Multi-omics and experimental analysis unveil the key components in 
Hepatic fibrosis is characterized by the aberrant accumulation of extracellular matrix (ECM), resulting in impaired liver function . It constitutes a fundamental pathological basis for chronic liver diseases including non-alcoholic fatty liver disease, alcoholic liver disease, and viral hepatitis . Chronic liver diseases pose a significant global health concern , with China experiencing a significant rise in the prevalence of cirrhosis due to chronic liver disease, exceeding 7 million cases and continuing to increase . This highlights the urgent necessity for efficacious preventive strategies against hepatic fibrosis. Although notable progress has been made in the field, the availability of effective pharmacological agents capable of reversing hepatic fibrosis remains limited . Consequently, there is an imperative to conduct comprehensive research into the underlying mechanisms of fibrosis and to identify new therapeutic targets. The primary pathological process underlying hepatic fibrosis is the activation of hepatic stellate cells (HSCs) , which typically maintain in a quiescent state within the normal liver. Upon liver injury, cytokines released by neighboring cells stimulate the activation of HSC, prompting their differentiation into myofibroblasts that exhibit heightened collagen secretion and deposition . This cascade of events culminates in structural and functional impairment of the liver, with the potential to progress to cirrhosis and hepatocellular carcinoma . Therefore, the inhibition of HSC activation has emerged as a crucial therapeutic approach for mitigating hepatic fibrosis . Scutellaria baicalensis Georgi, a traditional Chinese medicine frequently employed in the treatment of chronic liver diseases, has been shown to inhibit hepatic fibrosis . Specifically, the ethanol extract of Scutellaria baicalensis Georgi has demonstrated the ability to inhibit the activation of rat HSCs . Moreover, flavonoids derived from Scutellaria baicalensis Georgi, such as baicalein and wogonin, have exhibited significant anti-hepatic fibrosis effects . These findings suggest that Scutellaria baicalensis Georgi and its bioactive constituents show potential as therapeutic agents against fibrosis. However, further investigation is necessary to elucidate their precise mechanisms of anti-fibrosis. The aim of this study is to perform a comprehensive evaluation of the anti-fibrotic properties of the methanol extract of Scutellaria baicalensis Georgi (MESB) by employing a combination of diverse methodologies and omics approaches. This research identifies novel intervention targets and establishes a solid foundation for the clinical application of Scutellaria baicalensis Georgi in hepatic fibrosis. 

