The samples of Allium victorialis L. (AVL) (Figure 1A) were wild and grown in Hunchun City of Jilin province and were authenticated by Professor Xian Zhang, Agricultural College, Yanbian University, China. A voucher specimen (YBUCP20171212) was deposited in the Herbarium of the Agricultural College, Yanbian University, China.

Fresh AVL (1000 g) was soaked in ethanol for 24 h, followed by reflux extraction for 2 h. After filtration, the filter residue was collected and reflux extraction carried out 3 times. The powder of AVL was obtained by vacuum distillation and drying at 80°C. AVL extracts were analyzed by HPLC in supplement data.

The reagent strips for ALT/AST and TG were purchased from Changchun Huili Biotech Co., Ltd. (Changchun, China). Primary antibodies against GAPDH (ab8245), MPO (ab45977), lipin 1 (ab70138), LXRα (ab41902), lipin 2 (ab176347), CYP2E1 (ab28146), NLRP3 (ab4207), Sirt1 (ab110304), PPARγ (ab19481), SREBP1 (ab3259), F4/80 (ab6640), and Opti-MEM® were obtained from Abcam (Cambridge, MA, United States). Primary antibodies against ASC (sc514414), PPARα (sc9000), LXRβ (sc34341), IL6 (sc28343), IL1R1 (sc393998), and caspase 1 (sc622) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States). Primary antibodies against FXR (cs4173), p-AMPKα (cs2531), AMPKα (cs2532), p-AMPKβ1/2 (cs4181), AMPKβ1/2 (cs4150), LKB1 (cs3047), p-LKB1 (cs3482), p-ACC (cs11818), and ACC (cs3676) were purchased from Cell Signaling Technology (Beverly, MA, United States). Lipofectamine® 2000 Transfection Reagent was purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). The BCA Protein Assay Kit was obtained from Beyotime (Jiangsu, China). All other chemicals and reagents were obtained from Sigma-Aldrich (Shanghai, China).

Male C57BL/6 mice (body weight, 22–24 g) (SPF, SCXK [J] 2016-0003) purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. (Jilin, China) were housed under conditions of constant temperature (22 ± 2°C), relative humidity (50–60%), and light (12-h light−dark cycles) for 1 week. The animal experiment was handled in accordance with the Guide for the Care and Use of Laboratory Animals of Yanbian University (Resolution number, 201801022). After a week of acclimatization, all mice were randomly divided into six groups (six mice per group): normal group, EtOH group, EtOH plus AVL (100-, 200-, and 300-mg/kg) groups, and EtOH plus silymarin (100-mg/kg) group. The mice in the normal group were fed control Lieber–DeCarli liquid diet (TP 4030C, Trophic Animal Feed High-Tech Co., Ltd., China), and the mice in the other groups were fed Lieber–DeCarli liquid diet containing ethanol (TP 4030D, Trophic Animal Feed High-Tech Co., Ltd., China). The concentrations of ethanol were from 1 to 4% (v/v) for first 5 days, and then followed by 5% ethanol for 4 weeks. All mice were allowed free access to distilled water during the experiment. EtOH plus AVL or silymarin groups were daily gavaged with AVL (100, 200, and 300 mg/kg) or silymarin (100 mg/kg), and the normal and EtOH groups were daily administrated equal volumes of saline. Except for the normal group, the mice in the other groups were single gavaged with ethanol (5 g/kg) at the end of the fourth week. After the final administration, the mice were sacrificed under anesthesia. Finally, the serum and livers were collected for subsequent experiments. The detailed modeling procedures are described in Figure 1B.

