4-week-old C57BL/6J male mice were obtained from Beijing Vital River Laboratory Animal Technology Corporation (Beijing, China) and were housed at 23°C, with 50% humidity and on a 12 h light−dark cycle. After more than a month of adaptive feeding, 8-week-old mice were randomly assigned to three groups and fed with chow diet (CD), methionine and choline sufficient (MCS) diet (MCS Ctrl), which is a MCD control diet, and MCD diet. MCS and MCD diet are products of Research Diets, Inc (New Brunswick, NJ). In this experiment, we fed mice with MCD diet for 5 weeks to induce NASH model. After 5 weeks of feeding, the MCD group mice were randomly divided into two groups and were gavaged with double distilled water (MCD Ctrl) or theabrownin (MCD TB) (Lander Biotech, Xi’an, China) (200mg/kg body weight, dissolved in double distilled water) every day for another 4 weeks respectively, while continuing MCD diet feeding. CD group contains 5 mice, MCS Ctrl group contains 11 mice, MCD Ctrl group and MCD TB group contain 7 mice each. Body weights were recorded once a week during the experimental period. At the end of experiments, mice were sacrificed under anesthesia and blood samples and the liver were collected. Bloods samples were centrifuged at 12000 rpm and 4°C for 5 min. One part of liver tissue was fixed in the 10% neutral formalin for pathological observation and the rest of liver tissue was frozen in the liquid nitrogen and reserved at -80℃ for further research. All procedures were approved by the Institutional Animal Care and Use Committee at Shandong University and performed in conformance with the guide.

The levels of Triglyceride (TG) in serum and liver tissues were determined by Liquid Sample Triglyceride (TG) Content Assay Kit (Applygen Technologies Inc, #E1003) and High Fatty Sample Triglyceride (TG) Content Assay Kit separately (Applygen Technologies Inc, #E1025). The TG level of serum was determined following the manufacturer's instructions. Liver tissues were homogenized in lysis solution, and then let the lysate stand for 10 minutes. One part of the lysate was heated at 70°C for 10 minutes and then centrifuged at 2000rpm at room temperature for 5 minutes. The supernatant was used for enzymatic determination following the manufacturer's instructions. The remaining part of the lysate can be quantified by the BCA protein quantitative kit (Beyotime Biotechnology, #P0010). The final levels of liver TG were normalized by protein concentration.

The serum levels of Alanine aminotransferase (ALT) and Alanine aminotransferase (AST) were determined by using ALT kit (Nanjing Jiancheng Biotechnology, #C009-2-1) and AST kit (Nanjing Jiancheng Biotechnology, #C010-2-1) respectively.

Liver tissues were homogenized in phosphate-buffered saline (PBS) then centrifuged at 12000 rcf and 4 °C for 15 minutes. The supernatant was used for determination of Malondialdehyde (MDA) following the manufacturer's instructions. The remaining part of the lysate can be quantified by BCA protein quantitative kit (Beyotime Bio-technology, #P0010). The final levels of liver MDA were normalized by protein concentration.

Liver tissues were fixed in 10% neutral formalin, dehydrated and paraffin-embedded and cut into 5 μm thick sections. The sections of liver tissue were incubated with dihydroethidium solution (DHE, 1 μmol/L, Beyotime Biotechnology, # S0063) at 37°C in the dark for 30 min following the manufacturer's instructions. The fluorescence was measured under the excitation of 580 nm using a fluorescence microscope (EVOS FL, Thermo Fisher Scientific) (24).

Liver tissues were fixed in 10% neutral formalin, dehydrated and paraffin-embedded and cut into 5 μm thick sections for hematoxylin and eosin (H&E), Immunohistochemistry (IHC), Masson’s Trichrome and Picrosirius Red staining. Hepatic fibrosis was analyzed by Masson’s Trichrome, Picrosirius Red staining, and IHC staining for smooth muscle actin (SMA). After that, staining was observed under microscope. For IHC, the antibodies contained F4/80 (1:400) (Cell Signaling Technology, #70076), Ly-6G (1:400) (Santa Cruz Biotechnology, #sc-53515), IL-1β (1:400) (Cell Signaling Technology, #12242), SMA (1:400) (Santa Cruz Biotechnology, #sc-53142), Fibroblast growth factor 21 (Affinity Biosciences, #DF8947), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, #4370), p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling Technology, #4695). Image J software (National Institutes of Health, Bethesda, Maryland) was used for quantification.

