Shenling Baizhu powder alleviates non-alcoholic steatohepatitis by reducing PPARα-mediated NLRP3 inflammasome activation

Abstract

Introduction

Nonalcoholic steatohepatitis (NASH) is the progressive stage of nonalcoholic fatty liver disease characterized by varying degrees of inflammation, hepatic steatosis, liver cell damage and fibrosis, which can seriously harm people’s health. Shenling Baizhu (SL) powder exhibits a strong capacity to modulate cellular mechanisms, thereby demonstrating exceptional potential in addressing inflammatory and oxidative stress-related pathologies. However, the exact mechanism of SL in NASH has not been fully elucidated. We aimed to explore the therapeutic effect and potential mechanism of SL in NASH mice.

Methods

We fed mice the methionine-choline deficiency (MCD) diet to construct a model of NASH. The efficacy of SL in regulating the progression of NASH was evaluated by biochemical and histopathological analyses. The effects of SL on oxidative stress and inflammatory cytokines were examined. Transcriptomic analysis was performed to further explore the potential molecular mechanisms regulated by SL. Finally, the expression of key proteins was verified by Western blotting.

Results

The pathological phenotype of NASH was successfully established in mice. SL intervention demonstrated significant therapeutic benefits in reducing hepatic lipid accumulation, alleviating liver fibrosis, and decreasing inflammatory responses. Additionally, SL intervention was shown to ameliorate ER stress by attenuating the unfolded protein response (UPR). Gene expression profiling coupled with pathway analysis revealed that SL exerted its effects through the modulation of critical signalling pathways, including the PPAR signalling axis. Additionally, SL inhibited NLRP3 inflammasome activation and reduced the expression of downstream inflammatory cytokines in the liver.

Discussion

The findings indicate that SL mitigates NASH by activating the PPAR pathway and suppressing the ER stress-triggered UPR, thereby inhibiting NLRP3 inflammasome activation. These results highlight the therapeutic potential of SL for NASH management.

Graphical Abstract

1. Introduction

The incidence and mortality of cirrhosis due to nonalcoholic fatty liver disease (NAFLD) are increasing in 16 regions of the world, making it the primary cause of cirrhosis-related mortality (1). According to epidemiological statistics, NAFLD extensively and profoundly affects approximately one-quarter of the global population (1–3). Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, inflammation and fibrosis, which can develop into cirrhosis and HCC; thus, it has attracted much attention (4, 5). Recent data indicate a rising incidence of NASH (6), and while some drugs are believed to alleviate NASH (7, 8), very few have been approved for treating it (9), emphasizing the urgency of alternative treatment strategies.

The pathogenesis of NASH is complex and mostly involves inflammation, oxidative stress, lipid accumulation, insulin resistance and other factors (9, 10). Endoplasmic reticulum (ER) stress is a major component of oxidative stress and plays a crucial role in NASH (11–13).

Shenling Baizhu (SL) powder was first recorded in “Taiping Huimin Heji Jufang”, which was compiled during the Song Dynasty. This formula is composed exclusively of traditional Chinese medicines, such as Panax ginseng C.A. Mey., and Poria cocos (Schw.) Wolf, Atractylodes macrocephala Koidz., and Dioscorea bulbifera L. The entire formula can strengthen the spleen, benefit qi, infiltrate dampness and eliminate turbidity. It has been widely used to treat diseases for nearly a thousand years. SL exhibits significant efficacy in the management of gastrointestinal and liver lipid metabolism disorders (14–16), as it demonstrates pronounced ameliorative effects on inflammation, oxidative stress and lipid metabolism (17, 18). Studies have shown that SL significantly improves liver lipid deposition and oxidative stress damage, induced by a high-fat diet, in rat models of NAFLD (19). However, its potential to treat NASH and the underlying mechanisms remain unexplored. Given the pathophysiology of NASH, including excessive lipid accumulation, ER stress, and chronic inflammation, SL may play a beneficial role by targeting these processes.

