Gut microbiota contribution to hepatocellular carcinoma manifestation in non-alcoholic steatohepatitis

Table 1 Clinical studies investigating gut microbiota composition in patients with nonalcoholic fatty liver disease - induced hepatocellular carcinoma

Ref.	Participants (groups)	Exclusion criteria	Main findings	Other metabolites investigated
Behary et al[19]	Patients with NAFLD-HCC-cirrhosis n = 32; Patients with NAFLD-cirrhosis n = 28; Control group (non-NAFLD) n = 30.	Unspecified	Subjects with NAFLD-HCC and NAFLD-cirrhosis had reduced α-diversity indices compared to non-NAFLD controls; NAFLD-HCC was characterized by expansion of Proteobacteria compared to a non-NAFLD group; Expansion of Enterobacteriaceae in NAFLD-HCC compared to NAFLD-cirrhosis and controls; NAFLD-HCC was characterized by a reduction in Oscillospiraceae and Erysipelotrichaceae compared to non-NAFLD; NAFLD-cirrhosis was characterized by an expansion of Eubacteriaceae compared to both NAFLD-HCC and controls; Bacteroides caecimuris and Veillonella parvula, were both significantly enriched in NAFLD-HCC, compared to NAFLD cirrhosis and controls	Pyruvate carboxylase (pycA), responsible for the production of oxaloacetate from pyruvate, was overexpressed in NAFLD-HCC compared to NAFLD-cirrhosis and non-NAFLD control; Genes related to acetate synthesis (phosphate acetyltransferase) and butyrate/acetyl phosphate synthesis (phosphate butyryltransferase) were both overexpressed in NAFLD-HCC compared to NAFLD cirrhosis and non-NAFLD controls; The feces of NAFLD-HCC subjects were enriched in acetate, butyrate and formate compared to NAFLD-cirrhosis and controls; Fecal SCFA was NAFLD-HCC specific
Sydor et al[13]	Patients with NASH-non-HCC without cirrhosis n = 23; Patients with NASH-non-HCC with cirrhosis n = 11; Patients with NASH-HCC without cirrhosis n = 14; Patients with NASH-HCC with cirrhosis n = 19; Control group n = 20.	Unspecified	Bacteroidetes and, to a lesser extent, Actinobacteria were gradually decreased in abundance from controls to NASH-non-HCC to NASH-HCC; The abundance of Proteobacteria was significantly increased in NASH-HCC with cirrhosis; The abundances of Bacteroides and Bifidobacterium were decreased in NASH-non-HCC and NASH-HCC compared with controls; Lactobacillus showed a progressive increase in abundance from controls to NASH-HCC with cirrhosis; Abundance of Clostridium and Escherichia/Shigella remained unchanged; Lactobacillus-related ranks showed a progressive increase in abundance from controls to NASH-HCC with cirrhosis	Significant increase of BA associated with disease severity between healthy, NASH-non- HCC, and NASH-HCC; Individual and conjugated serum BA were associated with the abundance of Lactobacillus
Ponziani et al[20]	Patients with NAFLD-HCC with cirrhosis n = 21; Patients with NAFLD-non-HCC with cirrhosis n = 20; Control group n = 20.	Patients with CVH, AH, cholestatic disorders such as PBC or PSC, and inherited liver disorders leading to cirrhosis such as hemochromatosis, Wilson's disease, and alpha-1 antitrypsin deficiency; Patients who were taking drugs such as antibiotics, probiotics, prebiotics, PPIs, and laxatives during the last 6 mo; affected by diseases potentially influencing the gut microbiota composition; Patients with a history of cancer.	α-diversity was less diverse in patients with cirrhosis compared to controls; Cirrhosis patients showed enriched Proteobacteria, Bacteroidetes and Cyanobacteria compared to healthy controls; The gut microbiota of the HCC group was enriched with Bacteroides, Ruminococcaceae, Enterococcus, Phascolarctobacterium, and Oscillospira compared to patients with cirrhosis but without HCC and controls; Reduced abundance of Verrucomicrobiaceae, Bifidobacteriaceae, Akkermansia, Bifidobacterium, Dialister, Collinsella, and Adlercreutzia were seen in NAFLD-HCC compared with NAFLD-non-HCC.	Intestinal permeability was increased in all patients with liver cirrhosis, who had higher levels of plasma ZO1 and LPS compared to controls

AH: Autoimmune hepatitis; BA: Bile acids; CVH: Chronic viral hepatitis; HCC: Hepatocellular carcinoma; LPS: Lipopolysaccharides; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; PBC: Primary biliary cholangitis; PPI: Proton pump inhibitors; PSC: Primary sclerosing cholangitis; SCFA: Short-chain fatty acid.

Table 2 Animal models investigating gut microbiota composition in nonalcoholic fatty liver disease induced hepatocellular carcinoma

Ref.	Experimental animal	Participants (groups)	Main findings
Xie et al[12]	Mice	Mice with STZ-HFD induced NASH-HCC; Control group	STZ-HFD group exhibited lower α-diversity than controls; The most abundant species in both control group and STZ-HFD group were primarily from the Bacteroides genus; The most decreased in abundance in the STZ-HFD group were Parasutterella spp., Bacteroides acidofaciens, Odoribacter spp., Barnesiella spp., Moryella spp., Paraprevotella spp., Lactobacillus intestinalis, and Akkermansia spp; Atopobium spp., Bacteroides acidifaciens, Bacteroides spp., Bacteroides uniformis, Bacteroides vulgatus, Clostridium cocleatum, Clostridium xylanolyticum, and Desulfovibrio spp. were significantly positively correlated with LPS in plasma, liver and feces; As most Bacteroides and Desulfovibrio were LPS-producers, LPS concentration was significantly increased in the STZ-HFD group.
Carter et al[21]	Mice	Western diet only (high fat and fructose diet, no CCl4 injection); CCl4 only (CCl4 injection intraperitoneal once a week and normal diet); NASH-HCC (Western diet and CCl4 injection intraperitoneally once a week); Control group (normal diet, no CCl4 injection);	NASH mice display impaired intestinal barrier function, leading to increased leakage of bacterial byproducts such as LPS into the circulation; NASH mice had reduced alpha diversity; Expansion of Erysipelotrichales was only observed in NASH mice
Zhang et al[22]	Mice	HFHC-fed mice (NAFLD-HCC group); HFHC-fed mice; Normal diet-fed mice (control group).	The microbiota composition changed during NAFLD-HCC formation: Mucispirillum, Desulfovibrio, Anaerotuncus were sequentially increased; Gut bacterial metabolites alteration like TCA and IPA were increased in NAFLD-HCC mice; Lower bacterial diversity and increased bacterial richness were observed in HFHC-fed mice with HCC than HFLC diet-fed mice with only steatosis; LPS concentration was elevated in HFHC-fed mice compared to HFLC-fed mice.

HCC: Hepatocellular carcinoma; HFHC: High-fat/high-cholesterol; HFLC: High-fat/low-cholesterol; IPA: Indole-3-propionic acid; LPS: Lipopolysaccharides; NAFLD: Non-alcoholic fatty liver disease; STZ-HFD: Streptozocin-high-fat diet; TCA: Trichloroacetic acid.