Matrine (≥98% purity), Sophocarpine (≥98%), Oxysophocarpine (≥98%), and Oxymatrine (≥98%) were obtained from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), while the remaining chemicals were sourced from Sigma Chemical Co. (St. Louis, United States). HepG2 cells were cultured in a specialized medium obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Primary antibodies, including mouse/rabbit anti-TXNIP (DF7506), anti-NLRP3 (BF8029), anti-Caspase 1 (AF5418), anti-cleaved Caspase-1 (AF4005), anti-GSDMD (AF4012), anti-GSDMD-N (DF13758), anti-IL1β (BF8021), and anti-β-actin (AF7018), were procured from Affinity Biopharmaceutical Co., Ltd. (Nanjing, China). Horseradish peroxidase-conjugated goat immunoglobulin G (IgG) (ab150113) and rabbit anti-mouse IgG (ab150077) secondary antibodies were purchased from Abcam Plc. (Cambridge, United Kingdom). Various kits used in the experiment, including triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP) was acquired from Jiancheng Biotechnology Science, Inc. (Nanjing, China). Radioimmunoprecipitation lysis buffer (RIPA), phenylmethanesulfonyl fluoride (PMSF), and Bradford protein assay kits were purchased from Solaibo Technology Co., Ltd. (Beijing, China). The TRIeasy Plus Total RNA Kit reagents were obtained from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China), while BeyoFast SYBR Green One-Step qRT-PCR Kits were sourced from Beyotime Biotechnology (Shanghai, China). The levels of IL-1β, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA) kits from Boster Biological Technology Co. Ltd. (Wuhan, China).

S. moorcroftiana seeds were purchased from Lhasa in March of 2022 and their authenticity was confirmed by Professor Zhuoma Dongzhi of the College of Medicine at Tibet University in Lhasa, Tibet, China. A sample of the seeds, for which the voucher specimen (No. utibet-1539) was stored in the Herbarium of Herbarium of Department of Medicament, College of Medicine, Tibet University, Lhasa, China. The seeds, which weighed 5,005.1 g, were pulverized and extracted with 70% ethanol by refluxing. After filtering and concentrating under reduced pressure, 1,023.1 g of extract was obtained (20.44% yield). The extract was preserved in the Component Bank of Tibetan Medicine in Lhasa (No. CBTM-E396), Tibet, China for potential future research purposes.

Quantitative analysis of the plant metabolites of SMS was carried out utilizing HPLC. In this process, methanol was used to dissolve standard substances containing Matrine, Sophocarpine, Oxysophocarpine, and Oxymatrine at concentrations of 511.0, 170.0, 365.0, and 1,250.5 μg/mL, respectively. SMS at a concentration of 3.76 mg/mL, was also dissolved in methanol. The separation was performed on an Agilent 5 HC-C18 column (250 mm × 4.6 mm, particle size 5 μm) using an LC-2010A HT HPLC system (Shimadzu Corporation, Kyoto, Japan) with HPLC and detection at a wavelength of 210 nm. The mobile phase consisted of acetonitrile (A) and a 0.1% aqueous solution of phosphoric acid (B) with a linear gradient elution: 0–20 min with 3% A, 20–45 min with 3%–15% A, and 45–60 min with 15% A. The flow rate was maintained at 1.0 mL/min, and a 15-min column wash was conducted between injections. After injection, the column was equilibrated with 3% A for 30 min. The column temperature was set at 20 °C, and the injection volume was 5 μL. The identification of plant metabolites in SMS was achieved by comparing the retention times of corresponding standard compounds.

Male C57BL/6J mice (weighing 20 ± 2 g) were purchased from the Qingdao Institute for Food and Drug Control (approval number: SYXK (Lu) 2022 0816). These mice were acclimatized to controlled environmental conditions (temperature: 25 °C ± 2 °C, relative humidity: 55% ± 5%, and a 12/12 h light/dark cycle) for a period of 7 days, during which they had unrestricted access to food and water. The experimental protocols were ethically approved by the Institutional Animal Care and Use Committee of Qingdao University of Science and Technology (approval number: ACQUST-2020-053).