NASH models were successfully induced in mice by administering a methionine-choline-deficient (MCD) diet, as described in our previous study (20). By combining biochemical analysis, histopathological analysis, transcriptomic analysis, and Western blotting, we aimed to elucidate the mechanisms by which SL improves liver lipid metabolism, reduces inflammation, and relieves ER stress. Our findings shed light on SL as a natural therapeutic agent that offers a novel pharmacological approach rooted in traditional medicine to address NASH and lipid metabolism disorders.

2. Materials and methods

2.1. Materials and chemicals

SL was prepared following traditional Chinese medicine formulations, and the detailed composition of the powder is presented in Table 1. LC–MS/MS was used for ingredient identification (Supplementary Figure 1) and the molecular weights of the top 10 components (Table 1). Polyene phosphatidylcholine (PPC) capsules were sourced from Sanofi Pharmaceuticals (Beijing, China). Biochemical reagents for measuring hepatic and serum markers were obtained from the Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits used for cytokine detection were obtained from Multi Sciences Biotech (Hangzhou, China). The Antibodies against proteins, including NLRP3, ASC, Caspase-1, and β-actin, were obtained from Cell Signaling Technology (Massachusetts, USA). Anti-BIP, anti-IRE1, anti-PERK, and anti-ATF6 antibodies were provided by Bioss Antibodies (Beijing, China), and anti-PPARα antibodies were supplied by Abcam (Cambridge, USA). Staining kits for haematoxylin and eosin (HE), oil red O (ORO), and Masson’s trichrome were acquired from Servicebio Technology (Wuhan, China).

Table 1.

The composition of Shenling Baizhu power.

Chinese name	Botanical name	Proportion
Ren Shen	Panax ginseng C.A. Mey.	5
Fu Ling	Poria cocos (Schw.) Wolf	5
Bai Zhu	Atractylodes macrocephala Koidz.	5
Bai Bian Dou	Lablab purpureus subsp. purpureus	4
Shan Yao	Dioscorea bulbifera L.	5
Gan Cao	Glycyrrhiza glabra L. Preparata	3
Lian Zi	Nelumbinis plumula	3
Sha Ren	Amomum villosum Lour.	2
Yi Yi Ren	Coix lacryma-jobi L.	3
Jie Geng	Platycodon grandiforus	2

2.2. Animal modelling and treatment

Trophic Animal Feed High-tech (Nantong, China) provided the MCD diet (60 kcal% fat, 0.1% methionine, and no choline supplementation) and the methionine-choline sufficient (MCS) diet (10 kcal% fat). Male C57BL/6J mice at 5 weeks of age were purchased from HFK Bioscience (Beijing, China). The control experimental temperature was 24 ± 2 °C, and the relative humidity was 55 ± 5%, with a 12-h light/dark cycle. After the final administration, mice were anesthetized with isoflurane in oxygen (3% at 1 L/min for induction and 1% at 1 L/min for maintenance). At the end of the experiment, euthanasia was performed using an overdose of isoflurane (5% in oxygen at 1 L/min) followed by exsanguination. All procedures complied with the ethical guidelines approved by the Institute of Laboratory Animal Resources, Jinan University (Guangzhou, China) (Licence No. IACUC-20220114-06).

The dosage of SL was selected as we previously described (21). After 10 days of adaptive feeding, all the mice were randomly distributed into 5 groups, with 10 mice in each group: the MCS group was fed an MCS diet for 3 weeks; the MCD group was fed an MCD diet for 3 weeks; and the MCD+SL-L group was fed 15 g/kg/d SL by gavage and fed an MCD diet for 3 weeks; the MCD+SL-H group was given 30 g/kg/d SL by gavage and fed an MCD diet for 3 weeks; and the MCD+PPC group was orally given 120 mg/kg/d PPC and fed an MCD diet for 3 weeks. Both the MCS group and the MCD group were administered the same amount of ionized water by gavage.