Reagents and antibodies Carbon tetrachloride (CCl4) was obtained from Sinopharm Co., Ltd (Shanghai, China). MESB was purchased from Baoji Herbest Bio-Tech Co., Ltd (Baoji, Shanxi, China). The antibodies used as follows: COL1A1 (A24112, Abclonal, Wuhan, China; ET1609-68, HUABIO, Hangzhou, China), PAI-1 (AF7965, Beyotime, Shanghai, China; HA601152, HUABIO, Hangzhou, China), FN (A12932, Abclonal, Wuhan, China), cPLA2 (A0394, Abclonal, Wuhan, China), and GAPDH (A19056, Abclonal, Wuhan, China). Animal models and treatments Male wild-type C57BL/6, aged 6–8 weeks and weighing 15–20 g, were procured from the Experimental Animal Center of Chongqing Medical University. The mice were maintained under specific pathogen-free (SPF) conditions and provided with regular sterile food and water ad libitum. Except the control group, all mice were administered an intraperitoneal injection of a CCl4/corn oil solution (CCl4: corn oil = 1:4) at a dose of 1 ml/kg, twice per week for a duration of 8 weeks to induce hepatic fibrosis. At the fourth week of injections, the mice were randomly divided into four groups: the model group, the low-dose MESB group (10 mg/kg), the medium-dose MESB group (20 mg/kg), the high-dose MESB group (40 mg/kg). Upon conclusion of the experiment, the mice were anesthetized and sacrificed. The livers were subsequently excised, labeled, photographed, and sectioned into two portions. One portion was fixed in 4% paraformaldehyde and subsequently embedded in paraffin, while the other portion was immediately stored at -80 °C for subsequent analysis. All experimental protocols received approval from the Institutional Animal Care and Ethics Committee of Chongqing Medical University. Histologic analysis Liver tissue samples were fixed in 4% paraformaldehyde (PFA) for 48 h at 25 °C. Following fixation, the samples underwent dehydration in ethanol and subsequent embedding in paraffin for histopathological examination. Thin sections were stained with H&E and Masson’s trichrome for detailed analysis. The stained sections were then observed using light microscopy. Analysis of collagen area in the Masson’s trichrome-stained sections was performed utilizing ImageJ software. Serum biochemistry To assess the levels of alanine aminotransferase (ALT) and aspartate aminotransferase  enzymes, serum was isolated by centrifuging rat blood at 3000 rpm for 10 min following a standing for 2 h at room temperature. The quantification of ALT and AST serum levels was conducted using test kits procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The procedures outlined by the test kit manufacturer were meticulously followed during the measurement process. Spatial metabolomics Secure the liver tissue sample onto the positionable sample head using an embedding solution. Adjust the sample’s angle and orientation as necessary, and proceed with sectioning according to the microtome’s guidelines. The sections intended for mass spectrometry imaging should be 10 μm in thickness. Subsequently, label the sample information on indium tin oxide glass slides. Initiate the circulation spray matrix device (SunCollect, SunChrom) for the application of 1,5-naphthalenediamine. The nozzle was maintained at ambient temperature, and the spraying speed was configured to 1000 mm/min. The flow rate for each sprayed layer varied in a gradient fashion, ranging from 10 to 60 µl/min, with a total of 16 layers applied. Mass spectrometry imaging was conducted using the Thermo Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, USA) in conjunction with the AP/MALDI (ng) UHR ion source (MassTech, Columbia, MD). In the mass spectrometry imaging (MSI) experiment, the sample on a slide is scanned line by line using an AP-MALDI ion source instrument. The profile data acquired from each scan line are subsequently stored as RAW data files. High-resolution MSI image reconstruction and visualization are then performed using specialized MSI processing algorithms. Cell culture The LX-2 cell was purchased from Cellcook Biotech Co., Ltd. (Guangzhou, China). The cells were cultured at 37 °C and 5% CO2 with saturating humidity. The growth medium employed was high-glucose DMEM supplemented with 10% fetal bovine serum (VivaCell, Shanghai, China) and 1% penicillin/streptomycin solution. Cell viability assay The Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate cell viability. Initially, LX-2 cells were seeded at a density of 5 × 10 − 3 cells per well in a 96-well culture plate and allowed to adhere for 6 h. Subsequently, the culture medium was replaced with medium containing varying concentrations of MESB (0, 50, 100, 150, 200, 250, 300, 400 µg/ml). The cells were then incubated for 48 h. Following the incubation period, 10 µl of CCK-8 reagent was added to each well, and the absorbance was measured at a wavelength of 450 nm using a ThermoMax microplate reader (Biotek, EL, USA). EdU staining assay To achieve optimal cultivation of LX-2 cells in a 6-well plate, initially culture the cells overnight and restore them to their normal condition. Following a 48 h treatment with MESB, replace the cell culture medium and adjust the final concentration of EdU to 10 µM. Incubate the cells for an additional two hours to facilitate the EdU labeling. Subsequently, remove the culture medium and fix the cells using 4% paraformaldehyde. Permeabilize the cells by washing with 0.3% Triton X-100 in PBS. Proceed by adding 0.5 ml of freshly prepared Click reaction solution to each well, ensuring uniform distribution by gently shaking the culture plate. Incubate the plate at room temperature in the absence of light for 30 min. Finally, stain the nuclei with DAPI and observe them using a fluorescence microscope (Olympus IX73, Tokyo, Japan). Western blotting Combine the lysis buffer, which includes protease and phosphatase inhibitors, with the sample to ensure complete protein lysis. Following centrifugation, measure the protein concentration and incorporate the loading buffer. Denature the proteins by heating them in a 95 ℃ water bath. Prepare the electrophoresis buffer and load the samples into the gel wells to separate the proteins based on their molecular sizes via electrophoresis. Subsequently, prepare the membrane and transfer apparatus to facilitate the transfer of the separated proteins from the gel onto a polyvinylidene fluoride (PVDF) membrane. To prevent non-specific binding, block the membrane with non-fat milk. Incubate overnight with a diluted primary antibody. After washing, add HRP-conjugated secondary antibody and incubate for 1 h at room temperature. Wash again, add chromogenic substrate, and detect protein expression using an optical imaging system. qRT-PCR (qPCR) RNA was extracted with the Trizol method and reverse transcribed into cDNA. Gene amplification was done using specific primers and fluorescent dyes, and gene expression levels were calculated using the 2-ΔΔCt method. Data analysis was conducted with GraphPad Prism software. The primer sequences were as follows: COL1A1 (human) forward, 5’-GAGGGCCAAGACGAAGACATC-3’ and reverse, 5’-CAGATCACGTCATCGCACAAC-3’; and FN (human) forward, 5’-CGGTGGCTGTCAGTCAAAG-3’ and reverse, 5’-AAACCTCGGCTTCCTCCATAA-3’. Other primers used as previously reported . Metabolomics analysis The metabolomics analysis was conducted using a Thermo UHPLC-Q Exactive HF-X system. Samples were separated on an HSS T3 column (100 mm × 2.1 mm, 1.8 μm) before mass spectrometry detection. The mobile phases were 0.1% formic acid in water: acetonitrile (95:5, v/v) (solvent A) and 0.1% formic acid in acetonitrile: isopropanol: water (47.5:47.5:5, v/v) (solvent B). The sample injection volume was 2 µl with a flow rate of 0.4 mL/min. The mass spectrometer, equipped with an ESI source, operated in both positive and negative ion modes. Optimal conditions included an ISVF of -3500 V for negative mode and 3500 V for positive mode, with a full MS resolution of 60,000 and an MS/MS resolution of 7500. Detection was performed over a 70–1050 m/z range. Post-detection, LC/MS raw data was processed using Progenesis QI software (Waters Corporation, Milford, USA) and then uploaded to the Majorbio cloud platform ( https://cloud.majorbio.com ) for analysis. Component identification of MESB MESB components were identified using a UPLC-ESI-MS/MS system (UPLC: ExionLC™ AD; MS: Applied Biosystems 4500 Q TRAP). To prepare the sample, MESB powder was weighed and mixed with pre-cooled 70% methanol internal standard extract. After centrifuging at 12,000 rpm for 3 min, the supernatant was filtered through a 0.22 μm membrane for UPLC-MS/MS analysis. The UPLC analytical conditions were as follows: an Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used. The mobile phase consisted of solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The flow velocity was 0.35 mL/min. The effluent was connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. ESI source parameters were: source temperature 550 °C, ion spray voltage 5500 V (positive)/-4500 V (negative), gas I at 50 psi, gas II at 60 psi, curtain gas at 25 psi, and high collision-activated dissociation (CAD). DP and CE for each MRM transition were optimized. Specific MRM transitions were monitored based on the metabolites eluted during each period. 4D label free-based proteomics analysis Six dishes were seeded with equal cell amounts, split into two groups of three. One group was cultured with 0 µg/ml MESB and the other with 150 µg/ml MESB for 48 h. Subsequently proteins were extracted, quantified, and peptides dissolved in a mass spectrometry buffer for LC-MS/MS analysis using the EASY-nLC 1200 system and Q-Exactive HF-X (Thermo, USA). Raw mass spectrometry files were analyzed using Proteome Discoverer Software 2.2. Differential protein expression was determined from protein abundance data, with statistical significance assessed via Student’s t-test. Proteins with a p-value < 0.05 and a fold change > 1.2 were identified as differentially expressed. shRNA interference Inoculate 1.5 million cells into a 10 cm culture dish. Subsequently, combine 50 µg of shRNA with 50 µl of PEI transfection reagent (2 µg/µl), ensuring thorough mixing between the plasmid and the transfection reagent. Add the obtained mixture to the cells in the culture medium. Following transfection, incubate the cells for 6 hours and subsequently replace the medium. Continue culturing the cells for an additional 48 hours, after which they will be used for subsequent experiments, such as protein extraction. The target sequences of two shRNAs as follows: 5’-CCGACTTATTTGGAAGCAAAT-3’ (shcPLA2-1) and 5’-CCTTGTATTCTCACCCTGATT-3’ (shcPLA2-2). Molecular docking To perform molecular docking, first prepare the protein by retrieving the cPLA2 protein crystal structure from the RCSB PDB database. Use Schrödinger’s Protein Preparation Wizard to preprocess the protein, regenerate native ligand states, optimize H-bond assignments, minimize protein energy, and remove water molecules. Next, prepare the ligand baicalein and wogonin by converting their 2D structures into 3D chiral conformations using Schrödinger’s LigPrep module. Subsequently, the Schrödinger SiteMap module identified the optimal binding site, and the Receptor Grid Generation module defined the ideal enclosing box. Two ligands were then docked into the cPLA2 protein’s active sites using high-precision XP docking, followed by MM-GBSA analysis to estimate the binding free energy. Statistical analysis Statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were analyzed using Student’s t-test, while one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests was performed for multiple group comparisons. A p-value of less than 0.05 was considered to indicate statistical significance. 