The mice were divided into two groups: a control group (CON) with 10 mice and a model group with 50 mice. The CON group received a normal-fat diet comprising 30% wheat, 30% corn, 10% fish, 3% bone meal, and 7% soybean meal. In contrast, the model group was fed a high-fat diet composed of 80% NFD and 20% high-fat feed (containing 3% cholesterol, 7% lard, and egg yolk powder) for 8 weeks. Serum levels of TC, TG, ALT, and AST confirmed successful model group establishment using reagent kits. Subsequently, the model group was further divided into five subgroups (n = 10 each): the high-fat diet model group (HFD), a positive control group (administered Silymarin at 100 mg/kg), and low, medium, and high dosage groups (L: 52 mg/kg, M: 104 mg/kg, and H:208 mg/kg) of the SMS. While the CON and HFD groups received normal saline, the treatment group needed to be given SMS by intragastric administration at a fixed dose every day for 8 weeks. After a 12-h fasting period, blood samples were collected from the orbital vein and centrifuged to obtain serum samples. Liver samples were dissected, weighed, and half of them were stored at −80 °C, while the remaining half was preserved in a 4% polyformaldehyde solution for subsequent experiments.

The liver tissues were fixed in polyformaldehyde, dehydrated, paraffin-embedded, and sectioned to a thickness of 5 μm. Subsequently, the sections underwent H&E staining. Observations were made on these sections under a microscope (Nikon, Tokyo, Japan), and random areas were chosen for photography.

Immunohistochemical staining was conducted using 5 μm paraffin sections that were dewaxed in xylene. The sections were dehydrated and incubated with 3% H₂O₂ for 10 min, followed by washing with PBS. After blocking with PBS containing 5% fetal bovine serum (FBS), the sections were incubated overnight at 4 °C with primary antibodies against TXNIP, NLRP3, GSDMD, and IL-6 (1:200). This was followed by incubation with goat anti-rabbit IgG (H + L) HRP at 37 °C. After incubation with Streptavidin-HRP, 2,2′-Dimethylbenzidine (DAB) staining was performed, followed by hematoxylin counterstaining. Randomly selected fields were photographed at ×200 magnification using a light microscope (Nikon, Tokyo, Japan).

HepG2 cells, obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China), were cultured in minimum essential medium (MEM) with NEAA supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). The cells were maintained at 37 °C in a 5% CO₂ conditions, with sub-culturing performed every 2–3 days. Initially, the HepG2 cells were exposed to varying concentrations of SMS (12.5, 25, 50, and 100 μg/mL) for 24 h. Then, the cells received an additional treatment with free fatty acid (FFA, 1.0 mM) for an extra 24 h.

Cell viability was evaluated using the MTT assay. HepG2 cells were cultured in a 96-well plate at a concentration of 6 × 10^5 cells/mL (100 μL). The cells were exposed to SMS either alone or at varying concentrations (12.5, 25, 50, and 100 μg/mL), for 24 h. Additionally, the cells received an extra treatment with FFA (Sodium oleate (OA) and sodium palmitate (PA) were dissolved in a 20% bovine serum albumin (BSA) solution and mixed at a ratio of 2:1, 1.0 mM) for an additional 24 h. Subsequently, a solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 2.5 mg/mL, 50 μL) was added to each well. After a 4-h incubation, formazan crystals were dissolved in Dimethyl sulfoxide (DMSO), and the optical density (OD) was measured at a wavelength of 490 nm. The initial OD was recorded at the time of compound addition.

HepG2 cells were cultured in a 6-well plate at a concentration of 6 × 10^5 cells/mL (1 mL). Detection of ROS was conducted utilizing an ROS Assay Kit, following the manufacturer’s protocol. Diluted DCFH-DA (1:1,000) was added to each well, and the cells were incubated at 37 °C for 20 min in a cell culture incubator. After incubation, the cells were wished thrice with cell culture medium, and fluorescence was visualized using a laser confocal microscope (Olympus Corporation DP74, Tokyo, Japan).