Lysates prepared from frozen liver samples were used for western blot. The level of phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, #3033), NFκB p65 (Cell Signaling Technology, #8214), the stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) (Cell Signaling Technology, #9252), phospho-SAPK/JNK(Thr183/Thr185) (Cell Signaling Technology, #9251), IL-1β (Cell Signaling Technology, #12242), Phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, #4511), p38 MAPK (Cell Signaling Technology, #8690), FGF21 (Affinity Biosciences, #DF8947), Collagen 1 a 1 (Santa Cruz Biotechnology, # sc-293182),Collagen 3 a 1 (Santa Cruz Biotechnology, #sc-271249), Smooth muscle ac-tin (Santa Cruz Biotechnology, #sc-53142) and Tubulin (Cell Signaling Technology, #2125) were analyzed as described (25, 26).

Total RNA was isolated from liver tissues by using a commercial kit (RNAeasy™ Animal RNA Isolation Kit with Spin Column, Beyotime Biotechnology, # R0027), and 1 μg total RNA was reversed transcribed to cDNA with First-Strand cDNA Synthesis Kit (Accurate Biology, #AG11728) according to instruction book suggested by the manufacturer. Real-time PCR was performed with LightCycler 480 Real-Time PCR System (Accurate Biology, #AG11701) following the manufacturer's instructions. The sequences of primers used in this study are listed in Table 1 .

All experimental numeric data are presented as mean ± standard error of the mean (SEM). One-way ANOVA and least significance difference (LSD) method as a suitable post-hoc test was used to determine the differences among groups by using SPSS 26 (IBM, SPSS, USA) and a p-value of < 0.05 was considered statistically significant.

Liver steatosis is the initial manifestation of NAFLD. To investigate the effect of theabrownin on fat deposition in NASH mice, 8 weeks old mice were fed with CD, MCS or MCD diet for 5 weeks. In MCD diet feeding, mice were randomly divided into two groups and were gavaged with water or theabrownin for another 4 weeks separately. Body weight in MCD diet-fed mice significantly decreased compared to mice fed with MCS diet and CD ( Figure 1A ). Theabrownin didn’t influence the body weight of MCD-fed mice compared to control group ( Figure 1A ). In addition, theabrownin could not change the liver size ( Figure 1B ), liver weight ( Figure 1C ) and liver coefficient ( Figure 1D ), compared to MCD diet-fed control mice. Mice fed with MCD diet develop severe hepatic steatosis, which is manifested by increased hepatic triglyceride levels (27, 28). Similarly, in the current study, MCD diet can significantly increase the content of triglyceride (TG) in liver of mice ( Figure 1E ). However, theabrownin cannot reduce the level of TG in liver ( Figure 1E ), nor can it reduce the content of triglyceride in serum ( Figure 1F ). Moreover, the mRNA levels of lipogenic genes such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and sterol regulatory element-binding protein-1c (SREBP1c) in the liver of mice fed with MCD diet were lower than those fed with MCS diet ( Figure 1G ). It indicated that rather than the increase of de novo lipogenesis in liver, fats from adipose tissue lipolysis transported to liver may contribute to the high TG levels in MCD diet-fed mice (29). Consistently, in our study, almost no white adipose tissue was found in mice fed with MCD diet. It suggested that adipose tissue lipolysis may happen in MCD diet-fed mice. In addition, theabrownin did not change the mRNA levels of lipogenic genes in MCD diet-fed mice ( Figure 1G ). The mRNA level of carnitine palmitoyltransferase 1A (CPT1A), which was a key enzyme in for fatty acid oxidation, was not shown difference between MCS and MCD diet-fed mice and was not altered by administration of theabrownin ( Figure 1G ). This data indicated that theabrownin did not alleviate loss of liver mass and liver steatosis in NASH mice.