2.3. Surface microcirculation scan of the liver

After fasting for 12 hours prior to euthanasia, all the animals were anesthetized with pentobarbital (50 mg/kg). The mice were then exposed to normal temperature and natural light to measure liver blood flow. One frame per second is recorded for 60 seconds to generate the visual blood flow images.

2.4. Histological analysis

Liver tissues were coated with paraffin, sliced to 5 µm thickness, and stained with HE to evaluate overall histology. Lipid accumulation in tissues was assessed by ORO staining. The Masson method and α-SMA method were used to assess the degree of hepatic fibrosis. To assess the integrity and fine structure of ER, transmission electron microscopy (TEM) was used to compare ultrastructural changes among the different groups.

2.5. Biochemical detection

Blood samples were harvested, and the serum was isolated for subsequent analyses. The serum concentrations of ALT, AST, HDL-C and LDL-C were measured using corresponding kits. The supernatant of liver homogenate was collected after centrifugation, and the concentrations of TC, TG and NEFAs were determined by biochemical assay kits.

2.6. Oxidative stress index analysis

To assess liver oxidative stress, liver tissues were stained with dihydroethidium (DHE) according to the instructions of the kit. The reactive oxygen species (ROS) levels in the liver were subsequently evaluated by measuring the DHE fluorescence intensity under a fluorescence microscope. The homogenate was prepared by adding the sample homogenizer buffer of the kit to the homogenizer. The homogenate was then centrifuged at 12,000 rpm for 10 minutes to separate and collect the supernatant. The contents of SOD, MDA and GSH in the supernatant were subsequently determined following the kits’ instructions. Each experiment was repeated 6 times to ensure data reliability.

2.7. Cytokine analysis by ELISA

Total protein was extracted from the liver tissue homogenate, after which the supernatant was obtained by centrifugation. The levels of IL-1β, IL-6 and TNF-α in the liver were determined and quantitatively analysed following the instructions of the ELISA kits.

2.8. Liver transcriptomic analysis

Firstly, RNA integrity and concentration were assessed, and samples with RNA integrity number values >7.0 were then screened for clustering and quality control. A reference genome index was constructed and compared with the reference genome. The expression levels of the differentially expressed genes were analysed, and genes whose adjusted p value was <0.05 were defined as differentially expressed genes (DEGs). The overlapping differentially expressed genes identified from pairwise comparisons among the three groups were selected as the core gene set. Subsequently, functional annotation and pathway enrichment analyses were performed using GO-biological process (GO: BP) and the Kyoto Encyclopedia of Genes and Genomes (KEGG).

2.9. Western blot analysis

The total proteins from liver tissues were extracted with RIPA buffer, and the BCA method was used to determine the protein concentration. The target proteins were isolated and transferred to PVDF membranes using SDS–PAGE. After blocking, the membranes were incubated with primary antibodies at 4 °C overnight. After being washed with TBST, the membrane was incubated with secondary antibody for 1 h. The ECL detection system was used to visualize the protein bands, and ImageJ was used to quantify the protein expression levels.

2.10. Statistical analysis

GraphPad Prism 9.0 software was used for statistical analysis. Intergroup differences were tested by one-way ANOVA. The statistical results were expressed as the mean ± SD, and p < 0.05 was considered to indicate statistical significance.

3. Results

3.1. SL ameliorated lipid accumulation in MCD-induced NASH mice

Given that liver histopathological features are strong indicators for diagnosing NASH, we investigated the effects of SL on mice with NASH induced by 3 weeks of an MCD diet. After the mice were killed and fresh liver tissue was obtained, we assessed the extent of histopathological changes and lipid accumulation in the liver by HE and ORO staining (Figures 1A, B). Compared with those in the MCS group, the lipid droplets in the MCD group were irregularly arranged, and the size of the lipid droplets in hepatocytes was uneven and significantly increased, suggesting extensive degeneration of the liver tissue. After SL and PPC intervention, the above indices were compared with those in the MCD group. The above indices in the SL and PPC groups significantly improved. Notably, compared with the control group, the high-dose SL group exhibited significantly greater mitigation of lipid accumulation in the livers of NASH mice.

