Probiotics improve the balance of intestinal flora as well as stimulate bacterial products and metabolites, and may promote human health as well as preventive treatment of different diseases. In animal models of acute liver injury and cirrhosis, the use of probiotics has been reported to improve survival by correcting bacterial overgrowth, stabilizing mucosal barrier function, and reducing bacterial translocation and endotoxemia [16] . In patients with hepatic encephalopathy, the application of probiotics may delay the progression of hepatic encephalopathy, reduce plasma ammonia concentrations, and improve quality of life [17] . Currently, probiotic therapy for liver cancer is only based on animal models of liver cancer, and there are no specific data from clinical studies. Zhang et al. in a rat model of DEN-induced hepatocellular carcinoma found that administration of VSL#3 (including four species of Lactobacillus, three species of Bifidobacterium, and one species of Streptococcus thermophilus salivarius subgenera) reduced the incidence of hepatocellular carcinoma in rats by restoring intestinal homeostasis and attenuating hepatic and intestinal inflammation and prevented progression of cirrhosis to hepatocellular carcinoma, and that a high dose of VSL#3 reduced the mortality rate of DEN-induced hepatocellular carcinoma in rats [18] . In addition, Li et al. used a novel probiotic, Prohep (composed of Lactobacillus rhamnosus (LGG), Escherichia coli Nissle1917 (EcN), and heat-inac- tivated VSL#3 (1:1:1)), for the treatment of a mouse model of hepatocellular carcinoma, and found that the Prohep group reduces the growth of hepatic tumors by inhibiting angiogenesis through decreasing the production of IL-17 and other angiogenic factors. And limit excessive Th17 cell production in the intestine and reduce the migration of Th17 from the intestine to the tumor site case reduce inflammation occurs, and by increasing the production of SCFAs by bacteria to down-regulate pro-inflammatory cytokines, inducing the differentiation of regulatory T-cells and inhibit the Th17 polarization, through the above mechanisms effectively and significantly reduce the growth of subcutaneous hepatocellular carcinoma in mice [19] .

Prebiotics are oligosaccharides that cannot be digested and absorbed by the body but can be used as a source of nutrients for the intestinal flora. Prebiotics can promote the growth of intestinal flora, improve intestinal dysbiosis, and play a key role in inducing anti-tumor effects [20] . Based on chemical structure, inulin-type fructans (ITFs), one of the prebiotics, are indigestible carbohydrates. In a study of BALB/c mice transplanted with BaF3 cells, ITF treatment reduced BaF3 cell infiltration and attenuated hepatic inflammation, while increasing propionate levels in the mice’s portal veins. Propionate can inhibit the growth of BaF3 cells in vitro via a cAMP-dependent pathway. In addition, activation of free fatty acid receptor 2 (FFA2) upon activation of Gi/Gq-protein-coupled receptors bound to jasmonates reduces the proliferation of BaF3 and other human cancer cell lines [21] . It was shown that the fermentation of nutrients such as ITF into propionate counteracted the proliferation of malignant cells in liver tissue. In cirrhotic patients, anti-tumor immunity was significantly enhanced in patients given oral lactulose. In addition, oral lactulose can regulate the imbalance between oxidative and antioxidant systems in patients with hepatocellular carcinoma cirrhosis with hypersplenism after interventional therapy, attenuate liver injury, and improve anti-tumor immunity and prognosis. Therefore, given the antitumor properties of prebiotics, it is suggested that the modulation of gut flora by prebiotics may serve as a preventive measure against the progression of hepatocellular carcinoma and the potential of using mixtures of prebiotics and the corresponding prebiotics to be manufactured into synbiotics to enhance the efficacy of the probiotics and to selectively stimulate the growth of beneficial intestinal microorganisms is a novel strategy for further research to reduce the risk of cancer.

