Normal liver organoids (CLO) and NASH liver organoids (NLO) established in our previous study (Elbadawy et al., 2020a), were grown up from frozen stocked cryovials and reused for in vitro experiments. The culture medium and conditions were the same as previously described (Broutier et al., 2016; Elbadawy et al., 2020a). DRP1 inhibitor (Mdivi-1, M3108; Tokyo Kasei Kogyo Co., Ltd., Tokyo) and OPA1 inhibitor (MYLS22, S9885; Selleck chem.com, USA) were obtained commercially. To check ROS production by organoids, a solution of mitochondrial superoxide (mtSOX) Deep Red (Dojindo Laboratories, Kumamoto, Japan) was used. Primary antibodies were as follows: DRP1, MFF, and OPA1 were from Cell Signaling Technology, Inc., (Danvers, Massachusetts, United states), α-SMA was from DAKO (Glostrup, Denmark), β-actin was from MilliporeSigma (Burlington, Massachusetts, United states). Secondary antibodies were HRP-conjugated anti-rabbit IgG (Cayman, Ann Arbor, Michigan, United states) and HRP-conjugated anti-mouse IgG (MilliporeSigma). MCD diet and standard pelleted control diet were purchased from Oriental East Co., Ltd. (Tokyo, Japan).

Twenty-four male C57BL/6 mice of 7-weeks-old were purchased (Oriental East Co., Ltd.) and used for in vivo experiments. During acclimatization, the mice were housed in three mice per cage with a 12-h light-dark cycle in a temperature and ventilation-controlled room with ad libitum access to food and water until the experiment begins. NASH mice were fed an MCD diet to induce the NASH state, while the control mice were fed a standard pelleted mice diet. MCD was administered to mice for 12 weeks to induce a severe NASH state model (Itagaki et al., 2013). This experiment was conducted with the approval of the Tokyo University of Agriculture and Technology (TUAT) Animal Care and Use Committee and the Ethics Committee (R04-121).

The study was carried out in vitro and in vivo. The in vitro experiments were carried out on CLO and NLO generated in previous studies (Broutier et al., 2016; Elbadawy et al., 2020a) to compare morphology, microstructure, ROS production level, and protein expression level of mitochondria-related fission and fusion markers (DRP1, MFF, and OPA1). Further, the effects of Mdivi-1 on NLO size, morphology (dendritic shape formations), and expression level of NASH markers including Col1a1, Acta2, and DRP1 were investigated. The in vivo experiments were conducted on mice to elucidate the ameliorative effects of Mdivi-1 on NASH state using different analyses.

This experiment was conducted with the approval of the TUAT Animal Care and Use Committee and the Ethics Committee (R04-121). Twenty-four 7-weeks-old male C57BL/6 mice were divided into four groups (Figure 3A). The first group was assigned as a control group and fed a normal diet and administered vehicle (Mdivi-1 solvent). The second one was assigned as control-treated and fed a normal diet and administered Mdivi-1. The third group was assigned as NASH-non-treated and fed an MCD diet and administered vehicle. The fourth group was assigned as NASH-Mdivi-1-treated and fed an MCD and administered Mdivi-1. Mdivi-1 injection solution was prepared by dissolving the powder in DMSO and diluting it in physiological saline (Otsuka Pharmaceutical Factory, Inc.). Mdivi-1 was administered intraperitoneally to the assigned groups (second and fourth ones) at a dose of 1 mg/kg of body weight every other day for 8 weeks (Ding et al., 2022). MCD diet was administered to the NASH-assigned groups (third and fourth ones) for 8 weeks as indicated by the manufacturer. At the end of the experiments, mice were euthanized under isoflurane anesthesia, weighed, and livers were dissected and washed with PBS and weighed. Blood samples were collected and centrifuged at 2000 g for 15 min at 4°C and the serum was collected for liver functions-related biochemical analyses. Samples from liver tissues were picked up to generate organoids and check their sizes. Other samples were used for the analyses of histopathology, microstructure by Transmission Electron Microscopy (TEM), oil red o staining, Masson trichrome staining, the extractions of RNA and protein.

