Lactobacillus helveticus HY7804 Modulates the Gut-Liver Axis to Improve Metabolic Dysfunction-Associated Steatotic Liver Disease in a Mouse Model

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common type of liver disease worldwide. In a previous study, we confirmed that Lactobacillus helveticus HY7804 (HY7804) improves MASLD by suppressing the expression of mRNAs encoding genes related to hepatic lipogenesis, inflammation, and fibrosis in model mice. Here, we evaluated the ability of HY7804 to restore intestinal barrier function and modulate the gut microbiota, as well as improve MASLD symptoms. Mice fed an MASLD-inducing diet for 7 weeks received HY7804 (10⁹ CFU/kg/day), the Type strain, or positive control (Pioglitazone) during the same period. HY7804 alleviated physiological (p < 0.001) and blood biochemical indicators and reduced MASLD activity scores (p < 0.05) on histological analysis. In addition, HY7804 increased the expression of genes related to fatty acid oxidation (p < 0.001); decreased the expression of apoptosis-related genes (p < 0.001); rescued the expression of tight junction (TJ)-related genes (p < 0.05); and suppressed the expression of pro-inflammatory cytokines and TLR4/MyD88/NF-κB signaling (p < 0.01) in the intestine. Finally, HY7804 modulated the composition of the gut microbiota in MASLD-induced mice. HY7804 increased the abundance of MASLD-suppressive Bacteroidaceae and Bacteroides, which positively correlated with the expression of TJ- and fatty acid oxidation-related genes. By contrast, HY7804 decreased the abundance of bacteria related to the progression of MASLD, including Cloastridaceae, Clostridium, Streptococcaceae, Lactococcus, and Lachnospiraceae, which correlated with intestinal immune responses and MASLD symptoms. In conclusion, L. helveticus HY7804 may be suitable as a functional supplement that alleviates MASLD symptoms and improves intestinal health.

Keywords: Lactobacillus helveticus; MASLD; gut microbiota; intestinal inflammation; tight junction.

Figure 1

Effects of HY7804 on MASLD symptoms in MASLD-induced mice (n = 7 per group): (A) changes in body weight, (B) FER, (C) mass of liver tissues, (D) mass of epididymal fat, (E) mass of inguinal fat, (F) liver/body weight ratio, (G) epididymal fat/body weight ratio, and (H) inguinal fat/body weight ratio. The data are expressed as the mean ± SD. ^### p < 0.001 vs. the control group, ** p < 0.01, and *** p < 0.001 vs. the MASLD group (one-way ANOVA with post hoc analysis). MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type strain, Lactobacillus helveticus type strain KCTC 3545 + MASLD; FER, food efficiency ratio.

Figure 2

Concentration of biochemical indicators in mouse serum (n = 7 per group). (A) AST, (B) ALT, (C) triglycerides, (D) total cholesterol, (E) fasting glucose, (F) LDL-cholesterol, and (G) HDL-cholesterol. The data are expressed as the mean ± SD. ^## p < 0.01 and ^### p < 0.001 vs. the control group, * p <  0.05, ** p <  0.01, and *** p < 0.001 vs. the MASLD group (one-way ANOVA with post hoc analysis). MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type strain, Lactobacillus helveticus type strain KCTC 3545 + MASLD; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDL-cholesterol, low-density lipoprotein cholesterol; HDL-cholesterol, high-density lipoprotein cholesterol.

Figure 3

Effects of HY7804 on liver histology (n = 7 per group). (A) Liver tissue morphology. (B) Representative photomicrographs of hepatic histology (magnification ×100). (C) Steatosis score. (D) NAS. Data are expressed as the mean ± SD. ^### p < 0.001 vs. the control group, * p < 0.05 vs. the MASLD group (one-way ANOVA with post hoc analysis). MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type strain, Lactobacillus helveticus type strain KCTC 3545 + MASLD.

Figure 4

Effects of HY7804 on mRNA expression in the liver of the MASLD-induced mice (n = 7 per group). The expression of mRNA encoding genes related to lipogenesis, i.e., (A) Fasn, (B) Srebf1, (C) C/ebpα and (D) Pparγ. The expression of mRNA encoding genes related to lipid oxidation, i.e., (E) Cpt1a and (F) Ppargc1a. (G) The expression ratio of Bax/Bcl-2. The data are expressed as the mean ± SD. ^# p < 0.05 and ^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the MASLD group (one-way ANOVA with post hoc analysis). MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type strain, Lactobacillus helveticus type strain KCTC 3545 + MASLD.

Figure 5

Effects of HY7804 on the expression of mRNAs in the colon of MASLD-induced mice (n = 7 per group). The expression of mRNA encoding tight junction proteins (A) Ocln and (B) Cldn1. The expression of mRNAs encoding pro-inflammatory cytokines and components of the TLR/MyD88/NF-Κb pathway. (C) Il6, (D) Il1β, (E) Tnfα, (F) Tlr4, (G) MyD88, and (H) Nfκb1. Data are expressed as the mean ± SD. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, and *** p  < 0.001 vs. the MASLD group (one-way ANOVA with post hoc analysis). MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type strain, Lactobacillus helveticus type strain KCTC 3545 + MASLD.

Figure 6

Composition of the gut microbiota in the MASLD-induced mice (n = 7 per group). (A) α-diversity. (B) PCoA of β-diversity using a 3D plot. (C) Relative abundance at the phylum level. (D) Relative abundance at the family level. (E) Relative abundance at genus level. Black dots indicate the values of each samples. Yellow diamonds indicate the mean. Black lines indicate the median. In the graph, red represents CON, yellow represents MASLD, green represents PIO, blue represents HY7804, and purple represents Type_helveticus group. CON, control group mice; MASLD, mice fed an MASLD-inducing diet; PIO, pioglitazone + MASLD; HY7804, Lactobacillus helveticus HY7804 + MASLD; Type_helveticus, Lactobacillus helveticus type strain KCTC 3545 + MASLD.

Figure 7

Correlation (Pearson’s correlation coefficient) between the intestinal microbial flora and biochemical indicators. Red indicates a positive Pearson r value and blue indicates a negative Pearson r value. The number indicates the r value. p-value < 0.05 was considered statistically significant. (* p <  0.05, ** p < 0.01, and *** p < 0.001).

Figure 8

Flow chart of the animal experiments.