Probiotics and their beneficial effects on alcohol-induced liver injury in a rat model: the role of fecal microbiota

Abstract

Background: Current therapies for alcohol-induced liver injury are of limited efficacy and associated with significant side effects. With the proposed pathophysiology of alcohol-induced liver injury to be related to deranged gut microbiota, we hypothesized that probiotics would have beneficial effects in attenuating alcohol-induced liver injury.

Methods: Twenty-four male Sprague-Dawley rats were divided into 4 groups: control group, alcohol group, Lactobacillus plantarum group, and mixed-strain probiotics group. After 4 weeks, all rats were sacrificed, and blood samples were analyzed for ALT, lipopolysaccharide level (LPS), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Liver tissues were processed for histopathology, malondialdehyde (MDA) level and immunohistochemistry for toll-like receptors 4 (TLR-4). Stool samples were collected, and 16S rRNA sequencing was used to analyze the fecal microbiota.

Results: Liver histopathology showed the presence of significant hepatocyte ballooning in the alcohol group as compared with the control group, and the treatment with L. plantarum or mixed-strain probiotics alleviated these changes. Significant elevation of serum ALT, LPS, IL-6, and TNF-α, hepatic MDA levels, and hepatic TLR-4 expression were observed in alcohol-fed rats as compared with control rats. The administration of L. plantarum or mixed-strain probiotics restored these changes to the levels of control rats. The relative abundance of fecal bacteria at genus level showed a significant reduction in Allobaculum, Romboutsia, Bifidobacterium, and Akkermansia in the alcohol group as compared with the control group. In probiotics-treated rats, significant increases in Allobaculum and Bifidobacterium were observed, while the relative abundance of Romboutsia and Akkermansia was unchanged compared to the alcohol group. A reduction in alpha diversity was observed in alcohol-treated rats, whereas the improvement was noted after probiotic treatment.

Conclusions: The treatment with Lactobacillus, whether as single-, or mixed-strain probiotics, was beneficial in reducing the severity of alcohol-induced liver injury likely through the increase in beneficial bacteria, and the reduction of inflammatory responses, and oxidative stress.

Keywords: Alcohol-induced liver injury; Microbiota; Probiotics; Rat model.

Fig. 1

Liver histopathology in all groups. These are images of liver histopathology using H&E straining at 400x magnification (A, Control group; B, alcohol group; C, L. plantarum group; D, Mixed strain group). Black arrows indicate fat deposition inside hepatocytes and red arrows indicate hepatocyte ballooning

Fig. 2

Immunohistochemical study and percent positivity for TLR-4 in each group. A. Images of immunohistochemical study for TLR-4. These are images of immunohistochemical study for TLR-4 at 200x magnification (A, Control group; B, alcohol group; C, L. plantarum group; D, Mixed strain group). Red arrows indicate TLR-4 positive cells. B. Bar graph representing percent positivity of TLR-4 in each group. a = p-value< 0.05, ns = non-significant

Fig. 3

Bar graphs representing liver enzyme, inflammatory marker and hepatic MDA levels in all groups. A. Serum TNF-α levels in all groups. B. Serum IL-6 levels in all groups. C. Hepatic MDA levels in all groups. a = p-value < 0.05, b = p-value < 0.001, ns = non-significant

Fig. 4

Alpha-diversity (A) and Beta-diversity (B) of gut microbiota in all groups