Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting 12.5% of the global population (1). Non-alcoholic steatohepatitis (NASH) is the advanced stage of NAFLD, which typically increases the risk of developing into cirrhosis and hepatocellular carcinoma (HCC), ultimately leading to HCC (2). So far, NASH has become the fastest-developing cause of HCC. In clinical practice, changing lifestyle and drug therapy are the commonly used coping strategies mainly because there is currently no specific treatment method for NASH and the specific cause of NASH is not yet clear (3).

NASH is a chronic liver disease closely related to metabolic disorders, characterized by hepatocyte steatosis, inflammation, and fibrosis (4). Among them, inflammation is an important feature of NASH, which is a complex process caused by multiple factors. In brief, immune cells such as macrophages and T lymphocytes are activated by fatty acids and oxidative stress, releasing pro-inflammatory cytokines, leading to the progression of inflammation and fibrosis (5, 6). Secondly, abnormal fatty acid metabolism is one of the key factors in NASH inflammation. Once liver fatty acid metabolism is imbalanced, fatty acids accumulate excessively in hepatocytes, leading to the formation and enlargement of lipid droplets, thereby triggering an inflammatory response in the liver (7). Moreover, when exposed to excessive fatty acids and free radicals, hepatocytes can also result in increased oxidative stress, further increasing the inflammatory response and exacerbating the progress of NASH (8). In traditional Chinese medicine (TCM), spleen deficiency (SD) is the basis of NASH, which can be manifested as chest and rib swelling and pain, sighing, depression or irritability, lack of appetite, bloating, and loose stools (9). Mice gavaged with folium sennae were usually used as the SD model, exhibiting diarrhea, imbalance of short-chain fatty acid metabolism, and severe inflammation (10). However, it is not clear whether this severe inflammation will cause an inflammatory response in the liver and ultimately lead to metabolic disorders in the liver.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily comprised of three subtypes, namely, PPARα, PPARβ/δ, and PPARγ, and they play a crucial role in glucose and lipid metabolism and inflammation (11). Among them, PPARα can promote the uptake and oxidation of fatty acids, inhibit adipocyte differentiation, and also promote the reverse transport of cholesterol by regulating the expression of related genes (12, 13). Although abnormal PPARα signaling is often associated with abnormal liver lipid metabolism, its abnormal expression is also accompanied by inflammatory reactions in the liver (14). Recent studies have begun to consider the crosstalk between hepatocytes and immunocytes. It is known that lipotoxicity caused by lipid metabolism disorders in hepatocytes can induce inflammatory reactions as shown in our previous study (15). The administration of the PPARα/γ dual agonist tesaglitazar had anti-inflammatory effects in ob/ob mice by reducing the number of pro-inflammatory adipose tissue macrophages (16). The current study investigated the hypothesis that SD-induced liver inflammation leads to abnormalities in the PPARα pathway, thereby accelerating susceptibility to NASH.

It is believed that once the inflammatory response intensifies, it will further induce lipid metabolism disorders to form a vicious cycle, ultimately exacerbating the development of the disease (17). Similar to this point of view, our previous study proved that hepatic Zbtb18 protein transcriptionally activates FAO and CLTC expression, which inhibits NLRP3 inflammasome’s activity, alleviating inflammatory stress and insulin resistance. In this study, WT mice and PPARα knockout (PPARα^ko) mice gavaged with folium sennae (SE) were used as the SD model, a methionine–choline-deficient (MCD) diet was used as an incentive for NASH, and the molecular biology techniques and transcriptomics were utilized for detecting changes in inflammation, lipid metabolism, and PPARα expression.

The pandemic of NASH has forced us to consider countermeasures, and identifying susceptible populations of NASH and preventing their process in NASH are two of the feasible ways. TCM believes that SD, which is involved in the abnormal function of the spleen and liver as well as organs of the digestive system, is the key to the occurrence and development of NASH (18). An individual with SD is more likely to exhibit symptoms of digestive and metabolic abnormalities, which may trigger or accelerate NASH (19). Also, TCM research on NASH has always focused on SD (20). SE is a commonly used inducer for establishing SD models, and mice treated with its intervention may experience metabolic abnormalities, diarrhea, and other symptoms, which are typical manifestations of SD (21). This project outlines the increased susceptibility of SD individuals to NASH and points out one of its potential therapeutic targets—PPARα.

Inflammation is one of the important triggers in the progression of NAFLD to NASH (22). In this study, there are significant differences in metabolic and inflammatory pathways in the RNA-seq analysis of liver samples from NASH-SD and NASH patients, suggesting that metabolism and inflammation may be triggers for the development of SD toward NASH, as demonstrated by SD mice in our study. This is consistent with a recent study on increased inflammation and metabolic disorders in SD mice (10). However, SD-induced severe diarrhea in mice may also be associated with intestinal barrier dysfunction and dysbiosis of gut microbiota (23). For example, when transferred to sterile mice, specific microbial characteristics may be reshaped, and the progression of NASH can be delayed, indicating that mitochondrial dysfunction promotes the progression of NASH by exacerbating the gut–liver status as well as inflammation (24). Overall, the changes in the microenvironment of liver inflammation caused by different reasons will drive the activation of hepatic stellate cells and thereby accelerate the development of NASH, which is consistent with our research (25).