For analysis of the microstructure of CLO and NLO, and liver tissues from differentially-treated mice, TEM was used as described previously (Elbadawy et al., 2021c). Briefly, at an appropriate growth level, the CLO and NLO pellets or liver sections were fixed with 2.5% glutaraldehyde for 3 h at room temperature (RT) in 0.1% cacodylate (pH 7.4). Thereafter, organoids and liver tissues were washed with 0.1 M cacodylate (pH 7.4), incubated in 2% osmium tetroxide and 1.5% K₄Fe(CN)₆ in 0.1 M sodium cacodylate (pH 7.4) for 2 h at 4°C, and washed with distilled water. The organoids and tissues were then dehydrated with graded ethanol solutions (50%, 70%, 80%, 90%, 95%, and 99.5 up to 100%) and embedded in Epon. Ultrathin sections of 70–110 nm size were prepared with a diamond knife on a Leica UC7 ultramicrotome and transferred onto 50-mesh copper grids covered with a form bar and carbon film. The sections were post-stained with uranyl acetate for 15 min at RT and lead citrate. Sections were imaged using a transmission electron microscope (H-7500, Hitachi, Tokyo, Japan) using a TEM digital camera (NanoSprint500, Hitachi).

To measure ROS production in CLO and NLO, the Matrigel domes containing organoids were dissolved on ice using 5 mM EDTA/PBS solution for 90 min. The organoids solution was collected in a 15 mL tube and centrifuged (600 g/3 min/4°C), and the supernatant was aspirated. The organoids were washed once with PBS, trypsinized for 5 min in a water bath (37°C), filtered using a 70-μm cell strainer (Falcon, Cary, NC, United States), and seeded at 5 × 10⁴ cells/well. A special 2.5D culture medium (Elbadawy et al., 2020a) was added and the plates were incubated overnight in a 5% CO₂ incubator at 37°C. In next day, the medium was aspirated, a solution of mtSOX Deep Red was mixed with the medium to 10 μmol/L, and 1 μg/mL Hoechst (Fujifilm Wako Pure Chemicals Corporation) was added. The plates were then placed in a 5% CO₂ incubator at 37°C for 30 min. Thereafter, the solution was aspirated, PBS was added, and images were captured. To assess ROS production level in the liver sections of differentially treated mice, sections were washed with PBS, placed in a 10 μmol/L solution of mtSOX Deep Red in PBS with 1 μg/mL Hoechst, and placed in a 37°C, 5% CO₂ incubator for 30 min. Images of organoids and liver sections were then captured using a fluorescence microscope (BX52; Olympus Corporation) and the DP2-BSW program (Olympus Corporation). The fluorescence intensity of three different fields was quantified using ImageJ software (National Institutes of Health).

The protein expression was examined using Western blotting as described previously (Usui et al., 2018; Elbadawy et al., 2021b). Briefly, the Matrigel dome-containing organoids were melted on ice for 90 min using 5 mM EDTA/PBS. The organoid solutions were collected and centrifuged, and the supernatants were aspirated. Cell lysis buffer (MilliporeSigma) with 1% protease inhibitor (Sigma-Aldrich) was added to the organoids pellets and the minced fragments from precooled liver tissues in liquid nitrogen, which were pipetted and set on ice for 15 min. The protein lysates were then centrifuged at 12,000 g for 10 min, and the supernatant was collected, labeled, and kept at −80°C until analysis. Protein concentrations in lysates were measured using the DC protein assay kit (Bio-Rad Laboratories, Hercules, California, United States) at a wavelength of 650 nm and quantified.