Figure 1.

Liver pathological observation and biochemical parameters in mice. (A) Representative images of HE-stained sections (200 × and 400 ×). (B) Representative images of ORO staining (200 × and 400 ×). (C) Liver wet weight (n = 10 per group). (D) Liver TC levels (n = 10 per group). (E) Liver TG levels (n = 10 per group). (F) Liver NEFA levels (n = 10 per group). (G) Serum HDL-C levels (n = 10 per group). (H) Serum LDL-C levels (n = 10 per group). The data are presented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant.

3.2. SL ameliorated biochemical parameters in mice with NASH

We also examined lipid metabolism and biochemical markers in mice. Compared with the MCS group, the MCD group exhibited significantly higher liver wet weight (Figure 1C), TG (Figure 1E) and NEFA (Figure 1F) levels; conversely, compared with the MCD group, the SL-treated group demonstrated a notable reduction in these parameters. Compared with the MCS group, the MCD group had significantly higher serum NEFA levels. SL-H treatment reduced NEFA levels, while SL-L showed a decreasing trend but without statistical significance versus the MCD group. The concentrations of TC (Figure 1D), HDL-C (Figure 1G) and LDL-C (Figure 1H) were highest in the MCS group and lowest in the MCD group. Upon treatment with SL or PPC, these lipid parameters were elevated compared to the MCD group, with the MCD+SL-H group demonstrating superior efficacy compared to the MCD+ SL-L group. These findings suggest that SL improves lipid metabolism in a dose-dependent manner, with higher doses yielding greater therapeutic benefits. These data suggest that SL and PPC restore normal liver lipid metabolism and effectively reduce liver damage in a dose-dependent manner.

3.3. SL reduced liver fibrosis in mice with NASH

Masson tricolour staining revealed significant collagen deposition in the MCD group. PPC and SL interventions significantly reduced fibrotic areas, with a more significant improvement in the MCD+SL-H group (Figure 2A). Compared with that in the MCD group, the expression of collagen fibre α-SMA was decreased in both the SL and PPC groups, especially in the SL-H group, indicating that SL had a stronger anti-fibrotic effect (Figure 2B).

Figure 2.

Effects of different interventions on liver fibrosis in NASH mice. (A) Representative Masson’s trichrome staining (200 × and 400 ×). (B) Immunofluorescence staining for α-SMA (green), a marker of activated hepatic stellate cells, counterstained with DAPI (blue) to indicate nuclei. Merged images highlight the distribution of α-SMA-positive cells. Scale bar, 100 μm.

3.4. SL reversed liver surface microcirculation disturbance in mice with NASH

Mice in the MCD group had significantly enlarged livers with rough, greasy surfaces and blunt edges. These changes were notably reversed in both the low- and high-dose SL groups and in the PPC group (Figure 3A). Liver surface microcirculatory blood flow analysis revealed that blood flow was significantly reduced in the MCD group compared with the MCS group. The improvement was most significant in the MCD+SL-H group, indicating that the restoration of liver perfusion by SL or PPC was positively correlated with dose (Figure 3B). The results of the quantitative analysis also supported the above results, with those of the MCD+SL-H group more closely matching the levels of the MCS group (Figures 3C, D), suggesting that SL can reverse liver microcirculatory blood flow in mice with NASH, thereby inhibiting liver lipid accumulation.

Figure 3.

Liver surface blood flow scan. (A) Visual observation of the livers. (B) Scanning images of liver surface microcirculatory blood flow. The flux images (bottom row) represent blood PUs across the liver surface. The red and blue areas represent high- and low-perfusion areas, respectively. (C) The curves of liver microcirculatory blood flow in each experimental group were recorded, and the measurement time was 60 seconds. (D) Quantitative analysis of the average liver blood flow PU in each group (n = 10 per group). PU: perfusion units. The data are presented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant.