AML12 and HepG2 cells were generous gifts from Professor Dr. Jung Joon Lee of Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). The AML12 cells were incubated in DMEM/F-12 with a mixture of insulin–transferrin–selenium (1%), dexamethasone (40 ng/ml), GlutaMAX (1%), and nonessential amino acids, while HepG2 cells were incubated in DMEM. DMEM contains penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% fetal bovine serum (FBS) under the conditions of 5% CO₂ at 37°C. For MTT assay, AML12 cells were cultured in 96-well plates at a density of 1 × 10⁴ cells per well and treated with AVL (0–100 μg/ml) or EtOH (0–200 mM). The cells were cultured in 35-mm dishes at a density of 5 × 10⁶ cells per dish and were treated with EtOH (50 mM) with or without AVL.

The transfections of FXR and control plasmid were conducted using the Lipofectamine® 2000 Transfection Reagent according to the manufacturer’s protocols. The cells were cultured in a 24-well plate at a density of 6 × 10⁴ cells per dish, then transfected with Opti-MEM® containing 0.2 μg of FXR plasmid or a negative control and 1.5 μL Lipofectamine® 2000 Transfection Reagent at the confluence of 90–95%. The cells were incubated under the condition of 5% CO₂ at 37°C for 48 h and then Opti-MEM® substituted with DMEM containing 10% FBS and EtOH (50 mM) with or without AVL (12.5 μg/ml).

The serum samples were collected by centrifugation at 3000 rpm and 4°C for 30 min. The liver was homogenized with saline and the homogenate obtained. Serum AST/ALT and tissue TG were measured using Assay kit according to the manufacturer's instructions, which was provided by Changchun Meeting of Biological Co., Ltd. (Changchun, China).

Parts of the liver tissues were fixed with 10% formalin solution and embedded in paraffin. Frozen sections were prepared with Tissue-Tek® O.C.T. Compound. After dewaxing hydration, the sections were treated with hematoxylin and eosin (H&E) solution following the manufacturer's instructions. The frozen sections were fixed with the mixture of acetone and methanol or paraformaldehyde, and stained with Oil red O and Nile red stains, also following the manufacturer's instructions. For immunofluorescence (Han et al., 2019), the frozen sections were fixed with the mixture of methanol and acetone, dried at room temperature, washed with PBS, and then blocked with 5% goat serum. The slides were incubated with the primary antibody at 4°C overnight and incubated with Alexa Fluor Goat pAb with rat IgG at room temperature. The nuclei were stained with DAPI and observed under fluorescence microscope.

Protein was extracted from liver tissue or cells with RIPA lysis buffer. Equal amounts of protein were separated using 6–15% sodium dodecyl sulfate–polyacrylamide-gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (GE, Freiburg, Germany). The membranes were blocked with skim milk and then incubated with indicated primary antibodies overnight at 4°C, followed with a horseradish peroxidase–conjugated secondary antibody. Finally, the protein bands were visualized with BeyoECL Plus detection reagent (Beyotime, Nanjing, Jiangsu, China) and exposed to an X-ray film. Each band's densitometry was quantified using Bio-Rad Quantity One software.

Total RNA was isolated from liver tissue or cells using the Eastep Super total RNA Extraction Kit (Promega Biological Products Ltd., Shanghai, China) according to the manufacturer's instructions. Samples of RNA were reverse transcribed into complementary DNA (cDNA). Relative gene expression was assessed by real-time PCR, which was performed on an Agilent Mx3000P QPCR System in a mixture containing Power SYBR® Green PCR Master Mix (Life Technologies, Carlsbad, CA), and specific primers are listed in Table 1. GAPDH was used as a housekeeping gene to quantify relative fold difference, and the relative fold difference was quantified using the comparative threshold cycle (ΔΔCt) method.

All data in experiments were expressed as mean ± SD. The comparison between groups was evaluated by GraphPad Prism (GraphPad Software, San Diego, CA, United States). One-way analysis of variance and Tukey's multiple comparison tests were used to perform statistical analyses. Statistically significant differences between groups were defined as p-values no more than 0.05.

With the Lieber–DeCarli liquid diet containing 5% (vol/vol) ethanol and single ethanol gavage (5 g/kg), the liver index, serum ALT/AST levels, and hepatic TG levels were significantly increased compared to the normal group, while AVL treatments markedly decreased these alternations compared to the EtOH group (Figures 1C–E).