Electrophoresis was then performed on a 10% polyacrylamide gel (Fujifilm Wako Pure Chemicals Co., Ltd.). The protein solution was mixed with 4X SDS sample buffer (Bio-Rad Laboratories) at 3:1 (v/v) and heated at 95°C for 5 min. Equal amounts of protein (10 µg) were loaded into the gel, electrophoresed at 150 V, 400 mA for 60 min, and transferred to a PVDF membrane (WSE-4051; ATTO Corporation, Tokyo, Japan) for blotting. After blocking, the membranes loaded by protein lysates from non-treated CLO and NLO were incubated with primary antibodies (DRP1; 1:500, MFF; 1:500, and OPA1; 1:500). The membranes loaded by protein lysates from Mdivi-1-treated NLO were incubated with antibody to α-SMA (1:500). The membranes loaded protein lysates from liver tissues were incubated with antibody to α-SMA (1:500). Incubation was performed overnight at 4°C. The membranes were then washed three times with 0.1% TBS-T for 5 min and treated with HRP-conjugated anti-rabbit IgG (1:5000) or HRP-conjugated anti-mouse IgG (1:5000) for 1 hour at RT, followed by washing three times with 0.1% TBS-T again for 5 min. After treatment with Immobilon Forte Western HRP Substrate (MilliporeSigma), chemiluminescence of bands was observed using LAS3000 (Fujifilm Corporation, Tokyo, Japan) and photographed. The images were quantified using ImageJ software (National Institutes of Health).

Quantitative real-time PCR was performed as described previously (Elbadawy et al., 2020a). Briefly, RNA samples were prepared from Mdivi-1- or DMSO-treated NLO and minced fragments from precooled liver tissue in liquid nitrogen using the NucleoSpin RNA kit (MACHEREY-NAGEL, Düren, Germany). The RNA was then converted to cDNA using the ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). The PCR was then performed on cDNA using the QuantiTect SYBR I kit (Qiagen, Hilden, Netherlands) and the StepOnePlus Real-Time PCR system (Applied Biosystems, Waltham, MA, United States). Using the 2^−ΔΔCT method, values of cycle threshold (Ct) obtained in quantification were used for calculations of fold changes in mRNA abundance. The specific primers (Fasmac Corporation, Kanagawa, Japan) used for experiments were shown in Table 1.

Data are presented as mean ± SEM. Statistical evaluation was performed by one-way analysis of variance (ANOVA) and followed by the Bonferroni’s test. p values were considered significantly different at p < 0.05.

In the previous study, we generated CLO and NLO from C57BL/6 mice, which were fed a normal or MCD diet for 12 weeks (Elbadawy et al., 2020a). In the present study, we first compared the structures of the intracellular organelle between CLO and NLO using TEM (Figure 1A). As reported previously, CLO showed large spherical structures, while NLO showed small spherical structures with epithelial-mesenchymal transition and elongated dendritic- or stellate-like cell morphology (Figure 1B). The microstructures of NLO showed an increase in the rough endoplasmic reticulum (ER), fat droplets, and abnormal mitochondrial morphology (become swollen and spherical) compared with CLO (Figure 1B, Supplementary Figure S1).

To investigate the detailed relationship between the disease progression of NASH and mitochondria-related signals, we next compared the mitochondria-derived ROS production and expression level of mitochondria fission/fusion proteins (DRP1, MFF, and OPA1) between CLO and NLO (Figures 1A, C). We observed that ROS production was significantly increased in NLO compared with CLO (Figure 1D). Protein expression levels of DRP1, MFF, and OPA1 were also significantly increased in NLO compared with CLO (Figure 1E). These results suggest that abnormal functions and structures of mitochondria might increase ROS production in NLO.

To investigate the relationship between mitochondria-related molecules and fibrosis-related organoid morphology, NLO were treated with a mitochondrial mitogen (DRP1) inhibitor, Mdivi-1, or a fusion factor (OPA1) inhibitor, MYLS22 for 6 days (Figure 2A). In NLO, dendric-like cells and each organoid gradually increased after seeding the organoid cells. Mdivi-1 treatment significantly decreased the number of dendritic-like cells and increased the size of spherical structures (Figure 2B). On the other hand, MYLS22 treatment significantly increased the number of dendritic-like cells and had no effects on the size of spherical structures (Figure 2C). These results imply that DRP1 but not OPA1 has an important role for morphological changes of NLO.

