetic disorders.¹ NAFLD is characterized by simple steatosis (non-alcoholic fatty liver disease, NAFL) and non-alcoholic steatohepatitis (NASH), which are accompanied by liver cell damage, balloon like lesions, and lobular inflammation. Chronic inflammation can often lead to the development of liver fibrosis, which can progress to cirrhosis and hepatocellular carcinoma (HCC).² In addition, fibrosis and HCC can cause liver failure and even death. Currently, the global prevalence of NAFLD has increased from 25% in 2015 to 29.8%, which has become the leading cause of chronic liver disease worldwide. This rise can be attributed to the continuous increase in obesity rate and the incidence rate of type 2 diabetes mellitus (T2DM).³

Dopamine (DA) is a natural catecholamine formed by the decarboxylation of 3,4-dihydroxyphenylalanine (DOPA).⁴ Dopamine is essential in the formation of norepinephrine in noradrenergic nerves, as well as being a neurotransmitter in specific regions of the central nervous system. Dopamine produces positive chronotropic and inotropic effects on the myocardium, resulting in increased heart rate and cardiac contractility. This is accomplished directly by exerting an agonist action on beta-adrenoceptors and indirectly by causing release of norepinephrine from storage sites in sympathetic nerve endings.⁵ In clinical, dopamine is used to correction of hemodynamic imbalances present in the shock syndrome due to myocardial infarction, trauma, endotoxic septicemia, open-heart surgery, renal failure, and chronic cardiac decompensation as in congestive failure.⁶ Recent studies have shown that inhibition of dopamine receptor D2 blocking YAP signaling in macrophages, leading to the alleviation of liver fibrosis in NASH.⁷ Furthermore, the DA-DRD5-ARRB2-PP2A signaling axis can prevent S. aureus-induced inflammation and protect mice against S. aureus-induced sepsis and meningitis after DA treatment.⁸

In this study, researchers discovered the association between dopamine and NASH cell models. Dopamine showed remarkable improvements in treating NASH, particularly in reducing inflammation. The finding excites considerable attention from researchers. Further validation revealed that DA effectively alleviated the symptoms associated with NASH, such as steatosis and inflammation. However, it should be noted that the use of DA may also lead to transient drowsiness and mental fatigue. These experimental results suggest that the dopaminergic system may play an important role in the development of NASH. Furthermore, these findings indicate the potential effectiveness of drugs targeting the dopaminergic system for treating NASH. These findings provide a new direction for the study of the pathogenesis of NASH as well as the development of drugs.

Materials and methods

Materials and reagents

The following materials were used in the present study: Dulbecco modified eagle’s medium (DMEM)-high glucose (Sigma-Aldrich, USA), total cholesterol (TC), triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Institute, China), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Aladdin Bio-Chem Technology, China), antibiotics and fetal bovine serum (FBS) (Gibco, USA), isopropanol, oleate, palmitate, oil red O and defatted bovine serum albumin (St Louis, MO, USA), high fat diet (carbohydrates, 20%; fat, 60%; protein, 20%; D12492; Huafukang Bioscience; Beijing, China), methionine-choline deficient diet (TP3005G; TrophicDiet, Nantong, China), radio immunoprecipitation assay (RIPA) buffer (Wanleibio, China), polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA), efficient chemiluminescence (ECL) plus kit (Biosharp; Labgic), antibody to p-p65, p65, F4/80, NLRP3, ASC, Caspase1, IL-1β, IL6 and IL18 (Wanleibio, Shenyang, China), β-actin (Bioworld Technology, Co., Ltd China), GSMD (Wuhan ColorfulGene Biological Technology Co. Ltd, China), horseradish peroxidase (HRP)-linked secondary antibody (Sangon Biotech, China), FITC Goat Anti-rabbit IgG H&L/FITC; Bioss, Beijing, China).

Cell culture

HepG2 cells were purchased from Shanghai Institute of cell biology (Shanghai, China) and cultured in Dulbecco's Modified Eagle Medium containing 1% antibiotics and 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C. The mixture of oleic acid and palmitic acid (2:1) was dissolved in 1,640 containing 1% bovine serum albumin (BSA) under proper heating conditions to prepare free fatty acid (FFA) solution.