3.5. RNA-seq analysis demonstrated that SL modulates NASH mice via the PPAR signaling pathway

Next, transcriptomic analysis was performed on mice in the MCD, MCS and MCD+SL-H groups (hereafter referred to as the MCD_SL group). Principal component analysis (PCA) and heatmap analysis revealed significant differences between the MCD group, the MCS group, and the MCD_SL group, indicating that the MCD diet-induced NASH mice were successfully established (Figures 4A–D). To further elucidate the underlying mechanism by which SL acts on NASH mice, we performed RNA sequencing (RNA-seq). Among the MCS, MCD, and MCD_SL groups, 1219 genes were co-differentially expressed (Figure 4E). The KEGG enrichment pathway analysis performed specifically on these 1,219 overlapping DEGs revealed significant enrichment of multiple pathways, including the PPAR signaling pathway, the NF-κB signaling pathway and the PI3K-Akt signaling pathway. GO-biological process (GO: BP) analysis revealed significant enrichment of differentially expressed genes (DEGs) related to lipid metabolism and lipid binding processes (Figures 4F, G). In conclusion, SL may play a role in protecting the ER in NASH mice by regulating these key biological processes and signaling pathways.

Figure 4.

SL regulates the liver transcriptome of NASH mice. (A) Hepatic transcriptome PCA. (B) Volcano map of DEGs between the MCD group and the MCS group. (C) Volcano map of DEGs between the MCD_SL group and the MCD group. (D) Heatmap of differentially expressed genes. (E) Venn diagram of DEGs. (F, G) Based on the DEGs, KEGG and GO analyses were carried out, and the results are intuitively shown as bubble maps.

3.6. SL mitigated oxidative stress and restored ER integrity in the livers of mice with NASH

Disruption of endoplasmic reticulum homeostasis leads to ER stress, which is a well-established driver of NASH pathogenesis. Previous studies have shown that PPAR activation alleviates ER stress and mitigates NASH progression by reducing hepatic steatosis, fibrosis, and inflammation (22–24). Given that our RNA-seq analysis identified the PPAR signaling pathway as the predominant enriched pathway following SL intervention, we further evaluated the effect of SL on ER function. TEM revealed severe swelling of the ER, destruction of its structure ERand accumulation of large lipid droplets, in the MCD group. SL intervention, especially after high-dose SL treatment, significantly reduced lipid droplet deposition, alleviated ER swelling and restored its structural integrity (Figure 5A). We subsequently performed DHE staining and the determination of related oxidative stress indices, which revealed that the level of oxidative stress in the liver was more remarkable in the MCD group than in the control group, whereas SL and PPC markedly increased the level of oxidative stress in the livers of mice with NASH (Figures 5B, C). PPARα levels were significantly lower in the MCD group than in the control group, whereas the changes in BIP, IRE1, PERK, and ATF6 levels were reversed. However, these indicators were reversed in the MCD+SL group (Figures 5D, F). On the basis of these findings, we can further speculate that the remission of NASH from SL may negatively regulate the ER-associated UPR through the PPARα axis.

Figure 5.

SL improved oxidative stress levels and ER ultrastructure in the livers of mice with NASH. (A) TEM images of liver tissue. Red arrows represent the ER (9700x and 23000x). (B) DHE staining of reactive ROS levels (scale bar, 50 μm). (C–F) Relative expression levels of ROS, SOD, MDA and GSH (n = 10 per group). (G, H) The protein expression levels of PPARα, BIP, IRE1, PERK and ATF6 were detected by Western blotting (n=3). The data are presented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant.

