In a recent study published in eBioMedicine, researchers investigated whether probiotic supplementation with Lactobacillus acidophilus could help prevent non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC) development using tumourigenesis mouse models.
Study: Lactobacillus acidophilus suppresses non-alcoholic fatty liver disease-associated hepatocellular carcinoma through producing valeric acid. Image Credit: FOTOGRIN/Shutterstock.com
Background
The global incidence of NAFLD-HCC is increasing, but no preventive measures against it are currently available.
Given increasing evidence of its close correlation with gut microbial dysbiosis, researchers have begun to pursue studies investigating the prophylactic potential of probiotics, such as L. acidophilus, against NAFLD-HCC.
In humans, the mutual interplay between the liver and intestine through the portal vein circulation gets dysregulated during hepatocarcinogenesis or HCC, which enriches pathobionts in NAFLD-HCC patients and depletes their commensal probiotics, including Lactobacillus and Bifidobacterium.
L. acidophilus has been widely commercialized as an oral supplement against several diseases, such as lactose intolerance and colitis, to name a few.
Previous studies with human subjects have even hinted at its feasibility against NAFLD and non-alcoholic steatohepatitis (NASH). However, its anti-tumourigenic effects against liver malignancies remain largely unknown.
About the study
In the present study, researchers used NAFLD-HCC mouse models to investigate the anti-tumourigenic effects of L. acidophilus.
They developed NAFLD-HCC tumorigenesis in both conventional and germ-free mice by diethylnitrosamine (DEN) injection and feeding a high-fat-high-cholesterol (HFHC) or choline-deficient high-fat (CDHF) diet.
After daily administration of L. acidophilus or phosphate buffer saline (PBS) control for 28 weeks, the team sacrificed all HFHC-fed NAFLD-HCC mice and observed whether they had macroscopic tumors in the liver.
Further, they performed fecal metagenomic sequencing to evaluate the impact of L. acidophilus on the gut microbiota. Germ-free mice, given 34 weeks of treatment, helped testify to the sole beneficial effect of L. acidophilus.
The team also developed an orthotopic NAFLD-HCC allograft model by intrahepatic injection of murine HCC cells in conventional mice fed with an HFHC diet that helped further validate the beneficial effect of L. acidophilus treatment.
The team investigated the biological functions of L. acidophilus conditional medium (L.a CM) and metabolites in NAFLD-HCC human cells and mouse organoids.
Finally, they performed metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) to identify the functional metabolite of L. acidophilus.
Results
In the tumourigenesis mouse model, i.e., mice with NAFLD-HCC, L. acidophilus was among the top depleted species, suggesting its possible protective, anti-tumorogenic effect against NAFLD-HCC.
Fecal metagenomic sequencing results showed no differences in α- and β-diversity between L. acidophilus-treated test and untreated control mice, indicating that L. acidophilus had minimal effect on the overall microbiota composition.
However, it elevated the abundance of commensal microbes while reducing possible pathobionts.
The histological assessment also identified a marked reduction of steatosis and inflammation in the non-tumorogenic liver tissues of L. acidophilus-treated mice.
At the same time, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), two liver damage markers, and cholesterol decreased in L. acidophilus-treated mice, but their body and liver weights compared with control remained similar.
Indeed, L. acidophilus administration markedly reduced tumor number, size, and load without affecting tumor incidence.
Like the tumourigenesis mouse model, L. acidophilus-treated mouse allografts had smaller liver tumors with reduced weight and volume.
Fluorescence in situ hybridization experiment revealed that L. acidophilus was expressed abundantly in the colon but not in the liver tissues of gavaged mice.
It suggested that L. acidophilus exerted its protective effects via secreted molecules.
Further investigation attributed the anti-tumorigenic effect of L. acidophilus to a non-protein small molecule with a molecular weight of <3 kDa.
Untargeted metabolomic profiling by LC-MS on L.a CM with <3 kDa revealed valeric acid, a short-chain fatty acid (SCFA), was the top enriched metabolite in L.a CM. Targeted metabolomics also confirmed the significant enrichment of valeric acid in L.a CM.
Valeric acid suppressed the viability of NAFLD-HCC cells but not normal hepatocytes, with 100 μM being the minimal significant concentration needed.
RNA sequencing revealed that L. acidophilus-derived valeric acid
bound G protein-coupled receptors (GPRs)41/43 on hepatocytes to inactivate the oncogenic Rho-GTPase signaling pathway, thereby ablating NAFLD-HCC development.
Conclusions
In the present study, researchers identified that probiotic supplementation with L. acidophilus is a potential prophylactic of NAFLD-HCC.
They demonstrated that L. acidophilus exhibited robust anti-tumourigenic effects in vitro and NAFLD-HCC mice, and the metabolite responsible for the protective function of L. acidophilus is valeric acid.
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