The available evidence for the effectiveness of antibiotics in the treatment of hepatocellular carcinoma is limited to animal studies. The number and size of DMBA-HFD or DEN-CCL4-induced hepatocellular carcinoma tumors in mice were effectively reduced by oral administration of a mixture of antibiotics, including neomycin, ampicillin, vancomycin, and metronidazole [22] . In MYC transgenic mice with spontaneous hepatocellular carcinoma, the addition of an antibiotic mixture (ABX, consisting of vancomycin, neomycin, and imipenem) to the drinking water in order to deplete the intestinal commensal bacteria induced liver-selective anticancer effects, an increase in hepatic CXCR6 NKT cells, and an enhancement of interferon gamma synthesis, while the NKT cells mediated selective tumor suppression in the liver [23] . Despite the demonstrated antitumor efficacy of antibiotics in animal studies, it has been argued that the use of antibiotics in the early stages of treatment of malignancy is detrimental because antibiotics reduce the abundance of Bifidobacteria, Mucinophilic Acromobacteria, and Ruminalcoccus in the intestinal flora and increase the abundance of Mycobacterium phylum, and predispose to intestinal dysbiosis, which can affect the response to systemic therapy [24] . In addition, antibiotic therapy is not entirely appropriate for clinical management, as the potential harmful effects of long-term antibiotic use must be considered, including gastrointestinal dysfunction, immunocompromise, nephrotoxicity, and other antibiotic-related side effects. In addition, in hepatocellular carcinoma patients treated with immune checkpoint blockade (ICB), Fessas et al. [25] and Cheung et al. [26] reached opposite conclusions in their analysis of the correlation between antibiotics and patients’ survival, which may be due to the fact that the use of different antibiotic types increases or decreases the abundance and number of key strains of bacteria as well as the diversity of microbial functioning, causing alterations in the intestinal ecology, which, in turn, affects systemic or local metabolism, with significant impact on the immune system. Further exploration and development of low-risk antibiotics with clinical therapeutic potential for hepatocellular carcinoma is still needed.

FMT is the transplantation of intestinal flora from healthy people to the intestines of hepatocellular carcinoma patients, which can reduce the proliferation of hepatocellular carcinoma cells, improve liver function and alleviate the symptoms of hepatocellular carcinoma patients from promoting the repair of the intestinal mucosal barrier, altering the composition and function of the patient’s intestinal flora and improving the function of the immune system. A phase 1 randomized, placebo-controlled trial has now shown that by oral administration of FMT capsules in recurrent hepatic encephalopathy, an increase in the abundance of Ruminalococcaceae and Bifidobacteriaceae and a decrease in the abundance of Weillonella and the Aspergillus constituent, Sutterellaceae, in the microbiota after FMT is associated with an improvement in duodenal mucosal diversity and hepatic encephalopathy, and that oral administration of FMT capsules may be a safe and effective therapeutic option [27] . In addition, FMT has been shown to enhance the ability of immune checkpoint inhibitors to improve the efficacy of certain cancers, such as non-small cell lung cancer, colorectal cancer and melanoma [27] [28] . Enhancing the antitumor effect of ICB by FMT is therefore a potential strategy for hepatocellular carcinoma treatment. There is a lack of clinical research data on the treatment of hepatocellular carcinoma through FMT, and further studies are still needed. Although FMT as an emerging therapeutic approach shows great potential in the treatment of hepatocellular carcinoma, FMT may cause adverse reactions such as intestinal inflammation, infection, and allergic reactions, and further studies are needed to evaluate its safety and efficacy with the aim of providing better treatment options for hepatocellular carcinoma patients.

The importance of intestinal flora on the efficacy of ICB therapy has been well documented in preclinical models and patients. Intestinal flora modulate innate and adaptive immunity and influence anti-tumor immune responses in the tumor microenvironment [29] . It has been shown that some bacteria such as Ackermann’s slime mold, Bacteroidetes fragilis, Bifidobacterium spp. and E. faecalis spp. enhance the antitumor efficacy of ICB in animal models and cancer patients [30] . Zheng et al. found that among patients with sorafenib-treated refractory hepatocellular carcinoma, Aspergillus spp. predominated in fecal samples from treatment non-responders, whereas fecal samples from treatment responders were enriched in mucinophilic Acinetobacter spp. and Acomyclobacter spp. and the diversity of the intestinal flora in the body showed a close relationship with the favorable response to anti-PD-1 immunotherapy [31] . In a prospective study by Mao et al., fecal samples from patients with advanced unresectable hepatocellular carcinoma treated with PD-1 monotherapy after progression to first-line therapy (gemcitabine + cisplatin) were analyzed by macro-genomic sequencing, and differential enrichment of intestinal flora taxa was found between patients with and without clinical benefit, with energy metabolism-associated pathways being enriched in the intestinal flora of patients with clinical benefit, and those of patients with non-clinical benefit. Patients’ intestinal flora were enriched in amino acid metabolism-related pathways, with higher abundance enrichment of Trichoderma sp. GAM79 and Alistipes sp. Marseille-P147 being significantly associated with prolonged survival with immunotherapy, whereas the family of E. glomerulosum was associated with shorter PFS and OS, and the diversity of intestinal flora was found to correlate with irAE profiles [32] . These studies suggest that the intestinal microbiome modulates the response to cancer immunotherapy, and that interventions in the intestinal flora may be a novel therapeutic target and suggest that the intestinal flora of patients with hepatocellular carcinoma may be a potential biomarker for predicting a favorable response to ICI therapy and survival benefit.

In conclusion, current research suggests that gut flora therapy is potentially important for the treatment of hepatocellular carcinoma. However, further larger as well as robust clinical studies and explorations of the safety and limited availability of gut flora therapy are needed.