Although theabrownin has no effect on hepatic fat accumulation in MCD diet-fed mice, we wanted to figure out whether theabrownin influenced liver injury and inflammation in NASH. MCD diet can induce severe liver injury and inflammatory response (30). To determine whether theabrownin influenced liver function, we measured AST and ALT in serum. Consistent with previous studies, the ALT and AST levels in serum were significantly increased in MCD diet-induced NASH mice, while theabrownin reduced the high serum level of ALT ( Figure 2A ). Although theabrownin has no obvious effect on serum AST level, the decreasing trend of AST level was seen in MCD diet-fed mice treated with theabrownin ( Figure 2B ). The results indicated that theabrownin could alleviate liver injury. Except fat accumulation, NASH is also characterized by liver inflammation (6). Next, we investigated the effect of theabrownin on NASH related inflammation. Consistent with the above result, H&E staining of liver sections shows that theabrownin did not alleviate liver steatosis in MCD diet-fed mice. However, theabrownin significantly reduced the lobular inflammation in liver ( Figures 2C, D ). Moreover, IHC staining of the macrophage marker F4/80 and neutrophils marker Ly-6G showed that theabrownin significantly decreased macrophage and neutrophils infiltration ( Figures 2F–H ). Real-time Quantitative polymerase chain reaction assay showed that theabrownin significantly reduced the mRNA levels of pro-inflammatory genes monocyte-chemoattractant protein 1 (MCP1) and intercellular adhesion molecule 1 (ICAM-1) ( Figure 2E ). It further proved that theabrownin reversed the immune cells infiltration induced by MCD diet. We further confirmed the activation of NFκB and JNK by western blot analysis. Theabrownin reduced the increase of phosphorylation of NFκB p65 and JNK induced by MCD diet in liver ( Figures 3A–C ). Interleukin 1 beta (IL-1β), which is a key mediator of the inflammatory response, sends signals through IL-1 receptors widely expressed in different liver cell subsets to promote liver inflammation and fibrosis (6). In MCD diet-induced NASH mice, the level of IL-1β was remarkably enhanced, while theabrownin significantly reduced it ( Figures 2F , I ). It indicated that theabrownin decreased inflammatory response in NASH mice. These results suggest that theabrownin can improve liver injury and hepatic inflammation in MCD diet-fed mice.

Oxidative stress plays a crucial role in the pathogenesis and progression of NASH (31). Next, we investigated the effect of theabrownin on oxidative stress in the liver of NASH mice. Dihydroethidium (DHE) is a fluorescent probe for the detection of ROS generation. ROS production in the liver of MCD diet-fed mice much higher than that of MCS diet fed mice ( Figures 4A, B ). Theabrownin significantly reduced the ROS production in MCD diet-fed mice ( Figures 4A, B ). The levels of MDA in liver, which is considered a biomarker for oxidative damage of lipids, was dramatically increased in MCD diet fed mice, compared to MCS diet-fed mice, while theabrownin significantly decreased it in MCD diet-fed mice ( Figure 4C ). The results indicated that the theabrownin could downregulate oxidative stress in the NASH mice. Fibrosis is a hallmark of NASH (32). Next, we investigated the effect of theabrownin on NASH related fibrosis. Masson staining and Picrosirius Red staining in liver sections showed that MCD diet induced to develop liver fibrosis in mice, which can be alleviated by theabrownin ( Figures 5A–C ). The activation of extracellular signal-regulated kinase (ERK), which is a key component of MAPK signaling pathway, is associated with the development of liver fibrosis (33). MCD diet significantly increased the expression and the phosphorylation of ERK, while theabrownin significantly reduced the phosphorylation of ERK in liver, as shown from IHC staining ( Figures 5D–G ) and western blot ( Figures 5I, J ). Theabrownin also obviously reduced the high α-SMA level induced by MCD diet ( Figures 5D, H ). In addition, theabrownin significantly reduced the high mRNA levels of fibrogenesis genes collagen type I alpha 1 (Col1A1), fibronectin (Fn) and transforming growth factor-beta 1 (TGFβ1) in liver of MCD diet-fed mice ( Figure 6D ). Furthermore, the protein levels of Col1A1 and collagen type III alpha 1 (Col3A1) in the liver of MCD diet-fed mice were dramatically up-regulated, while theabrownin significantly decreased the levels of Col1A1 and Col3A1 ( Figures 6A–C ). Together, theabrownin reduced hepatic oxidative stress and fibrosis in MCD diet-fed mice.

p38, which is a mitogen-activated protein kinase (MAPK), is responsive to stress stimuli. p38 is a downstream molecule of FGF21 that can inhibit p38 phosphorylation (34). In order to uncover the therapeutic mechanism of theabrownin in treating NASH, we measured the levels of FGF21 mRNA and protein, as well as the phosphorylation of p38 in the liver. We found that the levels of FGF21 mRNA and protein in the liver of MCD diet-fed mice might be a little higher than that of MCS diet-fed mice ( Figures 7 A, F ), but there was no statistical difference. Theabrownin significantly increased the expression of FGF21 in the liver of MCD diet-fed mice, which can be seen by the results of FGF21 mRNA level, protein level, and immunohistochemical staining ( Figures 7A–F ). Meanwhile, theabrownin can reduce the high phosphorylation level of p38 induced by MCD diet in liver ( Figures 7E,G )