Gegen-Sangshen oral liquid and its active fractions mitigate alcoholic liver disease in mice through repairing intestinal epithelial injury and regulating gut microbiota

Abstract

Background: Liuweizhiji Gegen-Sangshen oral liquid (LGS), as a Chinese medicinal preparation, is developed from a Traditional Chinese medicinal formula consisting of six Chinese medicinal herbs, including Puerariae lobatae radix, Hoveniae semen, Imperatae rhizoma, Crataegi fructus, Mori fructus and Canarli fructus, and has been extensively utilized in the prevention and treatment of alcoholic liver disease (ALD) clinically. Previous study has demonstrated that LGS dose-dependently mitigated ALD in rat models. However, whether and how the main characteristic constituents of LGS (the flavonoid and polysaccharide fractions, LGSF and LGSP) contribute to the anti-ALD effect remains unclear. This study aimed to assess the anti-ALD effect of LGS and its main fractions (LGSF and LGSP) in a murine model of ALD and to explore the underlying mechanisms.

Methods: ALD mouse model was constructed using the chronic and binge ethanol feeding method. Biochemical determinations of AST, ALT, TC, TG, ADH, ALDH, HDL, LDL, IL-1β, IL-6, and TNF-α were performed using corresponding kits. Histopathological examination of liver and intestinal sections was conducted based on the H&E staining. Lipid accumulation in hepatocytes was evaluated by oil red O staining. Ethanol metabolism was assessed by determining the activity of ADH and ALDH enzymes. Intestinal barrier function was analyzed based on immunohistochemistry analysis of ZO-1 and occludin and immunofluorescence analysis of epithelial markers, Lgr5, Muc2, and Lyz1. Intestinal epithelial apoptosis was detected by TUNEL staining. Mouse fecal microbiota alterations were analyzed by 16S rRNA sequencing. An in vitro epithelial injury model was established by developing TNF-α-induced 3D-cultured intestinal organoids. In vitro culture of specific bacterial strains was performed.

Results: The results showed that LGS and its flavonoid and polysaccharide fractions (LGSF and LGSP) significantly alleviated ALD in mice through attenuating hepatic injury and inflammation, improving liver steatosis and promoting ethanol metabolism. Notably, LGS, LGSP, and LGSF mitigated intestinal damage and maintained barrier function in ALD mice. The intestinal barrier protection function of LGS, LGSP, and LGSF was generally more obvious than that of the positive drug meltadosine. Further study demonstrated that LGS, LGSP, and LGSF promoted intestinal epithelial repair via promoting Lgr5⁺ stem cell mediated regeneration in TNF-α-induced intestinal organoids. LGS and LGSF, other than LGSP, had a better effect on repair of epithelial injury in vitro. Moreover, LGS, LGSP, and LGSF remarkably alleviated gut dysbiosis in ALD mice via at least partially recovery of alcohol-induced microbial changes and induction of specific bacterial groups. In vitro culture of bacterial strains indicated that LGS, LGSP, and LGSF had a specific impact on bacterial growth. LGS and LGSP, but not the LGSF, significantly promoted the growth of Lactobacillus. Similarly, LGS and LGSP significantly increased the proliferation of Bacteroides sartorii, and LGSF had a minimal effect. LGS, LGSP and LGSF all promoted the growth of Bacillus coagulans, Bifidobacterium adolescentis, and Bifidobacterium bifidum. LGS and LGSP promoted the growth of Dubosiella newyorkensis, but the LGSF had no effect.

Conclusions: LGS exerts its anti-ALD effect in mice through regulating gut-liver axis, and its flavonoid and polysaccharide fractions, LGSF and LGSP, are responsible for its protective effect.

Keywords: Alcoholic liver disease; Gegen-Sangshen oral liquid; Gut microbiota; Gut-liver axis; Intestinal organoid.