Carbon tetrachloride (CCl4) was obtained from Sinopharm Co., Ltd (Shanghai, China). MESB was purchased from Baoji Herbest Bio-Tech Co., Ltd (Baoji, Shanxi, China). The antibodies used as follows: COL1A1 (A24112, Abclonal, Wuhan, China; ET1609-68, HUABIO, Hangzhou, China), PAI-1 (AF7965, Beyotime, Shanghai, China; HA601152, HUABIO, Hangzhou, China), FN (A12932, Abclonal, Wuhan, China), cPLA2 (A0394, Abclonal, Wuhan, China), and GAPDH (A19056, Abclonal, Wuhan, China). 

Male wild-type C57BL/6, aged 6–8 weeks and weighing 15–20 g, were procured from the Experimental Animal Center of Chongqing Medical University. The mice were maintained under specific pathogen-free (SPF) conditions and provided with regular sterile food and water ad libitum. Except the control group, all mice were administered an intraperitoneal injection of a CCl4/corn oil solution (CCl4: corn oil = 1:4) at a dose of 1 ml/kg, twice per week for a duration of 8 weeks to induce hepatic fibrosis. At the fourth week of injections, the mice were randomly divided into four groups: the model group, the low-dose MESB group (10 mg/kg), the medium-dose MESB group (20 mg/kg), the high-dose MESB group (40 mg/kg). Upon conclusion of the experiment, the mice were anesthetized and sacrificed. The livers were subsequently excised, labeled, photographed, and sectioned into two portions. One portion was fixed in 4% paraformaldehyde and subsequently embedded in paraffin, while the other portion was immediately stored at -80 °C for subsequent analysis. All experimental protocols received approval from the Institutional Animal Care and Ethics Committee of Chongqing Medical University. 

Liver tissue samples were fixed in 4% paraformaldehyde (PFA) for 48 h at 25 °C. Following fixation, the samples underwent dehydration in ethanol and subsequent embedding in paraffin for histopathological examination. Thin sections were stained with H&E and Masson’s trichrome for detailed analysis. The stained sections were then observed using light microscopy. Analysis of collagen area in the Masson’s trichrome-stained sections was performed utilizing ImageJ software. 

To assess the levels of alanine aminotransferase (ALT) and aspartate aminotransferase  enzymes, serum was isolated by centrifuging rat blood at 3000 rpm for 10 min following a standing for 2 h at room temperature. The quantification of ALT and AST serum levels was conducted using test kits procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The procedures outlined by the test kit manufacturer were meticulously followed during the measurement process. 

Secure the liver tissue sample onto the positionable sample head using an embedding solution. Adjust the sample’s angle and orientation as necessary, and proceed with sectioning according to the microtome’s guidelines. The sections intended for mass spectrometry imaging should be 10 μm in thickness. Subsequently, label the sample information on indium tin oxide glass slides. Initiate the circulation spray matrix device (SunCollect, SunChrom) for the application of 1,5-naphthalenediamine. The nozzle was maintained at ambient temperature, and the spraying speed was configured to 1000 mm/min. The flow rate for each sprayed layer varied in a gradient fashion, ranging from 10 to 60 µl/min, with a total of 16 layers applied. Mass spectrometry imaging was conducted using the Thermo Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, USA) in conjunction with the AP/MALDI (ng) UHR ion source (MassTech, Columbia, MD). In the mass spectrometry imaging (MSI) experiment, the sample on a slide is scanned line by line using an AP-MALDI ion source instrument. The profile data acquired from each scan line are subsequently stored as RAW data files. High-resolution MSI image reconstruction and visualization are then performed using specialized MSI processing algorithms. 

The LX-2 cell was purchased from Cellcook Biotech Co., Ltd. (Guangzhou, China). The cells were cultured at 37 °C and 5% CO2 with saturating humidity. The growth medium employed was high-glucose DMEM supplemented with 10% fetal bovine serum (VivaCell, Shanghai, China) and 1% penicillin/streptomycin solution. 

The Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate cell viability. Initially, LX-2 cells were seeded at a density of 5 × 10 − 3 cells per well in a 96-well culture plate and allowed to adhere for 6 h. Subsequently, the culture medium was replaced with medium containing varying concentrations of MESB (0, 50, 100, 150, 200, 250, 300, 400 µg/ml). The cells were then incubated for 48 h. Following the incubation period, 10 µl of CCK-8 reagent was added to each well, and the absorbance was measured at a wavelength of 450 nm using a ThermoMax microplate reader (Biotek, EL, USA). 

To achieve optimal cultivation of LX-2 cells in a 6-well plate, initially culture the cells overnight and restore them to their normal condition. Following a 48 h treatment with MESB, replace the cell culture medium and adjust the final concentration of EdU to 10 µM. Incubate the cells for an additional two hours to facilitate the EdU labeling. Subsequently, remove the culture medium and fix the cells using 4% paraformaldehyde. Permeabilize the cells by washing with 0.3% Triton X-100 in PBS. Proceed by adding 0.5 ml of freshly prepared Click reaction solution to each well, ensuring uniform distribution by gently shaking the culture plate. Incubate the plate at room temperature in the absence of light for 30 min. Finally, stain the nuclei with DAPI and observe them using a fluorescence microscope (Olympus IX73, Tokyo, Japan). 

Combine the lysis buffer, which includes protease and phosphatase inhibitors, with the sample to ensure complete protein lysis. Following centrifugation, measure the protein concentration and incorporate the loading buffer. Denature the proteins by heating them in a 95 ℃ water bath. Prepare the electrophoresis buffer and load the samples into the gel wells to separate the proteins based on their molecular sizes via electrophoresis. Subsequently, prepare the membrane and transfer apparatus to facilitate the transfer of the separated proteins from the gel onto a polyvinylidene fluoride (PVDF) membrane. To prevent non-specific binding, block the membrane with non-fat milk. Incubate overnight with a diluted primary antibody. After washing, add HRP-conjugated secondary antibody and incubate for 1 h at room temperature. Wash again, add chromogenic substrate, and detect protein expression using an optical imaging system. 

