Male C57BL/6J mice, which were 6–8 week-old and 18–22 g, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were raised under controlled conditions with 21°C ± 2°C, 50% ± 10% humidity, and 12 h light–dark cycles. All experimental operating procedures followed the guidelines of the Animal Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine (Approval ID: SH9H-2019-A574-1). Then, 10 mL/kg 10% CCl₄ peanut oil was intraperitoneally injected into the mice 3 times/week for 4 weeks to induce liver fibrosis. At the same time, Schizandrin C (Chishao Biotech Co., Ltd., Shanghai, China) at 50 mg/kg was orally administered once a day to the liver fibrosis model mice. The control mice were administered equivalent volumes of peanut oil and 0.5% carboxymethylcellulose solution (CMC-Na). Then, 4 weeks later, the mice were euthanized to obtain blood samples and liver tissues. The structure of Schizandrin C (C₂₂H₂₄O₆, CAS No: 61301-33-5- purity ≥98%) is presented in Figure 1A.

Blood was centrifugated at 1,000 g for 15 min to obtain serum. The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were analyzed using an automatic biochemical analyzer (Chemray 240, Kayto, Shenzhen, China) as per the manufacturer’s instructions.

After acquiring the liver tissues, the liver’s left lobes were separated, fixed in formaldehyde, embedded with paraffin, and sectioned. Then paraffin sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome staining in accordance with the standard protocol. The staining results were taken using a fluorescence microscope (Nikon Eclipse Ni, Japan).

Liver samples were crushed using a TL2020 grinding instrument (DHS Life Science & Technology, Beijing, China). Total RNA was isolated by Trizol reagent (Invitrogen, United States) as per the standard protocol. Quantitative real-time PCR analysis was performed using One-Step TB GreenTM PrimeScript PLUS RT-PCR Kit (TaKaRa, Japan) in a LightCycler 480II instrument (Hoffman-La Roche Ltd., Basel, Switzerland). The values were analyzed by using the 2^−ΔΔCT method. The primer sequences are presented in Supplementary Table S1.

After liver tissue sections were deparaffinized, the paraffin sections were blocked with bovine serum albumin (BSA) and incubated with antibodies against alpha-smooth muscle actin (α-SMA) (1:500, Servicebio, Cat# GB111364), type Ι collagen (collagen I) (1:1,000, Servicebio, Cat# GB11022-3), phospho (p)-nuclear factor kappa-B (NF-κB) p65 (1:200, Servicebio, Cat# GB13142-1), p-p38 MAP kinase (p38 MAPK) (1:200, Servicebio, Cat# GB13006-1), and p-extracellular signal-regulated protein kinase (ERK) (1:300, Servicebio, Cat# GB13004-1) overnight at 4°C. Then, the paraffin sections were treated with fluorescence-conjugated secondary antibodies (1:400, Servicebio, Cat# GB25303) and counterstained with 40, 6-diamidino-2-phenylindole (DAPI) (Servicebio, Cat# GB1012). The staining results were acquired using a Pannoramic MIDI scanner (3DHISTECH, Hungary) and viewed by using CaseViewer 2.4 (3DHISTECH, Hungary).

Liver total protein was isolated by RIPA buffer and quantified by a BCA protein assay kit (Thermo Fisher Scientific, United States) according to the manufacturer’s instructions. Proteins were separated through an SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes and then blocked in 5% skimmed milk. Afterward, the membranes were incubated with the primary antibody against IκB Kinase-β (IKKβ) (Cell Signaling Technology, Cat# 8943), NF-κB p65 (Cell Signaling Technology, Cat# 8242), p-NF-κB p65 (Cell Signaling Technology, Cat# 3033), ERK (Cell Signaling Technology, Cat# 4695), p-ERK (Cell Signaling Technology, Cat# 4370), p38 (Cell Signaling Technology, Cat# 8690), p-p38 (Cell Signaling Technology, Cat# 4511), and β-tubulin (Servicebio, Cat# GB122667) at 4°C overnight and then with the corresponding anti-rabbit secondary antibody (Cell Signaling Technology, Cat# 7074) or anti-mouse secondary antibody (Cell Signaling Technology, Cat# 7076) at room temperature for 1 h. Finally, the bands were determined using a chemiluminescence apparatus (Tanon 5200, China) and quantified using the Image J software (National Institute of Mental Health, United States).

First, 100 mg of liver tissues were treated with 0.75 mL of methanol in a glass tube with a Teflon-lined cap. After vortexing, 2.5 mL of methyl tert-butyl ether (MTBE) was added and incubated in a shaker for 1 h at room temperature. Afterward, the samples were added to 0.625 mL of MS-grade water, incubated for 10 min at room temperature, and centrifuged at 1,000 g for 10 min. The upper phase was gathered, and the lower phase was re-extracted using 1 mL of MTBE/methanol/water (10:3:2.5, v/v/v) for the organic phase. Combined organic phases were dried under nitrogen and re-dissolved in 100 μL of isopropanol for ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis by using a Vanquish UHPLC system (Thermo Fisher, Germany) with an Orbitrap Q ExactiveTM HF mass spectrometer (Thermo Fisher, Germany) in Novogene Co., Ltd. (Beijing, China).