Animals and treatments

The animal protocol was approved by China Pharmaceutical University Licensing Committee, and the investigation complied with the Guide for the Care and Use of Laboratory Animals by the U.S. National Institutes of Health. Seven-week-old mice male C57BL/6 J mice (22–27 g) were purchased from Animal Center of Yangzhou University and fed with an environment of pathogen-free conditions (controlled temperature at 24 °C ± 4 °C, and 12-h light/dark cycle). After one week of adaption, the mice were randomly divided into three groups of six mice each. Mice were fed with high fat diet or methionine-choline deficient diet. After 16 weeks of HFD diet and 4 weeks of MCD diet, dopamine 50 mg/kg was given intragastric administration. Mice from the control group were provided commercial feed (carbohydrates, 71.5%; fat, 10.2%; protein, 18.3%; 1,010,001; Xietongshengwu; Jiangsu, China).

Oil red O staining

HepG2 cells were washed twice with phosphate buffered saline (PBS). The cells were stained with 0.5% oil red O at 37 °C for 1 h, and then rinsed with 60% isopropanol for 2 min, and photos were taken under the microscope.

MTT assay

HepG2 cells were seeded at a density of 5 × 10³ cells/well in 96-well plates. Then, HepG2 cells were incubated for 24 h in Dulbecco's Modified Eagle Medium culture medium. After processing according to the conditions, 10 μL of MTT solution (5 mg/mL) was added to each well. The cells were incubated at 37 °C for 4 h, after which the medium was aspirated and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the crystals.

Immunofluorescence

F4/80 immunofluorescence staining were performed to measure the inflammatory infiltration of liver tissues. Paraffined sections of liver tissues were incubated with anti-F4/80 (WLH2545 1:100 dilution) primary antibody overnight at 4 °C and then labeled with a fluorophore-conjugated secondary antibody (bs-0295G-FITC Goat Anti-rabbit IgG H&L/FITC 1:100 dilution). The images of immunofluorescence were obtained using a fluorescence microscope and quantified using ImageJ software.

Histological analysis and staining

Hematoxylin and eosin (H&E) staining, oil red O (Servicebio, Wuhan, China) staining and picrosirius red staining is completed by Wuhan Servicebio Biotechnology Co., Ltd CD11b was detected by immunohistochemistry staining were performed to measure the inflammatory infiltration of liver tissues. Paraffined sections of liver tissues were incubated with anti-CD11b (WL01193 1:100 dilution primary antibody overnight at 4 °C and then labeled with a horseradish peroxidase (HRP)-linked secondary antibody (Sangon Biotech, 1:100 dilution). The images of immunofluorescence were obtained using a fluorescence microscope and quantified using ImageJ software.

Western blot analysis

Mice tissues and cells were lysed with NCM RIPA buffer supplemented with a protease and a Phosphatase Inhibitor Cocktail and centrifuged at 12,000 rpm for 15 min. Equal amounts of proteins were loaded onto 10% SDS-PAGE gels, and the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Thereafter, the membranes were blocked with 5% skim milk in TBST and immunoblotted with the indicated primary antibodies at 4 °C overnight. The next day, the membranes were washed and incubated with a horseradish peroxidase (HRP)-linked secondary antibody (1:15000 dilution) for 1.5 h at room temperature. Finally, the bands were detected using an enhanced chemiluminescence kit and quantified with a ChemiDoc MP Imaging System.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8. All data in this study were expressed as the mean ± SD. Differences were analyzed by Student t tests, one-way or two-way analysis of variance (ANOVA) as appropriate. A P value < 0.05 was considered significant.