RNA was extracted with the Trizol method and reverse transcribed into cDNA. Gene amplification was done using specific primers and fluorescent dyes, and gene expression levels were calculated using the 2-ΔΔCt method. Data analysis was conducted with GraphPad Prism software. The primer sequences were as follows: COL1A1 (human) forward, 5’-GAGGGCCAAGACGAAGACATC-3’ and reverse, 5’-CAGATCACGTCATCGCACAAC-3’; and FN (human) forward, 5’-CGGTGGCTGTCAGTCAAAG-3’ and reverse, 5’-AAACCTCGGCTTCCTCCATAA-3’. Other primers used as previously reported . 

The metabolomics analysis was conducted using a Thermo UHPLC-Q Exactive HF-X system. Samples were separated on an HSS T3 column (100 mm × 2.1 mm, 1.8 μm) before mass spectrometry detection. The mobile phases were 0.1% formic acid in water: acetonitrile (95:5, v/v) (solvent A) and 0.1% formic acid in acetonitrile: isopropanol: water (47.5:47.5:5, v/v) (solvent B). The sample injection volume was 2 µl with a flow rate of 0.4 mL/min. The mass spectrometer, equipped with an ESI source, operated in both positive and negative ion modes. Optimal conditions included an ISVF of -3500 V for negative mode and 3500 V for positive mode, with a full MS resolution of 60,000 and an MS/MS resolution of 7500. Detection was performed over a 70–1050 m/z range. Post-detection, LC/MS raw data was processed using Progenesis QI software (Waters Corporation, Milford, USA) and then uploaded to the Majorbio cloud platform ( https://cloud.majorbio.com ) for analysis. 

MESB components were identified using a UPLC-ESI-MS/MS system (UPLC: ExionLC™ AD; MS: Applied Biosystems 4500 Q TRAP). To prepare the sample, MESB powder was weighed and mixed with pre-cooled 70% methanol internal standard extract. After centrifuging at 12,000 rpm for 3 min, the supernatant was filtered through a 0.22 μm membrane for UPLC-MS/MS analysis. The UPLC analytical conditions were as follows: an Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used. The mobile phase consisted of solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The flow velocity was 0.35 mL/min. The effluent was connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. ESI source parameters were: source temperature 550 °C, ion spray voltage 5500 V (positive)/-4500 V (negative), gas I at 50 psi, gas II at 60 psi, curtain gas at 25 psi, and high collision-activated dissociation (CAD). DP and CE for each MRM transition were optimized. Specific MRM transitions were monitored based on the metabolites eluted during each period. 

Six dishes were seeded with equal cell amounts, split into two groups of three. One group was cultured with 0 µg/ml MESB and the other with 150 µg/ml MESB for 48 h. Subsequently proteins were extracted, quantified, and peptides dissolved in a mass spectrometry buffer for LC-MS/MS analysis using the EASY-nLC 1200 system and Q-Exactive HF-X (Thermo, USA). Raw mass spectrometry files were analyzed using Proteome Discoverer Software 2.2. Differential protein expression was determined from protein abundance data, with statistical significance assessed via Student’s t-test. Proteins with a p-value < 0.05 and a fold change > 1.2 were identified as differentially expressed. 

Inoculate 1.5 million cells into a 10 cm culture dish. Subsequently, combine 50 µg of shRNA with 50 µl of PEI transfection reagent (2 µg/µl), ensuring thorough mixing between the plasmid and the transfection reagent. Add the obtained mixture to the cells in the culture medium. Following transfection, incubate the cells for 6 hours and subsequently replace the medium. Continue culturing the cells for an additional 48 hours, after which they will be used for subsequent experiments, such as protein extraction. The target sequences of two shRNAs as follows: 5’-CCGACTTATTTGGAAGCAAAT-3’ (shcPLA2-1) and 5’-CCTTGTATTCTCACCCTGATT-3’ (shcPLA2-2). 

To perform molecular docking, first prepare the protein by retrieving the cPLA2 protein crystal structure from the RCSB PDB database. Use Schrödinger’s Protein Preparation Wizard to preprocess the protein, regenerate native ligand states, optimize H-bond assignments, minimize protein energy, and remove water molecules. Next, prepare the ligand baicalein and wogonin by converting their 2D structures into 3D chiral conformations using Schrödinger’s LigPrep module. Subsequently, the Schrödinger SiteMap module identified the optimal binding site, and the Receptor Grid Generation module defined the ideal enclosing box. Two ligands were then docked into the cPLA2 protein’s active sites using high-precision XP docking, followed by MM-GBSA analysis to estimate the binding free energy. 

Statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). Comparisons between two groups were analyzed using Student’s t-test, while one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests was performed for multiple group comparisons. A p-value of less than 0.05 was considered to indicate statistical significance. 