3.7. SL alleviated liver inflammation via inhibition of the NLRP3 inflammasome in mice with NASH

Immunofluorescence staining for F4/80 revealed obvious macrophage infiltration in the liver tissue of the MCD group, indicating an enhanced inflammatory response. Inflammatory infiltration was significantly reduced after SL treatment (Figure 6A). Liver function and inflammatory cytokine levels were markedly increased in the MCD group, but these markers were reversed by SL intervention (Figures 6B–F). Next, Western blotting was used to detect the expression of proteins related to the NLRP3 inflammasome. Compared with those in the MCS group, the expression levels of NLRP3, ASC, caspase-1 and cleaved caspase-1 in the MCD group were enhanced, indicating NLRP3 inflammasome activation. However, the expression of these indicators was notably inhibited by SL intervention. In summary, SL may exert an anti-inflammatory effect on NASH mice, possibly by inhibiting the activation of the NLRP3 inflammasome (Figures 6G, H).

Figure 6.

SL regulated the levels of inflammatory cytokines and activation of the NLRP3 inflammasome pathway in mice with NASH. (A) Immunofluorescence staining for the macrophage marker F4/80 (red) with nuclei counterstained using DAPI (blue), demonstrating macrophage infiltration in liver tissues (scale bar, 50 μm). (B, C) Serum ALT and AST levels (n = 10 per group). (D–F) Cytokine levels of IL-1β, IL-6, and TNF-α in liver tissues as measured by ELISA (n = 10 per group). (G, H) The protein expression levels of NLRP3, ASC, caspase-1 and cleaved caspase-1 were detected by Western blotting (n=3). The data are presented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant.

4. Discussion

NASH is a growing global epidemic that progresses strongly to liver fibrosis and liver tumours. To date, very few drugs are approved for treating NASH (11). Hence, there is an urgent need for further development of novel pharmacological agents for treating NASH. Emerging evidence shows that traditional Chinese medicine has broad application prospects in the management of NASH, with its mechanisms of action involving multiple targets (25). The aim of this study was to explore the molecular mechanism of SL in the treatment of NASH by combining transcriptomics. Our study demonstrated that SL can improve NASH by reducing the activation of the ER stress-triggered UPR and NLRP3 inflammasome through the activation of PPARα.

Previous studies have confirmed that SL is effective against NAFLD by regulating the gut microbiota-liver axis and lipid droplet dynamics, or by inducing autophagy through specific compounds (14, 19, 21, 26, 27). Animal models induced by an MCD diet mimic the histological features of NASH because they readily progress from steatosis to steatohepatitis and even to fibrosis (28). In this study, mice were fed an MCD diet for 3 weeks to establish a NASH mouse model that successfully reproduced the characteristics of human NASH, including liver inflammation and metabolic disorders. Polyene phosphatidylcholine capsule (PPC), a compound containing large amounts of phospholipids from soybeans, has been reported to exert favourable anti-fatty liver and anti-fibrotic effects by alleviating liver injury and metabolic inflammation (29, 30). We followed the previous research experience and PPC was used as a positive control drug to evaluate the efficacy of SL in the NASH mouse model. Different doses of SL were used in the NASH mouse model, and the optimal intragastric dose of SL for the treatment of NASH was determined to be 30 g/kg (31, 32). The results of HE staining, ORO staining and biochemical indices revealed that SL could effectively increase liver lipid accumulation in NASH mice and reduce fat vacuole occurrence and ballooning. Masson staining and α-SMA immunofluorescence staining confirmed that SL effectively improved liver fibrosis in mice with NASH. Moreover, SL significantly improved liver microcirculation in mice with NASH. Overall, these data demonstrated that SL could alleviate the characteristic manifestations of NASH in mice.