As shown in Figure 1F, the liver images of the normal mice show a smooth surface, soft texture, and sharp edges. In the EtOH group, the liver tissue of the mice appeared swollen, rough, and tarnished. With AVL or silymarin administration, the liver surface was smoothened and the swelling reduced; however, silymarin administration showed less change than AVL (300 mg/kg).

The H&E analysis showed that the liver in the EtOH group showed massive steatosis compared with that in the normal group, and more fibrous connective tissue in the central region of the hepatic lobules had caused irregular deformation and increased liver injury. All these obvious pathological changes were significantly improved with AVL treatment (Figure 1F). In addition, Oil red O and Nile red staining showed that EtOH could induce the formation of lipid droplets in the liver compared to the normal group; however, lipid droplets in the AVL or silymarin groups were less abundant and much smaller than those in the EtOH group (Figure 1F). These results indicate that AVL showed hepatoprotective effect against liver injury and steatosis induced by EtOH.

SREBP1 plays a critical role in alcoholic liver disease, and it could regulate the transcription of downstream signaling, such as FASN, SCD, and ACLY. CYP2E1 is a key pathway in the regulation of lipid peroxidation and oxidative stress induced by ethanol. EtOH elevated protein and mRNA levels of SREBP1, the protein level of CYP2E1, and the mRNA levels of FASN, SCD, and ACLY compared to the normal group. AVL treatment could significantly inhibit protein or mRNA expressions of SREBP1, CYP2E1, FASN, SCD, and ACLY compared to the EtOH group (Figures 2A,D), and silymarin showed no obvious regulating in CYP2E1 (Figure 2A). In immunohistochemical staining, AVL or silymarin obviously decreased the positive expressions of SREBP1 (in brown) compared to the ETOH group (Figure 2C).

Both lipin 1 and lipin 2 are the first central regulatory enzymes in the regulation of lipid metabolism in the liver (Song et al., 2018). As is shown in Figure 2B, alcohol intake increased the protein expression of lipin 1 and decreased the protein expression of lipin 2 compared to the normal group, whereas these changes were reversed by AVL treatment compared to the EtOH group (Figure 2B). In Figure 2E, EtOH stimulation obviously inhibited the immunofluorescence expression of lipin 2 (in green) compared to the normal group (Figure 2E), while AVL or silymarin treatment significantly increased the expressions of lipin 2 compared to the EtOH group (Figure 2E). These results suggest that AVL downregulates lipid synthesis and upregulates fatty acid oxidation, and eventually ameliorates hepatic steatosis.

FXR and liver X receptors (LXRs), which belong to the NRs supergene family, can regulate the lipid metabolism gene in metabolic diseases. As shown in Figure 3A, ethanol significantly decreased the protein expressions of FXR, LXRα, and LXRβ compared to the normal group, whereas AVL or silymarin treatments significantly increased the expressions of FXR, LXRα, and LXRβ compared to the EtOH group. Consistent with these changes, the results of the immunofluorescence staining showed that positive expressions of LXRα, LXRβ (in red), and FXR (in green) were significantly decreased in the EtOH group than in the normal group, AVL treatments significantly enhanced these expressions compared to the EtOH group, and silymarin had little effect on the expressions of these proteins (Figure 3B). These results indicate that AVL regulated lipid metabolism by FXR and LXRs activation.