Results

Establishment of NASH cell model in vitro

Free fatty acid (FFA) solution, composed of saturated fatty acid palmitic acid (PA) and unsaturated fatty acid oleic acid (OA), has been commonly used as a tool for establishing steatosis models in hepatocytes. Based on the previous study, lipopolysaccharide (LPS) can be administered separately to activate the intracellular NF-κB signal pathway by stimulating the Toll-like receptor 4 (TLR4), which is used to induce the assembly and activation of NLRP3 inflammasomes.^9–11 Therefore, the administration of LPS may be a promising method for the induced of NASH. The results of the MTT experiment on the effect of FFA and LPS on the survival rate of HepG2 cells revealed that concentrations of FFA at or below 1 mM slightly promoted cell proliferation, but without significant impact. However, concentrations of FFA at 2 mM significantly inhibited cell survival and exhibited noticeable toxicity (Fig. 1A). On the other hand, the selected concentration of LPS (0-1,000 ng/ml) did not significantly affect cell viability (Fig. 1B). The results of oil red staining indicated a notable correlation between intracellular lipid droplet accumulation and FFA concentration. After 24 h of treatment with 1 mM FFA, the cytoplasm was filled with lipid droplets stained red, indicating the presence of severe lipid deposition (Fig. 1C). The absorption value of lipid droplet precipitation detection at a wavelength of 500 nm is the same as that obtained from oil red staining (Fig. 1D).

Fig. 1.

Establishment of NASH cell model in vitro. A and B) Cell viability of HepG2 cells. C) HepG2 cells were stained with oil red O and viewed under a microscope at 400× magnification. D) Optical density value showing the lipid accumulation of HepG2 cells. E) Representative western blot image of NLRP3, ASC, Caspase1, IL-1β, IL6 and β-actin. F and G) Western blot analysis showing the protein expression levels of NLRP3, ASC, Caspase1, IL-1β, IL6. The data are presented as the mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^****P < 0.00001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

After treating the cells with 1 mM FFA for 18 h, different concentrations of LPS were administered as secondary shocks. The purpose was to establish an optimal NASH cell model by detecting the activation of inflammasome and the release of inflammatory factors. Western blot results indicated that the expression levels of both NLRP3 and ASC proteins, the constituents of the inflammasome, markedly increased upon stimulation by FFA and various concentrations of LPS. Additionally, the activated Caspase1 was significantly increased (Fig. 1E and F). In addition, inflammatory factors IL-1β and IL6 showed significant differences at a concentration of 100 ng/mL LPS (Fig. 1E and G). Based on the above experimental results, it was found that a HepG2 NASH cell model could be created by using a combination of 500 ng/mL LPS and 1 mM FFA.

Dopamine improves inflammation and lipid metabolism in NASH cell model

The effect of the DA on the survival rate of HepG2 cells was detected using an MTT assay. The results showed that after 24 h of treatment with different concentrations of the DA, the DA with concentrations of 200 μM had no significant effect on the survival of HepG2 cells, while the DA at concentrations of 400 and 800 μM significantly inhibited cell survival (Fig. 2A). The results of oil red staining showed that compared to the model group, there was a slight decrease in lipid droplets in the 200 μM DA group cells (Fig. 2B). Quantitative analysis indicated that DA could slightly decrease lipid deposition in the HepG2 NASH cell model (Fig. 2C). The Western blot results showed that compared to the control group, NLRP3 and ASC were significantly upregulated in the HepG2 NASH cell model, while after DA treatment, the upregulation of NLRP3 and ASC was significantly inhibited (Fig. 2D and E). The activation of NLRP3 inflammasomes leads to the maturation of Caspase 1, which can induce the production and secretion of inflammatory factor IL-1β and IL18. The experimental results showed that DA significantly downregulated the expression level of Caspase 1, IL-1β and IL18 in the HepG2 NASH cell model (Fig. 2D and E).

Fig. 2.

Dopamine improves inflammation and lipid metabolism in NASH cell model. A) Cell viability of HepG2 cells. B) HepG2 cells were stained with oil red O and viewed under a microscope at 400× magnification. C) Optical density value showing the lipid accumulation of HepG2 cells. D) Representative western blot image of NLRP3, ASC, Caspase1, IL-1β, IL6, IL18 and β-actin. E) Western blot analysis showing the protein expression levels of NLRP3, ASC, Caspase1, IL-1β, IL6, IL18. F) Immunofluorescence staining for GSMDM in the HepG2 NASH cell model and treated with 200 μM DA for 24 h. The data are presented as the mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

The activation of Caspase 1 in the NLRP3 inflammasome can lead to the cleavage and maturation of GSDMD. Afterward, the amino-terminal domain of GSDMD transfers to the cell membrane and leads formation of plasma membrane pores, leading to the release of intracellular inflammatory factors and the occurrence of pyroptosis.^12,13 Immunofluorescence assay was used to detect the expression level of GSDMD in HepG2 cells after treatment. The experimental results showed that compared with the control group, the model group treated with FFA and LPS significantly upregulated the expression of GSDMD, and the green fluorescence of most cells was concentrated near the cell membrane rather than uniformly distributed in the cytoplasm, indicating the formation of GSDMD membrane pores and the occurrence of pyroptosis. However, the green fluorescence in the cells was significantly weakened and evenly distributed after administration of DA. It shows that DA inhibits the occurrence of HepG2 pyroptosis (Fig. 2F).