Fig. 1

LGS and its active fractions (LGSP and LGSF) attenuated alcohol-induced hepatic injury. A Experimental design. B Food intake curve of mice. C Body weight change curve of mice. D Representative morphological images of the liver. E Liver index. F H&E-stained sections of mouse liver. G Pathologic scoring of H&E sections of the liver. H Oil red O staining of liver sections. I Positive area for oil red O staining. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001, vs. ALD group; * P < 0.05, ** P < 0.01 and *** P < 0.001, vs. control group; one-way ANOVA with a post hoc Tukey test

Fig. 2

LGS and its active fractions (LGSP and LGSF) alleviated hyperlipidemia and inflammation, and improved ethanol metabolism in ALD mice. A Serum level of AST; B serum level of ALT; C serum level of TG; D serum level of TC; E serum level of HDL; F serum level of LDL; G serum level of IL-6; H serum level of TNF-α; I serum level of IL-1β; J enzymatic activity of ALDH; K enzymatic activity of ADH; L serum level of GLP-1. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. ALD group; * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. control group; one-way ANOVA with a post hoc Tukey test

Fig. 3

LGS and its active fractions (LGSP and LGSF) mitigated intestinal injury, increased the expression of tight conjunction proteins, and promoted intestinal epithelial proliferation in ALD mice. A H&E staining of ileum. B TUNEL-stained ileum sections. Arrows indicate apoptotic cells. C The expression of occludin protein in ileum. D The expression of ZO-1 protein in ileum. E Immunofluorescence staining of Lyz1⁺ cells in ileum; red, Lyz1 positive staining; blue, DAPI staining. F Immunofluorescence staining of Muc2⁺ cells in ileum; red, Muc2 positive staining; blue, DAPI staining. Quantitative analysis of histopathological scores, TUNEL-positive cells, occludin or ZO-1 positively stained area, and number of Lyz1⁺ and Muc2⁺ cells are displayed in the supplementary file (Figs. S3, S7)

Fig. 4

LGS and its active fractions (LGSP and LGSF) protected against TNF-α-induced intestinal organoid injury in vitro through promoting epithelial cell differentiation and regeneration. A Intestinal organoid image under light microscopy. Scale bar = 100 μm. B Number of total organoids. C Number of injured organoids. D H&E-stained organoids. Arrow shows bud loss or reduced surface area. Scale bar = 50 μm. E EdU-stained organoids; scale bar = 20 μm. F TUNEL stained organoid sections; scale bar = 50 μm. G Lgr5 staining; red, Lgr5 positive staining; blue, DAPI staining; scale bar = 50 μm. H Lyz1 staining; red, Lyz1 positive staining; blue, DAPI staining; scale bar = 50 μm. I Muc2 staining; red, Muc2 positive staining; blue, DAPI staining; scale bar = 50 μm. Quantitative analysis of EdU⁺ and TUNEL⁺ immunofluorescence, and the number of Lgr5⁺, Lyz1⁺ and Muc2⁺ cells are displayed in the supplementary file (Fig. S8). ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. ALD group. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. control group; one-way ANOVA with a post hoc Tukey test

Fig. 5

LGS and its active fractions (LGSP and LGSF) altered gut microbial structure in ALD mice. A Shannon index. B Chao1 index. C PCoA analysis based on ASV level. D Percent of community abundance on phylum level. E Abundance of Actinobacteriota. F Abundance of Firmicutes. G Abundance of Bacteroidota. H Abundance of Verrucomicrobiota. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. ALD group; * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. control group; one-way ANOVA with a post hoc Tukey test

Fig. 6

LGS and its active fractions (LGSP and LGSF) mediated specific microbial changes in ALD mice. A LEfSe analysis of the microbial community in each group. The significantly altered microbial genera included the following: B Coriobacteriaceae-UCG-002; C Parasutterella; D Romboutsia; E Escherichia-shigella; F Akkermansia; G Faecalibaculum; H Lactobacillus; I Bacteroides; J Enterorhabdus; K Monoglobus; L Bacillus; M Bifidobacterium. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. ALD group; * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. control group; one-way ANOVA with a post hoc Tukey test

Fig. 7

LGS and its active fractions (LGSP and LGSF) promoted the in vitro growth of specific bacterial strains. A Growth curve of Lactobacillus. B Growth curve of Bacteroides sartorii. C Growth curve of Bacillus coagulans. D Growth curve of Bifidobacterium adolescentis. E Growth curve of Bifidobacterium bifidum. F Growth curve of Dubosiella newyorkensis. Glucose (Glu) was used as positive control throughout the experiments. *** P < 0.001 vs. medium group; one-way ANOVA with a post hoc Tukey test

Fig. 8

Schematic illustration of the protective effect of LGS and its active fractions (LGSP and LGSF) on ALD through modulating the gut-liver axis