MESB significantly alleviates CCl4-induced hepatic fibrosis in mice To evaluate MESB’s effect on hepatic fibrosis, a CCl4-induced mouse model was utilized with a four-week MESB treatment (Fig. A). Liver tissue changes, biochemical markers, and fibrosis-related gene expression was analyzed. H&E staining showed disrupted liver tissue architecture in the model group, including hepatocyte enlargement, necrosis, cytoplasmic vacuolation, pseudolobule formation, and significant fatty degeneration. Moreover, Masson staining showed significantly collagen fibres deposition in the model group. Administration of MESB markedly mitigated these histopathological alterations and attenuated deposition of collagen fibres (Fig. B, C). Throughout the experiment, there was a consistent trend in body weight across all treatment groups, with no statistically significant differences observed (Supplemental Fig. ). However, the liver-to-body weight ratio exhibited an increase in the model group, while the administration of MESB resulted in a significant reduction of this ratio in mice with hepatic fibrosis (Fig. D). Furthermore, MESB treatment led to a significant decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase  levels, with the highest improvement in liver function observed in the high-dose MESB group (H-MESB) (Fig. E, F). Since hydroxyproline constitutes 13.5% of collagen’s sequence and is seldom found in other proteins, it serves as an indicator of collagen . The administration of MESB significantly decreased serum hydroxyproline levels in fibrotic mice (Fig. A). Subsequent examinations demonstrated that MESB notably reduced the expression of pivotal fibrosis-related genes stimulated by CCl4, such as type I collagen (COL1A1), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) at both the transcriptional (Fig. B-D) and translational levels (Fig. E, F), confirming the anti-fibrotic properties of MESB. Additionally, qPCR analysis revealed a significant reduction in the expression of inflammatory mediators IL-6 and TNF-α in the liver tissues of fibrotic mice treated with MESB (Fig. G, H). MESB inhibits the proliferation and activation of hepatic stellate cells Hepatic stellate cells (HSCs) constitute a critical target for therapeutic strategies addressing hepatic fibrosis. In this study, we initially evaluated the impact of MESB on the proliferation of the human HSC line LX-2 using CCK-8 and EdU incorporation assays. The results of the CCK-8 assay revealed a significant, concentration-dependent reduction in the viability of LX-2 cells following MESB treatment (Fig. A). Complementary findings from the EdU incorporation assay corroborated that the proliferative activity of LX-2 cells significantly diminished with increasing concentrations of MESB (Fig. B, C), thereby indicating the inhibitory effect of MESB on LX-2 cell proliferation. Subsequently, the impact of MESB on the activation of LX-2 cells were investigated. qPCR results demonstrated a significant reduction in the mRNA levels of COL1A1 and FN following MESB treatment (Fig. D, E). Additionally, the mRNA expression of PAI-1, an essential regulator of the fibrosis process , exhibited a significant decrease with increasing concentrations of MESB (Fig. F), proving further evidence of MESB’s anti-fibrotic properties. Western blotting results confirmed these findings, showing a decrease in the protein levels of COL1A1, FN, and PAI-1 following MESB treatment (Fig. G, H). Proteomic analysis of hepatic stellate cells treated with MESB To elucidate the molecular mechanisms by which MESB inhibits hepatic fibrosis, a comprehensive analysis was performed using 4D label-free quantitative mass spectrometry-based proteomics (Fig. A). This investigation identified a total of 5,145 proteins, among which 349 exhibited statistically significant differential expression ( p < 0.05). Specifically, 183 proteins were upregulated, while 166 proteins were downregulated (Fig. B). Gene Set Enrichment Analysis (GSEA) revealed that treatment of LX-2 cells with MESB resulted in significant downregulation of critical gene sets implicated in the progression of hepatic fibrosis, including ECM, transforming growth factor-β (TGF-β) signaling, and focal adhesion (Fig. C-E). These findings align with the observed attenuation of the hepatic fibrosis, indicating that MESB exerts an overall molecular intervention. Subsequent analysis utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated a significant correlation between the anti-fibrotic properties of MESB and several biological pathways, including metabolic pathways, cell senescence, the p53 signaling pathway, the mTOR signaling pathway, and autophagy (Fig. F). Among these, the metabolic pathway demonstrated notably elevated enrichment, suggesting that MESB exerts its anti-fibrotic effects primarily through the modulation of cellular metabolic networks. MESB regulates arachidonic acid metabolism to alleviate hepatic fibrosis Using untargeted metabolomics and orthogonal partial least squares discriminant analysis (OPLS-DA), we compared the metabolic differences between the MESB treatment and control group. The analysis of scores plots showed distinct metabolic patterns between the groups and consistent response within each group (Supplementary Fig. A, B). Rigorous permutation tests confirmed the model’s validity, reducing the risk of false-positive results (Supplementary Fig. C, D). Subsequent analysis of metabolic pathway enrichment indicated that MESB is involved in the regulation of metabolic processes, particularly those related to lipid metabolism pathways that are crucial for the progression of hepatic fibrosis (Fig. A). Further analysis using KEGG topology analysis revealed notable alterations in glycerophospholipid metabolism and arachidonic acid metabolism ( p < 0.05) (Fig. B). Quantitative metabolomic analysis indicated an elevation in phosphatidylcholine (PCs) metabolites (Fig. C), concomitant with a reduction in lysophosphatidylcholine (LPCs) and arachidonic acid levels within the arachidonic acid metabolic pathway (Fig. D, E). The diminished arachidonic acid concentrations consequently led to a decreased biosynthesis of downstream inflammatory mediators, including 5-oxo-eicosatetraenoic acid (5-KETE) and leukotriene B4 (LTB4) (Fig. F, G). To investigate the role of arachidonic acid in HSC activation, varying concentrations of arachidonic acid were added to LX-2 cells. This treatment resulted in a concentration-dependent increase in the expression levels of FN, COL1A1, and PAI-1, demonstrating an exacerbation of HSC activation by arachidonic acid (Fig. H, I). Moreover, the treatment of MESB to LX-2 cells significantly decreased the expression of inflammatory cytokines IL-6 and TNF-α (Fig. J, K). These findings suggest that MESB modulates the arachidonic acid metabolic pathway and inflammatory responses, thereby inhibiting HSC activation and preventing excessive ECM deposition. MESB regulates arachidonic acid metabolism via inhibiting cPLA2 expression Previous studies have identified cytosolic phospholipase A2 (cPLA2) as the rate-limiting enzyme involved in the biosynthesis of endogenous arachidonic acid . In our study, we observed that treatment with MESB in LX-2 cells resulted in a significant reduction in cPLA2 protein expression (Fig. A, B), suggesting MESB modulates the arachidonic acid metabolic pathway by regulating cPLA2. To further investigate the role of cPLA2 in hepatic fibrosis, we designed shRNA interference vectors specifically targeting cPLA2 and verified their efficacy. Our experimental results showed that both shRNAs effectively inhibited cPLA2 protein levels (Fig. C, D). Subsequently, the downregulation of fibrosis markers COL1A1, FN, and PAI-1, which are closely linked to hepatic fibrosis, was observed to significantly decrease (Fig. C, D). These findings indicated that MESB may alleviate hepatic fibrosis by inhibiting cPLA2 expression and reducing arachidonic acid production. To further clarify the link between cPLA2 inhibition and alleviated fibrosis, we assessed cPLA2 levels in the liver tissues of various mouse groups. Compared to the control group, cPLA2 was significantly higher in the CCl4 model group (Fig. E, F). However, MESB treatment reduced cPLA2 expression. Results from both in vivo and in vitro studies collectively suggest that MESB mitigates hepatic fibrosis by downregulating cPLA2 expression. Arachidonic acid has been reported to activate the NF-κB pathway , which operates downstream of cPLA2 to modulate inflammatory responses. Consequently, we investigated the effect of MESB on the NF-κB pathway. Our results revealed a significant elevation in the phosphorylation levels of p65 in the CCl4 model group, which was subsequently reduced following MESB treatment (Fig. E, F). These findings further indicate that MESB suppresses ECM production and inflammatory responses by inhibiting cPLA2 expression, thereby alleviating hepatic fibrosis. Identification and spatial distribution of MESB’s key active components To determine the key active components of MESB, ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was utilized to identify 240 compounds by primary mass spectrometry, including four main flavonoid compounds: baicalein, baicalin, wogonin, and wogonoside (Fig. A, B). Due to the intricate nature of MESB, this study further integrated spatial metabolomics to investigate the core active components and their distribution patterns within fibrotic liver tissue. The section was initially stained with H&E to ascertain the morphology and orientation of fibrotic liver tissue (Fig. C). Subsequently, adjacent sections were coated with a 2,5-dihydroxybenzoic acid matrix, followed by scanning of each individual pixel using a mass spectrometer. The investigation identified the crucial constituents of MESB—baicalein, wogonin, and wogonoside—within the liver tissue (Fig. D, F, H). Moreover, spatial metabolomics imaging provided a detailed analysis of the distribution patterns and relative abundance of these three components within the liver (Fig. E, G, I), thereby confirming the targeted transport efficiency and therapeutic potential. Baicalin is absent in the spatial metabolomics analysis, potentially due to its metabolism into baicalein by intestinal microbiota prior to absorption . Baicalein and wogonin specifically inhibit cPLA2 expression To investigate the impact of baicalein, wogonin, baicalin, and wogonoside on cPLA2, Western blotting was employed. The results demonstrated a significant dose-dependent inhibition of cPLA2 protein expression by baicalein and wogonin. In contrast, baicalin and wogonoside did not exhibit a direct effect on cPLA2 expression (Fig. A, B). We hypothesized a potential direct interaction between baicalein or wogonin and cPLA2 protein. To verify this hypothesis, molecular docking analysis was performed. Initially, the crystal structure of cPLA2 was acquired from the RCSB Protein Data Bank (PDB) (PDB ID: 1CJY, Chain A) and subjected to structural refinements. Subsequently, ligand-receptor docking analyses was performed to determine and evaluate the most favorable binding conformations of each active compound. The results indicated that baicalein and wogonin exhibit strong interactions with the active site of cPLA2 (Fig. C, D). Specifically, baicalein forms a hydrogen bond with LYS281 and engages in hydrophobic interactions with MET308, while wogonin forms a hydrogen bond with LYS281 and establishes hydrophobic contacts with PHE388. The docking scores for baicalein and wogonin were − 5.669 and − 5.264, respectively, with calculated MM-GBSA binding free energies of -20.62 kcal/mol and − 28.57 kcal/mol. The results indicated that baicalein and wogonin exhibit inhibitory effects on cPLA2 may through direct binding interactions. 