Dopamine improves fibrosis in LX2 cell

In NASH, inflammatory factors and fibrosis-promoting factors secreted by liver cells can act on hepatic stellate cells nearby, thereby activating them and inducing their transformation into fibroblasts to promote fibrosis.^14,15 Therefore, we detected the expression of the fibrosis-promoting factor TGF-β1 in HepG2 NASH cell models. The results showed that TGF-β1 was significantly up-regulated in the model group, and 200 μM DA could significantly down-regulate TGF-β1 protein expression (Fig. 3A). Conditioned medium derived From HepG2 promotes fibrosis in LX2 cells (Fig. 3B). The immunofluorescence results showed that the expressions of Collagen I and α-SMA increased in LX2 cells after incubation with the conditioned medium from the HepG2 model group. However, the condition culture medium derived from HepG2 DA group failed to increase the expressions of Collagen I and α-SMA (Fig. 3C and D).

Fig. 3.

Dopamine improves fibrosis in LX2 cell. A) Representative western blot image of TGF-β1 and β-actin. B) Schematic diagram of the experimental procedure. C–F) Immunofluorescence staining for collagen I and α-SMA in LX2 cell and treated with 200 μM DA for 24 h. The data are presented as the mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

To investigate the effect of DA on fibrosis in LX2 cells, we initiated fibrosis model by inducing LX2 cells with 8 ng/mL TGF-β1. The immunofluorescence results showed that compared with the control group, TGFβ1 significantly increased the expressions of Collagen I and α-SMA in LX2 cells in the model group, while the administration of DA eliminated the up-regulation of Collagen I and α-SMA (Fig. 3E and F). These results suggest that DA could inhibit the accumulation of collagen I and α-SMA in LX2 cells TGFβ1-induced.

Dopamine attenuates MCD diet-induced liver injury in mice

Methionine and choline are essential for the synthesis of very-low-density lipoprotein (VLDL) and lecithin in the liver. In the absence of these nutrients, VLDL and lecithin synthesis may decrease, leading to a buildup of dietary triglycerides in the liver. This can result in the development of non-alcoholic fatty liver disease.¹⁶ According to the previously published method,¹⁷ DA was administered at a dosage of 50 mg/kg per day for a total of 4 weeks After two weeks of MCD feeding. DA could significantly reduce the ratio of liver weight to body weight (Fig. 4A), liver TG and TC content (Fig. 4B and C) in MCD diet-induced NASH mice. Compared with the MCD model group, the DA intervention group had a redder liver color (Fig. 4D). The results of liver H&E staining showed that the NAS index was reduced in the DA intervention group (Fig. 4E). Oil red O staining showed that DA significantly attenuates liver lipid accumulation (Fig. 4F).

Fig. 4.

Dopamine attenuates MCD diet-induced liver injury in mice. A) Ratio of liver weight to body weight. B and C) Liver TC and TG levels. D) Representative photograph of dissected liver tissues. E and F) Representative images of hepatic steatosis and lipid accumulation stained with H&E and oil red O. Scale bar, 100 μm. G) Immunohistochemistry staining for CD11b in inflammatory cell infiltration in liver tissues. Scale bar, CD11b 50 μm. H) Picrosirius red staining in liver tissues. Scale bar, 100 μm.The data are presented as the mean ± SD (n = 6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

CD11b is a membrane surface marker of M1 macrophages which can indicate the infestation of immune cells in liver tissue.¹⁸ Immunohistochemistry detection of CD11b showed that the inflammatory symptoms of NASH mice were significantly improved after DA intervention (Fig. 4G). Sirius red staining showed that DA could improve liver fibrosis in MCD diet-induced NASH mice (Fig. 4H). These results revealed that DA alleviates MCD diet-induced steatohepatitis in mice.