In the previous study (Elbadawy et al., 2020a), the protein expression analysis of fibrosis-related proteins, Col1a1 and Acta2, in NLO was confirmed. To confirm the effects of Mdivi-1 treatment on the expression of fibrosis-related genes in NLO, mRNA expression levels of fibrosis-related markers (Col1a1 and Acta2) were analyzed by real-time quantitative PCR. As expected, Mdivi-1 treatment significantly reduced expression levels of Col1a1 and Acta2 compared with vehicle treatment (Figure 2D). In NLO, the protein expression level of Acta2 protein (α-SMA) was also inhibited by Mdivi-1 treatment compared with vehicle treatment (Figure 2E).

High level of serum FFA is the main contributor to fatty liver disease and is elevated in NAFLD and NASH patients (Zhang et al., 2014). We thus checked the effect of Mdivi-1 on lipid accumulation in NLO after loading it with oleic acid. After treating NLO with 2 mM oleic acid, they showed a progressive lipid accumulation as visualized by LipidTOX staining compared with vehicle treatment (Supplementary Figures S2A, B). Treatment with Mdivi-1 significantly decreased the oleic acid-induced lipid accumulation in NLO (Supplementary Figures S2A, B).

To check the effects of Mdivi-1 in vivo, seven-weeks-old C57BL/6 mice were fed MCD diet for 8 weeks and Mdivi-1 was administered (Figure 3A). In MCD diet-fed mice, body and liver weight was significantly lower than control mice, which was not affected by Mdivi-1 administration (Supplementary Figure S3A). The serum concentration of ALT but not AST in NASH mice was inclined to be higher than control mice (Supplementary Figure S3B), while total cholesterol (T-CHO) and triglycerides (TG) were significantly lower (Supplementary Figure S3B). Mdivi-1 administration had no effects on these parameters. On the other hand, histological analysis showed that the accumulation of lipid droplets of the liver tissues from Mdivi-1-administered and MCD diet-fed mice was decreased compared with vehicle-administered and MCD diet-fed mice (Figure 3B). Observation by TEM also showed Mdivi-1 administration decreased accumulation of lipid droplets and abnormal mitochondrial morphology in the liver tissues from MCD diet-fed mice (Figure 3C). To investigate the inhibitory mechanisms of Mdivi-1, we next checked expression levels of lipid metabolism-related genes (Lpl, Ldlr, Pnla3, and Tm6sf2). Among these genes, Mdivi-1 significantly decreased Tm6sf2 expression, which was significantly upregulated in the liver tissues of MCD diet-fed mice (Figure 3D).

We next checked whether Mdivi-1 prevents fibrosis in NASH mice. Masson trichrome staining showed that collagen fibers were observed in the peri-central vein and sinusoids in the liver tissues from MCD diet-fed mice, which was significantly inhibited by Mdivi-1 administration (Figures 4A, B). To confirm the inhibitory mechanisms by Mdivi-1, we checked expression levels of α-SMA. Western blotting analysis also showed that the expression level of α-SMA was higher in the liver tissues from MCD diet-fed mice, which was slightly prevented by Mdivi-1 administration (Figure 4C). Mdivi-1 also decreased Acta2 but not Col1a1 expression, which was slightly upregulated in the liver tissues of MCD diet-fed mice (Supplementary Figure S4).

To analyze whether the long-term Mdivi-1 administration to mice affects mitochondria-derived ROS production in liver tissues from MCD diet-fed mice, the liver tissues from each group of mice were stained for mtSOX Deep Red. ROS production was significantly higher in the liver tissues from MCD diet-fed mice, which was significantly inhibited by Mdivi-1 administration (Figure 5A).

To finally check the effects of Mdivi-1 on the capacity of organoid formation, the efficacy of organoids was evaluated by organoid size at day 5 after seeding the same number of cells (Figure 5B). Organoid size was smaller in the liver tissues from MCD diet-fed mice compared with control mice, which was significantly improved by Mdivi-1 administration (Figure 5B). Considering these results, Mdivi-1 administration might prevent the development of NASH diseases through maintenance of functional hepatocytes in NASH mouse.