A Thermo Accucore C30 column (150 × 2.1 mm, 2.6 μm) was used, and the samples were injected with a linear gradient flow rate of 0.35 mL/min for 20 min. The temperature of the column was set at 40°C. Acetonitrile/water (6/4) coupled with 10 mM ammonium acetate and 0.1% formic acid was used as mobile phase buffer A, and acetonitrile/isopropanol (1/9) coupled with 10 mM ammonium acetate and 0.1% formic acid was used as buffer B. The solvent gradient was initially 30% B for 2 min and then 43% B for 5 min, 55% B for 5.1 min, 70% B for 11 min, 99% B for 16 min, and 30% B for 18.1 min.

Raw data were processed by using Compound Discoverer 3.01 (CD3.1, Thermo Fisher) for peak alignment, peak picking, and quantitation. Then, peak intensities were normalized to the total spectral intensity and used to calculate the molecular formula on account of additive ions, molecular ion peaks, and fragment ions. The data were matched for accurate qualitative and relative quantitative results using LIPID MAPS, LipidBlast, and HMDB databases. If data were not distributed normally, area normalization was attempted used for normal transformation. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were operated using MetaboAnalyst 5.0 (www.metaboanalyst.ca). Potential differential metabolites were picked with variable importance in projection (VIP) > 1 and p < 0.05, or fold change (FC) ≥ 2, or FC ≤ 0.5 (Supplementary Table S2).

The hepatic hydroxyproline (HYP) content was measured using a hydroxyproline content detection kit (Fushen Biotech, China).

Hepatic stellate cell lines LX-2 (CVCL_5792) and HSC-T6 (CVCL_0315) were acquired from FuHeng Cell Center (Shanghai, China). Both cell lines were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and maintained in an incubator with 5% CO₂ at 37°C. Cell Counting Kit-8 (CCK-8, Fushen Biotech, China) was used to screen suitable concentrations of Schizandrin C as per the manufacturer’s instructions, and TGF-β1 at 20 ng/mL was used to activate HSCs.

The values are shown as mean ± SD. Multiple comparisons were conducted with one-way ANOVA, followed by the Student–Newman–Keuls post hoc test. All data were analyzed by GraphPad Prism 8.0 (GraphPad Software Incorporated, United State).

To evaluate the effect of Schizandrin C on liver fibrosis, male C57BL/6J mice were raised, and CCl₄ was used to establish a liver fibrosis model (Figure 1B). As shown in Figure 1C, serum ALT, AST, and TBIL were significantly decreased upon treatment with Schizandrin C in CCl₄ mice. In addition, the hepatic hydroxyproline content was raised notably in model group and reduced by treatment with Schizandrin C (Figure 1D). H&E and Masson staining results displayed that Schizandrin C markedly attenuated the degree of liver necrosis and collagen deposition stimulated by CCl₄ (Figure 1E). Overall, Schizandrin C can attenuate liver fibrosis induced by CCl₄ in C57BL/6J mice.

As shown in Figure 2A, the mRNA levels of α-SMA and collagen Ⅰ activated by CCl₄ were significantly decreased upon treatment of Schizandrin C. Immunofluorescence staining results also showed that the high protein levels of α-SMA and collagen Ⅰ in CCl₄ mice liver were downregulated by treatment with Schizandrin C (Figure 2B). To further identify the effect of Schizandrin C on HSC activation, different concentrations of Schizandrin C were treated in LX-2 and HSC-T6 cells to assess the toxicity of Schizandrin C on cells (Supplementary Figures S1A, B). Then, LX-2 and HSC-T6 cells were activated by TGF-β1, and Schizandrin C at 20, 40, and 80 μM was used to treat the cells. As shown in Figures 2C, D, Schizandrin C at 20, 40, and 80 μM depressed the mRNA levels of α-SMA and collagen Ι raised by TGF-β1 both in LX-2 and HSC-T6 cells. Thus, Schizandrin C can attenuate HSC activation in vivo and in vitro.