Dopamine attenuates HFD-induced steatohepatitis in mice

The HFD diet induced NASH is a common method to induce NASH mouse models. In this model, C57BL/6 mice are fed with a high-fat diet for 10–12 weeks, which leads to changes in hyperlipidemia, hyperinsulinemia and abnormal glucose tolerance.¹⁶ Based on the previous studies,¹⁷ the mice were fed the high-fat diet for 16 weeks, and then intraperitoneal injection of DA at a dosage of 50 mg/kg was given every three days for 8 weeks. Ratio of liver weight to body weight (Fig. 5A), liver TG and TC contents (Fig. 5B and C) were significantly decreased in HFD-induced NASH mice after DA intervention. Furthermore, compared with the model group, it was found that the liver was ruddier and smoother (Fig. 5D) after 8 weeks of DA intervention. Histological examination of hepatic sections stained with H&E and Oil-red-O showed that there were extensive micro-vesicular cellular vacuoles, reflecting intrahepatic fat accumulation in HFD-induced NASH mice. However, this symptom was markedly ameliorated by 50 mg/kg DA (Fig. 5E and F). These results suggest that DA attenuates HFD-induced hepatic injury and metabolic disorders in mice.

Fig. 5.

Dopamine attenuates HFD-induced steatohepatitis in mice. A) Ratio of liver weight to body weight. B and C) Liver TC and TG levels. D) Representative photograph of dissected liver tissues. E and F) Representative images of hepatic steatosis and lipid accumulation stained with H&E and oil red O. Scale bar, 100 μm. The data are presented as the mean ± SD (n = 6). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

Dopamine inhibit p65 signaling pathway in NASH

To determine the specific mechanism of dopamine on the HepG2-NASH cell model, we used protein blotting to detect the activation level of the p65 signaling pathway in each group. The experimental results showed that the phosphorylation of p65 was significantly up-regulated in the model group compared with the control group. However, the administration of DA significantly inhibited the increase of phosphorylation level of p65 (Fig. 6A). After conducting the experiment, cellular immunofluorescence was employed to observe the localization of p65 in the nucleus. The results demonstrated that the expression level of p65 significantly increased in the model group compared to the control group, and the fluorescence signal intensity in the nucleus was notably higher than in the cytoplasm. However, the entry of p65 into the nucleus was effectively inhibited after the administration of DA (Fig. 6B). Recent studies have found that DA could block the NF-KB pathway by activating the DRD5-ARRB2-PP2A signaling axis to inhibit the occurrence of inflammation. SKF38393, as a DRD5 agonist, has the same anti-inflammatory and blocking effects of NF-KB signaling pathway as DA. Similarly, this study also demonstrated that SKF38393 inhibited the p65 signaling pathway (Fig. 6A and B). This suggests that dopamine is likely to inhibit the p65 signaling pathway through DRD5.The experimental results showed that compared with the control group, the level of p-p65 in the MCD and HFD modeling groups was significantly increased. In the DA group, p-p65 levels were significantly reduced compared to those in the modeling group (Fig. 6C and D). These results suggest that dopamine improves NASH by inhibiting the activation of p65 signaling pathway.

Fig. 6.

Dopamine inhibit p65 signaling pathway in NASH. A) Representative western blot image of p-p65, p65 and β-actin. B) Immunofluorescence staining for p65 in the HepG2 NASH cell model and treated with 200 μM DA or 40 μM SKF38393for 24 h. Scale bar, 50 μm. C and D) Representative western blot image of p-p65, p65 and β-actin. The data are presented as the mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001. The data were analyzed with one-way ANOVA or two-tailed Student’s t-test.