As the metabolic centre of the human body, the liver plays an important role in maintaining metabolic homeostasis (33). We subsequently conducted liver transcriptomic analysis to further investigate the molecular mechanisms underlying the effects of SL against NASH in mice. The results indicated that the pathways through which SL was enriched in NASH mice included PPAR signaling pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, etc. Studies have shown that peroxisome proliferator-activated receptors (PPARs) play a key regulatory role in metabolism and inflammation (34). Abnormal expression of PPARs may lead to hepatic steatosis, NASH, fibrosis and HCC (35). The PPAR family includes three isoforms, namely, PPARα, PPARβ/δ and PPARγ (36). Among them, PPARα is expressed primarily in liver tissue and plays a central role in lipid metabolism (37, 38). Accumulating evidence has demonstrated that PPARα activation improves liver steatosis, inflammation, and fibrosis, serving as a potential therapeutic approach for treating NASH (39). As expected, our data revealed that SL intervention increased PPARα expression in the livers of NASH mice, indicating that SL may exert beneficial effects through PPAR activation. Notably, it is widely believed that PPARα serves as a crucial regulator for modulating lipid peroxidation. The ER (ER) is present inside cells and promotes the proper folding of thread-like peptides and proteins (40). The inability of the ER to form the correct protein 3D structure causes misfolded or unfolded proteins to accumulate within the ER, perturbs ER homeostasis, and causes ER stress (11, 41). Chronic ER stress has been shown to drive NAFLD progression (42, 43). The unfolded protein response (UPR) can trigger an inflammatory cascade, inflammasome activation, and hepatocyte apoptosis if ER stress is unresolved (44). Therefore, we next observed the ER structure in the livers of NASH mice. With SL intervention, most of the swollen ER was recovered, and lipid droplet aggregation was significantly reduced. Furthermore, the degree of oxidative stress has also been alleviated accordingly. Collectively, these findings suggest that SL can effectively mitigate oxidative stress and ER stress in the livers of NASH mice.

More recently, it has been reported that activating PPARα reduces ER stress-mediated UPR, thereby alleviating NASH progression (24). Three membrane-spanning UPR sensors, IRE1, PERK and ATF6, are responsible for sensing unfolded proteins within the ER lumen and relaying signalling cascades from the ER to the cytoplasm (45). The UPR mitigates ER stress by decreasing protein synthesis and upregulating the production of ER folding enzymes (46). Immunoglobulin binding protein (BIP) is a critical ER protein that is highly expressed in the ER. Under physiological conditions, BIP binds to and inhibits the transcriptional activity of all three UPR sensors (47). In response to ER stress, misfolded proteins interact with BIP, initiating its dissociation from the three UPR sensors. This dissociation activates the sensors and triggers their downstream signalling pathways, which are crucially involved in either restoring ER function or activating apoptotic pathways (48). To determine whether SL can trigger the UPR to improve NASH, the key proteins regulating the UPR were detected by Western blotting. As expected, we found that SL can upregulate PPARα and downregulate the expression levels of BIP, IRE1, PERK and ATF6, indicating that SL may exert an anti-NASH effect by activating PPARα and negatively regulating ER stress.

ER stress may induce the activation of the NLRP3 inflammasome in hepatocytes, leading to liver injury (49, 50). The NLRP3 inflammasome consists of NLRP3, ASC, and Caspase-1 proteins (51) and plays a significant role in the progression of NASH (52, 53). The NLRP3 inflammasome can mediate pyroptosis, inflammation and fibrosis in hepatocytes to affect the progression of NASH (54, 55). Therefore, the NLRP3 inflammasome is dependent on ER stress, and inhibiting its activation and the subsequent inflammatory cascade may be a potential strategy to treat NASH. In agreement with the findings of other studies (56, 57), our data revealed that the NLRP3 pathway was activated in the livers of NASH mice. On the other hand, the liver levels of IL-1β, IL-6 and TNF-α and the expression of NLRP3 family proteins in NASH mice were significantly inhibited by SL intervention. Taken together, these findings suggest that SL may inhibit NLRP3 activation by suppressing ER stress, thereby alleviating liver inflammation in NASH mice.