To evaluate MESB’s effect on hepatic fibrosis, a CCl4-induced mouse model was utilized with a four-week MESB treatment (Fig. A). Liver tissue changes, biochemical markers, and fibrosis-related gene expression was analyzed. H&E staining showed disrupted liver tissue architecture in the model group, including hepatocyte enlargement, necrosis, cytoplasmic vacuolation, pseudolobule formation, and significant fatty degeneration. Moreover, Masson staining showed significantly collagen fibres deposition in the model group. Administration of MESB markedly mitigated these histopathological alterations and attenuated deposition of collagen fibres (Fig. B, C). Throughout the experiment, there was a consistent trend in body weight across all treatment groups, with no statistically significant differences observed (Supplemental Fig. ). However, the liver-to-body weight ratio exhibited an increase in the model group, while the administration of MESB resulted in a significant reduction of this ratio in mice with hepatic fibrosis (Fig. D). Furthermore, MESB treatment led to a significant decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase  levels, with the highest improvement in liver function observed in the high-dose MESB group (H-MESB) (Fig. E, F). Since hydroxyproline constitutes 13.5% of collagen’s sequence and is seldom found in other proteins, it serves as an indicator of collagen . The administration of MESB significantly decreased serum hydroxyproline levels in fibrotic mice (Fig. A). Subsequent examinations demonstrated that MESB notably reduced the expression of pivotal fibrosis-related genes stimulated by CCl4, such as type I collagen (COL1A1), fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) at both the transcriptional (Fig. B-D) and translational levels (Fig. E, F), confirming the anti-fibrotic properties of MESB. Additionally, qPCR analysis revealed a significant reduction in the expression of inflammatory mediators IL-6 and TNF-α in the liver tissues of fibrotic mice treated with MESB (Fig. G, H). 

Hepatic stellate cells (HSCs) constitute a critical target for therapeutic strategies addressing hepatic fibrosis. In this study, we initially evaluated the impact of MESB on the proliferation of the human HSC line LX-2 using CCK-8 and EdU incorporation assays. The results of the CCK-8 assay revealed a significant, concentration-dependent reduction in the viability of LX-2 cells following MESB treatment (Fig. A). Complementary findings from the EdU incorporation assay corroborated that the proliferative activity of LX-2 cells significantly diminished with increasing concentrations of MESB (Fig. B, C), thereby indicating the inhibitory effect of MESB on LX-2 cell proliferation. Subsequently, the impact of MESB on the activation of LX-2 cells were investigated. qPCR results demonstrated a significant reduction in the mRNA levels of COL1A1 and FN following MESB treatment (Fig. D, E). Additionally, the mRNA expression of PAI-1, an essential regulator of the fibrosis process , exhibited a significant decrease with increasing concentrations of MESB (Fig. F), proving further evidence of MESB’s anti-fibrotic properties. Western blotting results confirmed these findings, showing a decrease in the protein levels of COL1A1, FN, and PAI-1 following MESB treatment (Fig. G, H). 