Discussion

The theory of “two-hit” is generally accepted to clarify the pathogenesis of NAFLD. The “two-hit” theory is characterized by two steps: intrahepatic lipid accumulation (hepatic steatosis), and inflammatory progression to nonalcoholic steatohepatitis (NASH).^19,20 To establish the NASH cell model, LPS and FFA were selected as the first and second signals, respectively.⁹ Expression of NLRP3 inflammasome related proteins was used as a key indicator in the model. After treating HepG2 cells with various concentrations of FFA, significant lipid droplets were observed within the cells. To better simulate the impact of lipid accumulation, the concentration that had the most significant effect within the safe range was chosen for subsequent experiments. The optimal concentration for inducing inflammation was determined by combining LPS at different concentrations. The analysis results showed that LPS stimulation at 500 ng/mL, combined with FFA, significantly activated the expression of NLRP3 and ASC proteins, which are components of inflammatory bodies. Moreover, the effector protein IL-6, Caspase 1 and IL-1β was also significantly upregulated.

In clinical practice, dopamine is used to correction of hemodynamic imbalances present in the shock syndrome due to myocardial infarction, trauma, endotoxic septicemia, open-heart surgery, renal failure, and chronic cardiac decompensation as in congestive failure.⁶ In recent years, many studies have found that the dopaminergic system involves in inflammatory diseases, particularly neuroinflammatory diseases development.^21,22 The results of this study indicate that different concentrations of DA treatment were associated with significant downregulation of the NLRP3 and ASC proteins, which belong to the inflammasome. Furthermore, Caspase 1, IL 6, IL-1β and IL-18 were significantly reduced, thereby indicating that DA blocked the synthesis and activation of the NLRP3 inflammasome.

In a normal liver, hepatic stellate cells maintain a non-proliferative and static phenotype.²³ However, after liver injury, hepatic stellate cells become activated and transform from fat storage cells to fibroblasts, exhibiting characteristics such as proliferation, an inflammatory response and chemotaxis. This process is characterized by an enhanced production of extracellular matrix, including fibrosis markers such as collagen I and α-SMA.²⁴ The experimental results demonstrate that 200 μM DA can significantly reduce the protein expression of TGF-β1 in HepG2 cells. These findings suggest that dopamine may inhibit the progression of fibrosis in hepatic stellate cells. In addition, administration of DA abolished the TGF-β1-induced upregulation of Collagen I and α-SMA, indicating that DA inhibits TGFβ1-induced transformation of LX2 cells into fibroblasts. Similarly, animal experiments also showed that DA administration could significantly improve liver fibrosis induced by MCD diet.

In the construction of NASH model, three methods are usually used: high-fat and high-sugar diet (e.g. HFD; HFHC; MCD), nutrient deficiency diet (e.g. MCD; CD), or drug induction (e.g. CCl4). High-fat and high-sugar diets usually require prolonged feeding (> 4 months), whereas nutrient deficient diets require shorter feeding periods. In this study, HFD diet containing 60% fat was fed for up to 6 months. In addition, MCD feeding for 6 weeks was used to further verify the effect of DA. Recently, a Western diet plus CCl4-induced model was reported to induce liver fibrosis at stage 3 in mice after 12 weeks of exposure and liver cancer after 24 weeks of exposure.^25–27 This model has unique advantages over simple high-fat, high-sugar feeding and nutrient-deficient feeding in that it encapsulates the progression of human fatty liver disease (from simple steatosis to inflammation, fibrosis, and cancer) in a consistent manner. The effect of DA on cancer progression from NASH was not further explored in this study. Although DA is effective in the treatment of NASH, it also has significant adverse effects, including drowsiness, listlessness, and even death in HFD high-fat model. The reason for this phenomenon may be due to the unreasonable dosage of DA. On the other hand, it may be due to the complexity of the dopaminergic system, including the diversity of dopamine receptors and the differences in the expression of dopamine receptors in different tissues. Nevertheless, this study indicates that DA has a role in ameliorating NASH symptoms and provides a direction for future research on the application of dopaminergic system in liver diseases.

Conclusion

This study indicates that DA has a role in ameliorating NASH symptoms and provides a direction for future research on the application of dopaminergic system in liver diseases.

Author contributions

Peng Ma and Xu Wang participated in conceptualization, methodology, investigation, formal analysis, and original draft preparation. Xiuqi Wen and Lingyun Pu participated in conceptualization, methodology, validation, supervision. Yu Ou participated in conceptualization, methodology, validation, resources mobilization, supervision, manuscript review, and editing. All authors read and approved the final manuscript.

Funding statement

This work was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.