In NASH patients, not only severe inflammation and lipid deposition but also a tendency for liver fibrosis was observed (26). Similar results were observed in SD mice, indicating that individuals with SD are more susceptible to NASH. Meanwhile, NASH treatment strategies either seek to alleviate metabolic disorders and cell damage or directly target the accompanying inflammation and fibrosis, with targets for reducing the activation of inflammatory cascade reactions including nuclear receptor agonists (27). In this study, we observed that the liver of SD mice exhibited not only elevated levels of inflammation and a tendency toward fibrosis but also abnormal PPARα signaling, in which PPARα is one of the nuclear receptors. When entering the nucleus, PPARα can bind to promoters that are associated with β-oxidation-related factors such as ACADM and CPT1A and also enhance their transcriptional activity and ultimately drive the process of fatty acid β-oxidation (28). Also, activated PGC-1α can regulate fatty acid β-oxidation through synergism with transcription factors such as PPARα, indicating the crucial role that PPARα/PGC1α signaling plays in liver fatty acid metabolism homeostasis (29). PPARα is downregulated in many liver diseases such as NAFLD and NASH (30). In this study, we found for the first time that PPARα is downregulated in the liver of SD mice. Thus, the abnormal expression levels of PPARα and PGC1α occurred simultaneously with fibrosis and elevated inflammatory response in SD mice, indicating that this signaling may be the reason for the increased susceptibility of SD mice to NASH.

In further research, we investigated whether PPARα deficiency increased the susceptibility to NASH, using PPARα^ko mice and WT mice with MCD+SD intervention as the study subjects and conducting histopathological observations. H&E staining and Oil Red staining are commonly used to observe fat deposition in mouse liver, while Sirius staining and immunohistochemistry for detecting αSMA expression are commonly used to observe the severity of liver fibrosis (31). In our study, PPARα^ko mice treated with MCD+SD intervention showed more severe fat deposition and fibrosis in the above liver pathological analysis compared with WT mice, but there was no difference between the two in the CD diet. It is known that PPARα is highly expressed in the liver and participates in regulating fatty acid metabolism (32). After binding to fatty acid ligands, PPARα stimulates the transcription of genes containing PPARα response elements in its enhancer, with the most significant being those genes involved in lipid metabolism and energy homeostasis (33). Previously, Li’s research showed that there was no significant difference in the liver between normally fed PPAR^ko mice and WT mice, which is consistent with our research results (34). The absence of PPARα exacerbates the NASH performance caused by MCD+SD intervention, which is related to the functions of PPARα in regulating lipid metabolism homeostasis and inflammatory response. Meanwhile, our results also indirectly reflect that the absence of PPARα increases the susceptibility to NASH. In addition, only a preliminary exploration of the mechanism has been conducted, and the number of clinical samples used was limited, which cannot be representative of all situations. Further studies are needed in the future.

Overall, the lack of PPARα accelerated the vicious cycle between lipid metabolism and inflammatory response in NAFLD with SD. Similar results have been found in sepsis in which the metabolic and inflammatory responses to bacterial infection were impaired in the absence of PPARα, which leads to an enhancement in mortality due to bacterial sepsis (35). Our previous studies also exhibited that the ingredients of spleen-tonifying drugs can improve NAFLD or NASH by regulating PPAR signaling, which indirectly confirms the conclusion of this topic (36, 37). Unfortunately, there was no separate comparison between the MCD group and the MCD+SD group when setting up the groups, which will also be our next research direction. In addition, only a preliminary exploration of the mechanism has been conducted, and the number of clinical samples used was limited, which cannot be representative of all situations. Further studies are needed in the future.

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: https://www.ncbi.nlm.nih.gov/, PRJNA1061326.

The studies involving humans were approved by the First Affiliated Hospital of Guangzhou University of Chinese Medicine Institutional Review Board and Ethics Committee affiliated to Guangzhou University of Chinese Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The animal study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine affiliated to Guangzhou University of Chinese Medicine. The study was conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

JH: Writing – review & editing, Writing – original draft, Investigation, Funding acquisition, Formal analysis. JL: Writing – review & editing, Writing – original draft, Investigation, Formal analysis. YP: Writing – original draft, Writing – review & editing, Formal analysis. TQC: Writing – original draft, Visualization, Investigation. JG: Writing – original draft, Visualization, Investigation. SD: Writing – original draft, Software, Investigation. KZ: Writing – original draft, Software, Investigation. SH: Writing – original draft, Investigation, Data curation. JC: Writing – original draft, Investigation, Data curation. QY: Writing – original draft, Investigation. MQ: Writing – original draft, Investigation. TTC: Writing – original draft, Investigation. XW: Writing – original draft, Investigation. CM: Writing – original draft, Investigation. ZZ: Writing – original draft, Investigation. YZ: Writing – original draft, Investigation. XT: Writing – original draft, Investigation. YQP: Writing – review & editing, Conceptualization, Funding acquisition. LZ: Writing – review & editing, Writing – original draft, Resources, Formal analysis. CZ: Writing – review & editing, Writing – original draft, Resources, Formal analysis. YG: Writing – review & editing, Writing – original draft, Resources, Funding acquisition, Formal analysis, Conceptualization.