To elucidate the molecular mechanisms by which MESB inhibits hepatic fibrosis, a comprehensive analysis was performed using 4D label-free quantitative mass spectrometry-based proteomics (Fig. A). This investigation identified a total of 5,145 proteins, among which 349 exhibited statistically significant differential expression ( p < 0.05). Specifically, 183 proteins were upregulated, while 166 proteins were downregulated (Fig. B). Gene Set Enrichment Analysis (GSEA) revealed that treatment of LX-2 cells with MESB resulted in significant downregulation of critical gene sets implicated in the progression of hepatic fibrosis, including ECM, transforming growth factor-β (TGF-β) signaling, and focal adhesion (Fig. C-E). These findings align with the observed attenuation of the hepatic fibrosis, indicating that MESB exerts an overall molecular intervention. Subsequent analysis utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated a significant correlation between the anti-fibrotic properties of MESB and several biological pathways, including metabolic pathways, cell senescence, the p53 signaling pathway, the mTOR signaling pathway, and autophagy (Fig. F). Among these, the metabolic pathway demonstrated notably elevated enrichment, suggesting that MESB exerts its anti-fibrotic effects primarily through the modulation of cellular metabolic networks. 

Using untargeted metabolomics and orthogonal partial least squares discriminant analysis (OPLS-DA), we compared the metabolic differences between the MESB treatment and control group. The analysis of scores plots showed distinct metabolic patterns between the groups and consistent response within each group (Supplementary Fig. A, B). Rigorous permutation tests confirmed the model’s validity, reducing the risk of false-positive results (Supplementary Fig. C, D). Subsequent analysis of metabolic pathway enrichment indicated that MESB is involved in the regulation of metabolic processes, particularly those related to lipid metabolism pathways that are crucial for the progression of hepatic fibrosis (Fig. A). Further analysis using KEGG topology analysis revealed notable alterations in glycerophospholipid metabolism and arachidonic acid metabolism ( p < 0.05) (Fig. B). Quantitative metabolomic analysis indicated an elevation in phosphatidylcholine (PCs) metabolites (Fig. C), concomitant with a reduction in lysophosphatidylcholine (LPCs) and arachidonic acid levels within the arachidonic acid metabolic pathway (Fig. D, E). The diminished arachidonic acid concentrations consequently led to a decreased biosynthesis of downstream inflammatory mediators, including 5-oxo-eicosatetraenoic acid (5-KETE) and leukotriene B4 (LTB4) (Fig. F, G). To investigate the role of arachidonic acid in HSC activation, varying concentrations of arachidonic acid were added to LX-2 cells. This treatment resulted in a concentration-dependent increase in the expression levels of FN, COL1A1, and PAI-1, demonstrating an exacerbation of HSC activation by arachidonic acid (Fig. H, I). Moreover, the treatment of MESB to LX-2 cells significantly decreased the expression of inflammatory cytokines IL-6 and TNF-α (Fig. J, K). These findings suggest that MESB modulates the arachidonic acid metabolic pathway and inflammatory responses, thereby inhibiting HSC activation and preventing excessive ECM deposition. 

Previous studies have identified cytosolic phospholipase A2 (cPLA2) as the rate-limiting enzyme involved in the biosynthesis of endogenous arachidonic acid . In our study, we observed that treatment with MESB in LX-2 cells resulted in a significant reduction in cPLA2 protein expression (Fig. A, B), suggesting MESB modulates the arachidonic acid metabolic pathway by regulating cPLA2. To further investigate the role of cPLA2 in hepatic fibrosis, we designed shRNA interference vectors specifically targeting cPLA2 and verified their efficacy. Our experimental results showed that both shRNAs effectively inhibited cPLA2 protein levels (Fig. C, D). Subsequently, the downregulation of fibrosis markers COL1A1, FN, and PAI-1, which are closely linked to hepatic fibrosis, was observed to significantly decrease (Fig. C, D). These findings indicated that MESB may alleviate hepatic fibrosis by inhibiting cPLA2 expression and reducing arachidonic acid production. To further clarify the link between cPLA2 inhibition and alleviated fibrosis, we assessed cPLA2 levels in the liver tissues of various mouse groups. Compared to the control group, cPLA2 was significantly higher in the CCl4 model group (Fig. E, F). However, MESB treatment reduced cPLA2 expression. Results from both in vivo and in vitro studies collectively suggest that MESB mitigates hepatic fibrosis by downregulating cPLA2 expression. Arachidonic acid has been reported to activate the NF-κB pathway , which operates downstream of cPLA2 to modulate inflammatory responses. Consequently, we investigated the effect of MESB on the NF-κB pathway. Our results revealed a significant elevation in the phosphorylation levels of p65 in the CCl4 model group, which was subsequently reduced following MESB treatment (Fig. E, F). These findings further indicate that MESB suppresses ECM production and inflammatory responses by inhibiting cPLA2 expression, thereby alleviating hepatic fibrosis. 

To determine the key active components of MESB, ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was utilized to identify 240 compounds by primary mass spectrometry, including four main flavonoid compounds: baicalein, baicalin, wogonin, and wogonoside (Fig. A, B). Due to the intricate nature of MESB, this study further integrated spatial metabolomics to investigate the core active components and their distribution patterns within fibrotic liver tissue. The section was initially stained with H&E to ascertain the morphology and orientation of fibrotic liver tissue (Fig. C). Subsequently, adjacent sections were coated with a 2,5-dihydroxybenzoic acid matrix, followed by scanning of each individual pixel using a mass spectrometer. The investigation identified the crucial constituents of MESB—baicalein, wogonin, and wogonoside—within the liver tissue (Fig. D, F, H). Moreover, spatial metabolomics imaging provided a detailed analysis of the distribution patterns and relative abundance of these three components within the liver (Fig. E, G, I), thereby confirming the targeted transport efficiency and therapeutic potential. Baicalin is absent in the spatial metabolomics analysis, potentially due to its metabolism into baicalein by intestinal microbiota prior to absorption . 

To investigate the impact of baicalein, wogonin, baicalin, and wogonoside on cPLA2, Western blotting was employed. The results demonstrated a significant dose-dependent inhibition of cPLA2 protein expression by baicalein and wogonin. In contrast, baicalin and wogonoside did not exhibit a direct effect on cPLA2 expression (Fig. A, B). We hypothesized a potential direct interaction between baicalein or wogonin and cPLA2 protein. To verify this hypothesis, molecular docking analysis was performed. Initially, the crystal structure of cPLA2 was acquired from the RCSB Protein Data Bank (PDB) (PDB ID: 1CJY, Chain A) and subjected to structural refinements. Subsequently, ligand-receptor docking analyses was performed to determine and evaluate the most favorable binding conformations of each active compound. The results indicated that baicalein and wogonin exhibit strong interactions with the active site of cPLA2 (Fig. C, D). Specifically, baicalein forms a hydrogen bond with LYS281 and engages in hydrophobic interactions with MET308, while wogonin forms a hydrogen bond with LYS281 and establishes hydrophobic contacts with PHE388. The docking scores for baicalein and wogonin were − 5.669 and − 5.264, respectively, with calculated MM-GBSA binding free energies of -20.62 kcal/mol and − 28.57 kcal/mol. The results indicated that baicalein and wogonin exhibit inhibitory effects on cPLA2 may through direct binding interactions. 