The present study has several limitations. Transcriptomic analysis revealed enrichment of the PPARα pathway following SL intervention, suggesting its potential involvement in the anti-NASH effects of SL. However, current data only show a correlation, and causality remains to be established using PPARα agonists/antagonists or gene silencing. Although SL modulated NLRP3 inflammasome-related pathways, direct evidence of ASC speck formation or Caspase-1 activity, the morphological gold standard for pyroptosome assembly and direct functional evidence of pyroptosis activation, respectively, is lacking. Thus, whether SL exerts anti-NASH effects via PPARα activation or pyroptosis inhibition requires further validation. Future studies should employ pharmacological and genetic interventions to clarify the role of PPARα.

5. Conclusions

In conclusion, this study reveals the significant potential of SL in alleviating NASH. These findings indicate that the mechanism of action of SL involves activating the PPARα pathway, negatively regulating ER stress-induced UPR, and inhibiting NLPR3 inflammasome activation. These actions reduced liver lipid accumulation, inflammation, and fibrosis, demonstrating that SL effectively modulates the progression of MCD diet-induced NASH.

Glossary

MCS

methionine-choline sufficient

MCD

methionine-choline deficiency

SL

Shenling Baizhu Powder

L

low

H

high

PPC

polyene phosphatidylcholine capsule

HE

hematoxylin and eosin

ORO

oil red O

TC

total cholesterol

TG

triglycerides

NEEA

non-esterified fatty acids

HDL-C

high-density lipoprotein cholesterol

LDL-C

low-density lipoprotein cholesterol. PCA, principal component analysis

DEGs

differentially expressed genes

KEGG

Kyoto encyclopedia of genes and genomes

RNA-seq

RNA sequencing

GO

BP, GO-biological process

PPAR

peroxisome proliferator-activated receptor

NF-κB

nuclear factor kappa-B

PI3K-Akt

phosphatidylinositol 3-kinase (PI3K)-Akt

TEM

transmission electron microscopy

DHE

dihydroethidium

ROS

reactive oxygen species

SOD

superoxide dismutase

MDA

malondialdehyde

GSH

glutathione

BIP

immunoglobulin binding protein

IRE1

inositol requiring protein-1

PERK

PKR-like ER kinase

ATF6

activating transcription factor-6. α-SMA, α-smooth muscle actin

DAPI

4′,6-diamidino-2-phenylindole. ALT, alanine aminotransferase

AST

aspartate aminotransferase

IL-1β

interleukin-1β

IL-6

interleukin-6

TNF-α

tumor necrosis factor-α

NLRP3

NOD-like receptor thermal protein domain associated protein 3

ASC

apoptosis-associated speck-like protein containing a CARD

Funding Statement

The authors declared that financial support was received for this work and/or its publication. This research was supported by the National Nature Science Foundation of China (No. 82374230, 82405209, 82574885, 82274393), Guangdong Basic and Applied Basic Research Foundation, China (No. 2021A1515110774), Guangzhou Region Traditional Chinese Medicine Major Science and Technology Project (No.2025CX006), the China Postdoctoral Science Foundation (No. 2024M751135), Fundamental Research Funds for the Central Universities, China (No. 21624359), the Traditional Chinese Medicine Bureau of Guangdong Province, China (No. 20251076, 20231085), the Construction Project of National Famous and Old Chinese Medicine Expert Inheritance Studio (No.(2022)75).

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PRJNA1482749 (Bioproject, NCBI).

Ethics statement

The animal study was approved by the Institute of Laboratory Animal Resources, Jinan University (Guangzhou, China). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

SH: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing, Investigation. YD: Formal analysis, Project administration, Software, Writing – review & editing. DP: Conceptualization, Data curation, Software, Visualization, Writing – original draft. FZ: Investigation, Writing – review & editing, Validation. JP: Investigation, Writing – review & editing, Software. QS: Formal analysis, Validation, Writing – review & editing. LL: Methodology, Visualization, Writing – review & editing. YL: Investigation, Software, Writing – review & editing. QY: Funding acquisition, Supervision, Writing – review & editing. MP: Conceptualization, Investigation, Methodology, Project administration, Writing – review & editing. YZ: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1797561/full#supplementary-material