The oral administration of Lacticaseibacillus casei Shirota alleviates acetaminophen-induced liver injury through accelerated acetaminophen metabolism via the liver-gut axis in mice

Abstract

Acetaminophen is a widely used antipyretic and analgesic drug, and its overdose is the leading cause of drug-induced acute liver failure. This study aimed to investigate the effect and mechanism of Lacticaseibacillus casei Shirota (LcS), an extensively used and highly studied probiotic, on acetaminophen-induced acute liver injury. C57BL/6 mice were gavaged with LcS suspension or saline once daily for 7 days before acute liver injury was induced via intraperitoneal injection of 300 mg/kg acetaminophen. The results showed that LcS significantly decreased acetaminophen-induced liver and ileum injury, as demonstrated by reductions in the increases in aspartate aminotransferase, total bile acids, total bilirubin, indirect bilirubin, and hepatic cell necrosis. Moreover, LcS alleviated acetaminophen-induced intestinal mucosal permeability, decreased serum IL-1α and lipopolysaccharide levels, and elevated serum eosinophil chemokine (eotaxin) and hepatic glutathione levels. Furthermore, analysis of the gut microbiota and metabolome showed that LcS reduced the acetaminophen-enriched levels of Cyanobacteria, Oxyphotobacteria, long-chain fatty acids, cholesterol, and sugars in the gut. Additionally, the transcriptomic and proteomic results showed that LcS mitigated the decrease in metabolic and immune pathways as well as glutathione formation during acetaminophen-induced acute liver injury. This is the first study showing that pretreatment with LcS alleviates acetaminophen-enriched acute liver injury, and it provides a reference for the application of LcS.IMPORTANCEAcetaminophen is the most frequently used antipyretic analgesic worldwide. As a result, overdoses easily occur and lead to drug-induced acute liver injury, which quickly progresses to liver failure with a mortality of 60%-80% if not corrected in time. The current emergency treatment for overused acetaminophen needs to be administered within 8 hours to avoid liver injury or even liver failure. Therefore, developing preventive strategies for liver injury during planned acetaminophen medication is particularly important, preferably nonpharmacological methods. Lacticaseibacillus casei Shirota (LcS) is a famous probiotic that has been used for many years. Our study found that LcS significantly alleviated acetaminophen-induced acute liver injury, especially acetaminophen-induced liver injury toward fulminant hepatic failure. Here, we elucidated the function and potential mechanisms of LcS in alleviating acetaminophen-induced acute liver injury, hoping it will provide preventive strategies to people during acetaminophen treatment.

Keywords: Lacticaseibacillus casei Shirota; acetaminophen; glutathione; liver injury; liver-gut axis.

Fig 1

LcS alleviates APAP-induced ALI and gut injury. (A) Animal experimental procedure of the LcS pretreatment assay. (B) Body weight of mice during the experiment. LcS decreased the APAP-induced (C) increases in liver function indicators, including AST, TBA, TBil, and IBil. (D) Elevations in the serum cytokine eotaxin and decline in IL-1α. (E) Increases in the hepatic histological activity index (HAI) score. (F) Increases in the histological scores of the ileum and serum lipopolysaccharide (LPS). (G) Representative images of hepatic hematoxylin-eosin (HE) staining; the scale bars represent 200 µm and 20 µm at 5× and 40× magnification, respectively. (H) Representative images of ileal HE staining; the scale bar represents 100 µm. The data are shown as the means ± SEMs; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 2

LcS alleviates APAP-induced gut microbiota dysbiosis. (A) Differences in α-diversity: observed species, Chao1 and Shannon indices. The values are expressed as the medians with interquartile ranges. (B) Differences in β-diversity determined by PCoA and NMDS. (C) LcS alleviated some APAP-induced alterations in the gut microbiota. The data are shown as the means ± SEMs; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3

LcS alleviates APAP-induced gut metabolome dysbiosis. (A) OPLS-DA score plots of metabolome profiles. (B) Top 10 metabolites contributing to the separation of the APAP group from the Ctrl group based on VIP values. (C) Top 10 metabolites contributing to the separation of the LcS + APAP group from the APAP group based on VIP values. (D) LcS alleviated some APAP-induced alterations in gut metabolites. The data are shown as the means ± SEMs; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4

LcS alleviates APAP-induced changes in the liver transcriptome and proteome. LcS alleviates the APAP-induced alterations in gene transcription (A) and protein expression (B) in the liver. All these data were normalized to those of the Ctrl group. LcS alleviates APAP-induced pathway alterations in the transcriptome (C) and proteome (D) of the liver.

Fig 5

LcS alleviates the APAP-induced alterations in gene transcription (A) and pathways (B) in the terminal ileum. The gene transcription data were normalized to those of the Ctrl group.

Fig 6

LcS improves the APAP-induced downregulation of the glutathione pathway. (A) LcS improves the APAP-induced downregulation of the transcription of genes in the glutathione pathway, as demonstrated by RT-qPCR. (B) LcS alleviated the APAP-induced downregulation of the expression of proteins in the glutathione pathway, as exhibited by proteomics. (C) LcS increased the APAP-induced decrease in glutathione in the liver, as shown by ELISA. (D) General view of the role of LcS in alleviating the APAP-induced downregulation of glutathione-related genes. The data are shown as the means ± SEMs; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 7

Spearman’s rank correlations between the APAP-induced LcS-alleviated indicators. (A) The correlations among the APAP-induced LcS-alleviated gut microbes, metabolites with liver function indicators, inflammatory cytokines, and serum LPS. The correlations among the APAP-induced LcS-alleviated liver function indicators, inflammatory cytokines, serum LPS gut microbes, metabolites with altered proteins (B), and indicators in the glutathione metabolism (C) of the liver. The results with P values < 0.05 are shown in the heatmap. The color key and circle size indicate the strength of the correlation (r value). The red color indicates positive correlations; the blue color indicates negative correlations.

Fig 8

General view of the beneficial effects of LcS on ALI.