According to the principles of traditional Chinese medicine, the fundamental pathogenesis of hepatic fibrosis involves damp-heat, blood stasis and pathogenic toxins. The ancient herbal book, “Compendium of Materia Medica” (Bencao Gangmu; 1956), records that Scutellaria baicalensis Georgi possesses properties for clearing heat, drying dampness, purging fire and detoxification. In clinical practice, Scutellaria baicalensis Georgi is frequently utilized in the treatment of chronic liver diseases within the framework of traditional Chinese medicine. Despite experimental evidence supporting the anti-fibrotic effects of Scutellaria baicalensis Georgi , its interactions with metabolic pathways remain largely unexplored. In this study, we employed multi-omics analysis and experimental validation to investigate the relationship between MESB and cPLA2-mediated arachidonic acid metabolism. Previous research has demonstrated that CCl4-induced hepatic fibrosis is associated dysregulated arachidonic acid metabolism . In the present study, we observed a substantial increase in several phosphatidylcholine metabolites following MESB treatment, while levels of arachidonic acid and its downstream inflammatory mediators, including LTB4, and 5-KETE, were markedly reduced. Arachidonic acid functions as a crucial precursor in the biosynthesis of various inflammatory mediators , which play a pivotal role in the processes of inflammation and fibrosis development . LTB4 facilitates the differentiation of fibroblasts into myofibroblasts . Additionally, 5-KETE induces the migration of basophils, contributing to the inflammatory process . Accumulating evidence demonstrated inflammation and fibrosis frequently co-occur in numerous chronic liver diseases . In the context of sustained hepatic injury, inflammatory mediators released by Kupffer cells and other sources perpetually activate HSCs , resulting in progressive fibrosis. Activated HSCs not only secrete ECM components but also produce a variety of pro-inflammatory factors, thereby exacerbating liver dysfunction. Consequently, mitigating inflammation may serve as a therapeutic strategy to alleviate hepatic fibrosis . cPLA2, a member of the phospholipase A2 family, catalyzes the hydrolysis of membrane phospholipids, resulting in the release of arachidonic acid . Studies have demonstrated that cPLA2 is upregulated in conditions such as hepatic fibrosis, non-alcoholic fatty liver disease and cirrhosis , suggesting a significant association with the progression of fibrosis. In a bleomycin-induced model of pulmonary fibrosis, the knock down of cPLA2 has been shown to yield superior therapeutic effects compared to the inhibition of the TGF-β pathway . Nevertheless, there is a paucity of direct research investigating the role of cPLA2 in the progression of hepatic fibrosis. In this study, we observed that the knock down of cPLA2 significantly inhibits the expression of hepatic fibrosis-related markers, including COL1A1, FN, and PAI-1, suggesting the potential of cPLA2 as a target for anti-fibrotic therapy. Furthermore, our results indicated that MESB significantly inhibits cPLA2 protein expression. These findings lay the groundwork for development of anti-hepatic fibrosis drugs that target cPLA2-mediated arachidonic acid metabolic pathways. The primary active constituents of Scutellaria baicalensis Georgi are baicalein, wogonin, baicalin, and wogonoside . In the component analysis of MESB, these four flavonoids were identified. However, spatial metabolomics of liver tissue revealed the presence of only baicalein, wogonin, and wogonoside. It has been documented that baicalin, the glucoside form of baicalein, can be converted to baicalein via gut microbiota metabolism . This metabolic conversion likely accounts for the absence of baicalin in the liver tissue. Interestingly, our findings revealed that baicalein and wogonin significantly inhibit cPLA2 protein expression, whereas baicalin and wogonoside do not exhibit such effects. Subsequent analyses indicated that baicalein and wogonin can directly bind to the cPLA2 protein. This interaction may represent the molecular mechanism through which MESB inhibits cPLA2-mediated arachidonic acid metabolism, thereby suppressing hepatic fibrosis. Existing studies indicate that the oral bioavailability of baicalein and wogonin is relatively low , similar to most flavonoids, possibly due to their limited solubility . Nonetheless, spatial metabolomics analysis of liver tissue reveals the presence of baicalein and wogonin, with baicalein exhibiting higher concentrations. This suggests that these compounds can accumulate in liver tissue, thereby facilitating their hepatoprotective effects. Recent pharmacokinetic studies on baicalein and wogonin indicate their presence in the body for up to 24 h . This finding is consistent with the timing of MESB intervention, as our study employed gavage over a 24-hour period to maintain the sustained presence of the drug within the body. In comparison to baicalein, wogonin attains its maximum concentration in the body more rapidly; however, this peak concentration is lower than that observed for baicalein . This may contribute to the relatively lower ion signal of wogonin compared to baicalein in spatial metabolomics analyses of liver tissue. Tissue distribution studies have demonstrated that, 30 min after the oral administration of baicalein, the compound exhibits its highest concentration within liver tissue, surpassing levels observed in heart, spleen, lung, kidney, and brain tissues, with the exception of stomach and intestinal tissues . Furthermore, tissue distribution studies have indicated that, within 10 to 120 min post-oral administration, wogonin concentrations are higher in kidney and liver tissues than in other tissues . These findings further substantiate the capacity of baicalein and wogonin to accumulate at the site of hepatic lesions, thereby facilitating their hepatoprotective effects in instances of liver damage or fibrosis. 

In conclusion, our findings elucidated the anti-fibrotic effects of MESB within a CCl4-induced hepatic fibrosis. Specifically, in the HSC line LX-2, the study explored the mechanisms by which MESB modulates arachidonic acid metabolism and inhibits inflammation through the regulation of cPLA2 (Fig. ). These results provided novel insights into the clinical applications of Scutellaria baicalensis Georgi in the treatment of hepatic fibrosis. 

Below is the link to the electronic supplementary material. Supplementary Material 1: Body weight of mice Supplementary Material 2: Comparative analysis of two groups. (A-B) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots of negative (A) and positive (B) ion modes. (C-D) OPLS-DA permutation tests of negative (C) and positive (D) ion modes 