To further explore the effect of Schizandrin C on lipid metabolism, lipidomics was performed, and PCA was used to display the clustering separation among the original data of the three groups. As shown in Figures 3A, B, the QC samples clustered closely with no drift. In addition, samples of the CCl₄ model group were markedly separated from those of the control and Schizandrin C groups, suggesting that CCl₄ stimulated the liver lipid profile change and Schizandrin C can affect lipid metabolism in CCl₄ mice. In addition, OPLS-DA was performed to determine the lipid variations between the model and control groups, and Schizandrin C and model groups in positive and negative ion modes. After 1,000 permutations, the intercept value of R2Y and Q2 was 0.999 and 0.958, 0.997 and 0.954, respectively, in positive and negative ion modes between the model and control groups (Supplementary Figures S2A, B). In addition, the intercept value of R2Y and Q2 was 0.994 and 0.821, 0.988 and 0.747, respectively, in positive and negative ion modes between the Schizandrin C and model groups (Supplementary Figures S2C, D). The values of R2Y and Q2 indicated that OPLS-DA models conformed to the true situation of sample data with a low risk of overfitting and reliability. As shown in Figures 3C–F, OPLS-DA score plots suggested that the model and control groups, and Schizandrin C and model groups separated notably in positive and negative ion modes. These results displayed that Schizandrin C can affect the liver lipid profile in CCl₄ mice.

Then, an S-plot was further employed to identify altered lipids that significantly contributed to the differences between the model and normal groups or Schizandrin C and model groups (Supplementary Figures S3A–D). Varied lipids with VIP>1, p < 0.05, FC ≥ 2, or FC ≤ 0.5 were further selected for analysis. Those lipid species which were differential simultaneously in model vs. control groups and Schizandrin C vs. model groups were considered pivotal potential biomarkers for the Schizandrin C treatment of liver fibrosis (Figure 4A). A total of 36 lipid species, including ten triacylglycerols (TAGs), eight phosphatidylethanolamines (PEs), six phosphatidylcholines (PCs), three sphingomyelins (SMs), two acylcarnitines (ACars), two ceramides (Cers), two glucosylceramides (GlcCers), two phosphatidylglycerols (PGs), and one phosphatidylinositol (PI) were differentially regulated in the liver. What is more, PE (20:0/20:1), PE (20:0/20:3) and PI (16:1/18:2) levels were markedly decreased in model group, but notably increased by Schizandrin C treatment (Figure 4B). In addition, OxPE (16:0–22:6+4O), SM (d14:0/29:0), TAG (16:0-18:0-22:6), TAG (16:0-18:2–22:5), TAG (18:1-18:2-22:5), and TAG (18:1-22:4-22:6) levels were significantly increased in the model group but reversed by Schizandrin C supplementation (Figures 4B, C). Finally, the effect of Schizandrin C on lipid metabolism-associated gene expressions was determined. It is obvious that the mRNA levels of CD36 molecule (Cd36), fatty acid synthase (Fasn), sterol regulatory element-binding transcription factor 1 (Srebf1), acetyl-coenzyme A carboxylase alpha (Acaca), carnitine palmitoyltransferase 1a (Cpt1a), long-chain acyl-CoA dehydrogenase (Lcad), lipoprotein lipase (Lpl), microsomal triglyceride transfer protein (Mttp), and apolipoprotein B (Apob) were markedly increased, and treatment with Schizandrin C notably decreased Cd36, Fasn, stearoyl-coenzyme A desaturase 1 (Scd1), Srebf1, Acaca, Cpt1a, acyl-coenzyme A oxidase 1 (Acox1), Lcad, monoglyceride lipase (Mgl), Mttp, and Apob (Figure 4D). Together, these results indicated that Schizandrin C can reverse lipid disorder induced by CCl₄.

As shown in Figure 5A, the mRNA level of interleukin 6 (IL-6) was stimulated by TGF-β1, and treatment with Schizandrin C concentration-dependently suppressed it in both LX-2 and HSC-T6 cells. Furthermore, Schizandrin C notably reduced the mRNA levels of inflammation factors induced by CCl₄ in the liver, including Il-6, transforming growth factor beta 1 (Tgfβ-1), tumor necrosis factor (Tnfα), and cyclooxygenase-2 (Cox-2) (Figure 5B). Then, inflammation-related NF-κB signaling pathway was further detected. It is notable that Schizandrin C decreased the protein levels of IKKβ, NF-κB p65, and p-NF-κB p65, which were activated by CCl₄ (Figures 5C, D). In addition, the phosphorylation level of NF-κB p65 was inhibited by treatment with Schizandrin C (Figure 5E). Immunofluorescence staining results also displayed that high p-NF-κB p65 expression in the nucleus was attenuated by treatment with Schizandrin C in CCl₄ liver (Figure 5F). These results suggested that Schizandrin C can improve liver inflammation and inhibit the NF-κB signaling pathway in CCl₄ mice.

As the mitogen-activated protein kinase (MAPK) signaling pathway is closely associated with lipid metabolism, inflammation, and liver fibrosis, relative protein levels were then detected. Western blot analysis showed that the total protein levels of p38 and ERK were basically unchanged, but phosphorylation levels of ERK and p38 were higher in CCl₄ liver, and treatment with Schizandrin C reduced the protein levels of p-ERK and p-p38 (Figures 6A,B). The same trends can be seen in immunofluorescence staining images (Figures 6C, D). Taken together, Schizandrin C can inhibit the p38/ERK MAPK signaling pathway in CCl₄ mice.