To evaluate lipid accumulation in liver tissue and HepG2 cells, liver sections underwent fixation using 4% formaldehyde and subsequent staining with Oil Red O. After isopropanol washing, the sections were counterstained with hematoxylin and examined using a microscope (Olympus Corporation DP74, Tokyo, Japan). HepG2 cells were stained using the Oil Red O kit designed for cultured cells (Solarbio, Beijing, China). Random areas were selected for photographic documentation via light microscopy (Olympus BX53, Tokyo, Japan).

Protein specimens were initially gathered from HepG2 cells and the liver tissues of mice. The isolation procedure utilized RIPA buffer, supplemented with 1% PMSF. The Bradford protein assay kit was used to quantify the protein quantities. Following this, protein samples of equal amounts were differentiated using 10%–15% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These were then transferred onto NC membranes. The membranes were treated with 5% skim milk in TBST for a duration of 2 h at room temperature. Primary antibodies specific were applied to the membranes for 1.5 h at room temperature. After washing the membranes thrice with TBST, secondary antibodies were introduced for 1 h at room temperature. The protein bands were visualized using an Enhanced chemiluminescence kit (ECL). A ChemiDoc image analyzer (Tanon 4,600, Shanghai, China) was used to ascertain protein concentrations, and ImageJ software (NIH, Bethesda, MD, United States) was utilized for image analysis and manipulation.

The process of isolating RNA from HepG2 cells utilized the TRIeasy Plus Total RNA Kit. This was followed by reverse transcription and qRT-PCR, facilitated by the BeyoFast SYBR Green One-Step qRT-PCR Kit, to measure the mRNA levels of TNF-α, IL-6, IL-18, IL-1β, and TXNIP. β-actin mRNA served as a control in these experiments. The entire procedure was repeated three times, and the Livak method was used to assess mRNA expression levels. ΔCt represents the difference between the Ct value of the target gene and the Ct value of the reference gene. ΔΔCt​ is the difference between the ΔCt values of the experimental groups and those of the control group. The primer sequences are provided in Table 1. m R N A     e x p r e s s i o n   l e v e l s = 2 − Δ Δ C t L i v a k     m e t h o d

The lipid deposition was detected by measuring the levels of TG, TC, LDL-C, HDL-C in both serum and HepG2 cells using a reagent kit. Oxidative stress was detected by measuring the levels of SOD, CAT, GSH-PX and the activity of MDA in both serum and HepG2 cells using a reagent kit. Liver function was assessed by measuring the levels of AST, ALT, and AKP in serum using reagent kits. The levels of IL-1β and IL-18 in serum homogenates and HepG2 cells, as well as IL-6 and TNF-α in liver tissues were detected using ELISA kits. Additionally, the mRNA levels of TNF-α, IL-6, IL-18, and IL-1β in HepG2 cells were determined using qRT-PCR, as mentioned earlier.

The HepG2 cells, after appropriate preparation, were rinsed with PBS and then fixed with 4% paraformaldehyde for 30 min. Subsequently, the cells were treated with 0.1% Triton X-100 for 10 min and then blocked using a solution of PBS containing 5% FBS at ambient temperature for another 30 min. The primary antibody was applied and allowed to incubate overnight at 4 °C. This was followed by an incubation period with FITC-conjugated goat anti-mouse IgG or rabbit anti-rabbit IgG secondary antibody at room temperature for 1 h, in conjunction with a 5-min incubation with DAPI. The immunostaining was then observed using a fluorescence microscope (Olympus Corporation DP74, Tokyo, Japan).

The HepG2 cells, after appropriate preparation, were washed with PBS to remove the culture medium and then fixed with 5% glutaraldehyde for 1 h at room temperature. Post the glutaraldehyde exposure, gradient concentrations of ethanol were successively dehydrated for 15 min. The samples were then subjected to a 12-h freeze-drying period in a vacuum freeze-dryer. Pretreatment steps were performed on the cells using ion sputter coating technology to stabilize the cell surface and mitigate charge effects. The structure of the cell membrane was then examined by SEM.