PPARα and PPARγ, members of the ligand-activated NRs transcription factor superfamily, are involved in lipogenesis, and in energy and glucose homeostasis. In the EtOH group, the protein expression of PPARα was deceased and that of PPARγ increased compared to the normal group, whereas AVL treatments significantly increased the expression of PPARα and decreased the expression of PPARγ compared to the EtOH group (Figure 3A). The regulation of AVL on PPARα expression was also supported by immunohistochemistry staining, which showed positive expression (in brown) (Figure 3B). Moreover, alcohol intake decreased mRNA expressions of the PPARα-regulated genes—ACOX1, mabp, and CPT2, while AVL administrations obviously ameliorated these changes caused by EtOH. These results demonstrate that AVL regulated PPARα-mediated fatty acid oxidation (Figure 3C). In addition, AVL significantly regulated AMPKα/β, LKB1, and ACC phosphorylation and Sirt1 compared to the ETOH group (Figure 3D). The effect of AVL on Sirt1 was also verified by the immunohistochemistry staining (Figure 3B). Thus, AVL regulated AMPK/LKB1/ACC and Sirt1 signaling pathway against ALD.

Inflammation plays a key role in the development of hepatic fibrosis. Inflammasomes are large intracellular multiprotein complexes involved in the development of inflammatory disorders, and NLRP3 is a danger-signal sensor to a regulatory node of inflammatory diseases. The protein expression of NLRP3 significantly increased in the EtOH group than in the normal group (Figure 4A). EtOH resulted in a marked inflammatory response in the liver as evidenced by increased expression levels of NLRP3 and ASC, which promoted the inflammatory response with releases of inflammatory cytokines, including IL6, caspase1-p10, IL1R1, and IL1β. Caspase-1 is a protease associated with inflammatory reaction and produces mature IL1β and IL18. Thus, we found AVL and silymarin administrations significantly decreased the expressions of NLRP3, ASC, IL6, caspase1, IL1R1, and IL1β. However, silymarin showed no significant decrease of ASC compared to the EtOH group (Figure 4A). The mRNA expressions of NLRP3, IL1β, IL18, IL1α, and TNF-α were significantly decreased by AVL (Figure 4B). Immunofluorescence staining also indicated that AVL significantly decreased the expressions of MPO, NLRP3, and F4/80 compared to the EtOH group (Figure 4C). These results demonstrated that AVL inhibits the release of inflammatory factors and the related inflammatory response, and further ameliorated ethanol-induced hepatic inflammation.

EtOH (50, 100, and 200 mM) did not significantly reduce the cell viability of AML12 compared with the negative control (Figures 5A,B). AVL (6.25–100 µM) significantly reduced the cell viability of AML12 compared with the negative control without AVL (Figure 5B). Consequently, ETOH (50 mM) and AVL (3.125, 6.25, and 12.5 µM) were chosen in the subsequent experiments.

AVL could significantly regulate protein expressions of SREBP1 and lipin 1/2 compared to the EtOH group (Figure 5C), which was supported by the immunofluorescence staining of lipin 1/2 (Figures 5F,G). In Oil red O staining, AVL obviously decreased the lipid droplets induced by EtOH (Figure 5E). AVL treatments could inhibit NLRP3, IL1α, TNF-α, and IL18 at the gene level (Figure 5D). These results demonstrate that AVL could inhibit lipid droplets by regulating the expression of SREBP1, lipin 1/2, and inflammation in vitro.

The LXRs and FXR could regulate the lipid metabolism gene in metabolic and digestive diseases. As we expected, AVL treatments could reverse the reductions of FXR, LXRα, and LXRβ in AML12 cells stimulated with EtOH (Figure 6A), which were further verified by immunofluorescent staining of FXR and LXRα (Figures 6B,C).

To further confirm that AVL ameliorates alcoholic fatty liver by activating FXR mediation on lipid accumulation, HepG-2 cells were transfected with FXR (NR1H4) shRNA and then treated with EtOH, and with or without AVL. The FXR deficiency was appropriately achieved by FXR shRNA (Figure 6D,E). EtOH-mediated decrease of LXRα and LXRβ was strengthened by FXR shRNA, which was also attenuated by AVL interference (Figures 6D,E). FXR deficiency also resulted in the decreasing of PPARα (Figure 6D), and the increase of SREBP1 (Figure 6E). These results suggest that FXR is necessary for AVL to ameliorated EtOH-induced liver lipid deposition, and FXR activation might be the potential therapeutic target for AVL against ALD.