Primary liver cancer, particularly hepatocellular carcinoma (HCC), accounts for about 80% of all liver cancer cases. It has been the third most common cause of cancer mortality globally up until 2020 (Sung et al., 2021). HCC is often associated with Hepatitis B Virus (HBV) infection. It represents a critical global health problem due to its significant morbidity and death rates, particularly in regions with prevalent HBV infection. This trend underscores the critical need to understand the complex mechanisms underlying HBV-related HCC and identify potential biomarkers for its effective management and treatment (Beckebaum et al., 2003, Castelnau et al., 2006). The role of the liver in the gut-liver axis is crucial. It involves a complex interplay with the gut that is essential for nutrient metabolism, immune responses, and the biotransformation of bacterial metabolites (Tripathi et al., 2018). The effectiveness of this axis is contingent upon healthy gut microbiota, effective intestinal permeability, and healthy liver activity. The gut microbiota is recognized as the main component of human microecology, whereby it represents a symbiotic relationship with humans (Su et al., 2022). It shows variations influenced by clinical factors such as age, body mass index, dietary preferences, and exercise habits (Liu et al., 2023, Zhang et al., 2019). Liver conditions such as nonalcoholic fatty liver disease (NAFLD) and liver cirrhosis are frequently linked to changes in intestinal microbiota, with growing evidence suggesting that gut bacterial products could contribute to the progression of liver cancer (Chen et al., 2011, Qin et al., 2014, Ren et al., 2019, Shen et al., 2017, Zhang et al., 2017). In summary, the gut microbiota might be mechanistically correlated with HCC pathogenesis.

Traditional Chinese Medicine (TCM) has showcased significant benefits in the treatment of liver cancer, effectively alleviating clinical symptoms at different stages of the disease. TCM helps boost the effectiveness of other therapeutic modalities, decreasing the toxic side effects of radiotherapy and chemotherapy, promoting quality of life, and potentially delaying or even preventing the recurrence and metastasis of liver cancer, thus prolonging life expectancy (Hu et al., 2016). Bielong Ruangan decoction (BLRG decoction), originally named "Shugan Lipi II Formula", is an important component in the treatment regimen of liver fibrosis/cancer. Developed from "Shugan Lipi Decoction", the formula consists of ingredients like Taoren (dry seed of Prunus persica (L.) Batsch), Ezhu (the dry root of Curcuma phaeocaulis Val.), Biejia (carapace of Trionyx sinensis Wiegmann), earthworm (the dry whole body of Pheretima aspergillum (E. Perrier)); Fuling (dry sclerotia of Poria cocos (Schw.) Wolf), Chaihu (the dry root of Bupleurum chinense DC.), Baishao (dry root of Paeonia lactiflora Pall.) and Gancao (dry root and stem of Glycyrrhiza uralensis Fisch.), functioning to break blood stasis, eliminate accumulations, and disperse masses (Lei et al., 2000, Wang et al., 2018). Preliminary studies have found that BLRG decoction effectively combats chronic liver injury and fibrosis, enhances cellular immunity, and exhibits anti-liver cancer properties. It also significantly improves the conditions of spleen deficiency and blood stasis (Wang et al., 2010, Wu et al., 2017). Furthermore, BLRG decoction combined with entecavir has shown more efficacy than entecavir alone in treating hepatic fibrosis in chronic hepatitis B, improving TCM syndrome scores and the degree of hepatic fibrosis, with confirmed clinical effectiveness and good safety (Yang et al., 2023). Notably, a previous study has reported that Jiawei Xiaoyao San, another TCM formula that exerts a certain therapeutic effect on liver cancer, could considerably alter the intestinal flora composition of the incidence of liver cancer, elevate the proportion of beneficial bacteria, and decrease the proportion of pathogenic bacteria (Li et al., 2022). Therefore, it could be hypothesized that gut microbiota might also mediate the effects of BLRG decoction on HBV-related HCC.

Herein, an orthotopic liver tumor model was established in mice, and the effects of BLRG on tumor growth, intestinal barrier function, immune-related factors, gut microbiota, and serum metabolomics were investigated in mice models. Differential bacteria and serum metabolites were analyzed. Moreover, fecal microbiota transplantation and BLRG administration were performed to examine the co-effects on mouse models.