In order to investigate the relationship between TXNIP and pyroptosis, HepG2 cells were transfected with TXNIP-siRNA, procured from GenePharma Co., Ltd. (Suzhou, China), in accordance with the instructions provided by the manufacturer. The transfection process was initiated when the cell density was within the range of 60%–80%. After transfection, the cells were incubated at 37 °C for a duration of 4–6 h, following which they were transferred to complete culture medium. The expression of mRNA was examined within a period of 24–72 h, while protein expression was analyzed within a timeframe of 48–96 h.

The experimental data underwent analysis using GraphPad Prism 7 software, developed by GraphPad Software in San Diego, CA, United States of America. The data were presented as mean ± Standard deviation (SD). For continuous data, a one-way ANOVA was applied, followed by Tukey’s post hoc test for inter-group comparisons. Statistical significance is indicated as ^* P < 0.05, ^** P < 0.01, ^# P < 0.05, ^## P < 0.01 and ^§ P < 0.05, ^§§ P < 0.01.

The HPLC spectra representative of SMS are shown in Figure 1B. The primary constituents of SMS are detailed in Table 2. The chemical formulas for Matrine, Sophocarpine, Oxysophocarpine, and Oxymatrine are illustrated in Figure 1. As per the regression equation of the curve, the concentrations of Matrine, Sophocarpine, Oxysophocarpine, and Oxymatrine in S. moorcroftiana seeds are 8.26, 1.41, 3.59, and 45.89 mg/g, respectively.

The effect of SMS on improving accumulation of lipids was confirmed by evaluating liver histopathology. Liver histopathology revealed significant hepatic injury in the HFD group compared to the CON group. However, H&E staining indicated orderly ordering of hepatocytes and reduced fat infiltration after SMS treatment (Figure 2B). After 8 weeks of treatment, a significant decrease in liver weight and index was observed in the SMS group (Figures 2C,D). Treatment with SMS also reduced the levels of ALT, AST, and AKP compared to the FFA group in a dose-dependent manner,The levels of ALT, AST and AKP in L, M and H groups were significantly affected (Figures 2E–G). Collectively, these findings suggest that SMS exhibits a protective effect against liver injury of NAFLD in vivo.

Lipid accumulation plays a significant role in the initiation and progression of liver injury in NAFLD. Oil Red O staining demonstrated a notable increase in orange lipid droplets in the group subjected to HFD. Subsequently, the abundance of these lipid droplets decreased upon treatment with varying concentrations of SMS. The quantitative analysis of Oil Red O staining results indicated a significant reduction in orange lipid droplets with increasing drug concentration (Figures 3A,B). In addition, SMS treatment effectively inhibited the levels of TC, TG, and LDL-C in H group compared to HFD (Figures 3C–E). Furthermore, SMS treatment led to an elevation in HDL-C levels (Figure 3F). In conclusion, SMS administration alleviated hepatic lipid deposition in vivo.

Oxidative stress and inflammation play a significant role in both the onset and progression of liver injury in NAFLD (Li J. et al., 2021). The MDA activity increased in the HFD group and diminished after SMS treatment. Furthermore, the levels of SOD and GSH-Px exhibited a notable rise compared to the HFD group (Figures 4A–C). Compared with HFD group, SMS treatment resulted in dose-dependent inhibition of expression levels of TNF-α, IL-6, IL-18, and IL-1β, with significant improvement in group H (Figures 4D–G). In conclusion, these findings indicate that SMS mitigates liver oxidative stress and inflammation in vivo.

The impact of SMS on pyroptosis was assessed by analyzing snap-frozen liver tissue sections from mice via IHC for TXNIP, NLRP3, GSDMD, and IL-1β (Figure 5). The staining indicated that SMS treatment led to a dose-dependent reduction in the levels of TXNIP, NLRP3, GSDMD, and IL-1β compared to the HFD group. Overall, the SMS extract suppresses pyroptosis in non-alcoholic fatty liver disease through the TXNIP/NLRP3 pathway.

In cell experiments, SMS (200 μg/mL) significantly inhibited cell proliferation (Figure 6A). However, SMS at lower concentrations had no effect on cell proliferation. Consequently, subsequent studies will use concentrations of SMS ranging from 12.5 to 100 μg/mL. Established in vitro lipid deposition cell culture model using FFA treatment in HepG2 cells FFA treatment group. The results showed that significantly elevated expression levels of TG, TC, and LDL-C in the FFA group were reversed by SMS in a dose-dependent manner, and concentrations of 50 and 100 μg/mL were significantly improved (Figures 6B–D). Additionally, treatment with SMS increased levels of HDL-C (Figure 6E). Oil Red O staining showed that there were obvious orange lipid droplets increased in the FFA treatment group compared to the CON group (Figure 6F). However, orange lipid droplets were decreased after treated with various concentrations SMS. Taken together, these results indicate that SMS reduces lipid deposition in vitro.

To further confirm the antioxidative and anti-inflammatory effects of SMS. The antioxidative effect of SMS was assessed by evaluating intracellular ROS levels through fluorescence staining. Treatment with SMS following FFA induction significantly mitigated ROS levels in HepG2 cells (Figure 7A). Furthermore, the antioxidative impact of SMS was validated by evaluating the levels of the lipid peroxidation product (MDA) and antioxidant enzymes (CAT, SOD, and GSH-Px) using assay kits. Concordant with the intracellular ROS levels, MDA concentrations increased in the FFA group and decreased following SMS administration. Moreover, the levels of CAT, SOD, and GSH-Px were significantly elevated compared to the FFA group (Figures 7B–E). The anti-inflammatory effect of SMS was examined using qRT-PCR. Treatment with SMS in a dose-dependent manner suppressed the expression of TNF-α, IL-6, IL-18, and IL-1β mRNA in comparison to the FFA group (Figures 7F–I). Collectively, these findings demonstrate that SMS mitigates oxidative stress and inflammation in vitro.

To determine the protective effect of SMS on cell injury induced by FFA. Cell viability significantly increased compared with FFA group (Figure 8A), indicating the protective effect of SMS on FFA-induced HepG2 cells. The morphology of FFA cultured HepG2 cells was compared using SEM. The results showed that the surface of cell membrane in the CON group remained intact. FFA group exhibited a lot of membrane pores, while the SMS (100 μg/mL) group showed a significant reduction in the number of membrane pores compared to the FFA group (Figure 8B). Assessed the effect of SMS on release of maker of pyroptosis (Lactate dehydrogenase, LDH) and inflammatory factors (IL-18, IL-1β). Treatment with SMS inhibited the levels of LDH, IL-18, and IL-1β compared to the FFA group in a dose-dependent manner (Figures 8C–E). Taken together, these results indicate that reduce the number of membrane pores and release of inflammatory factors in vitro.

To investigate the impact of SMS on pyroptosis, HepG2 cells induced by FFA were exposed to varying concentrations of SMS and subsequently subjected to immunofluorescence staining (Figures 9A–C). Representative images from each group confirmed that SMS alleviated NAFLD by inhibiting pyroptosis, the levels of TXNIP, NLRP3, and GSDMD proteins were upregulated, and this escalation was mitigated by SMS. qRT-PCR findings revealed a dose-dependent inhibition of TXNIP mRNA expression following SMS treatment compared to the FFA group (Figure 9D). Western blot analysis demonstrated that the markedly elevated expression levels of TXNIP, NLRP3, caspase 1, cleaved-caspase 1, and pyroptosis-related proteins in the FFA group were dose-dependently reversed by SMS (Figure 9E). In addition, studies on the active compounds of SMS have shown that four alkaloids can effectively reduce the expression of LDH and have a certain anti-pyroptosis effect (Supplementary Figure S1A–C). These results collectively indicate that SMS suppresses pyroptosis via the TXNIP/NLRP3 pathway in vitro.

HepG2 cells were subjected to TXNIP silencing using siRNA to assess its impact on pyroptosis. The findings exhibited successful delivery of siRNA-TXNIP into HepG2 cells, resulting in decreased levels of TXNIP mRNA and protein (Figures 10A–C). The silencing of TXNIP significantly reduced the expression of TC, TG, IL-18, and IL-1β in FFA-induced HepG2 cells compared to the Con group, and the therapeutic effect of SMS was abolished (Figures 10D,E). These outcomes indicate that SMS attenuates the expression of inflammatory markers and mitigates lipid accumulation by suppressing TXNIP expression.