Combined intake of blueberry juice and probiotics ameliorate mitochondrial dysfunction by activating SIRT1 in alcoholic fatty liver disease

Abstract

Background: Mitochondrial dysfunction has been implicated as a significant factor in the liver disease process. Blueberry juice and probiotics (BP) synergistically improve liver function in alcoholic fatty liver disease (AFLD), although the mechanism for this effect was unclear. This study aims to investigate the effect and specific mechanisms of BP on AFLD.

Methods: C57/BL6 mice were randomly divided into seven groups: CG (control), MG (AFLD model), BJ (MG mice treated with blueberry), BJB (MG mice treated with BP), SI (AFLD mice treated with SIRT1 siRNA), BJSI (SI mice treated with blueberry), and BJBSI (SI mice treated with BP). The mice were fed an alcohol liquid diet for 10 days to establish the AFLD model, and subjected to BP and SIRT1 siRNA intervention for 10 days. Liver pathology was performed on day 11, and biochemical and molecular analyses of liver mitochondria were employed on day 12.

Results: BP significantly ameliorated hepatic mitochondrial injury, mitochondrial swelling, and hepatic necrosis in AFLD. BP alleviated hepatic mitochondrial dysfunction by increasing the expression of succinate dehydrogenase and cytochrome c oxidase, increasing respiratory control rate and the ADP/O ratio, and facilitating the synthesis of energy-related molecules. Besides, BP increased the expression of glutathione and superoxide dismutase, and inhibited malondialdehyde expression and reactive oxygen species activity. BP-induced sirtuin 1 (SIRT1), which activates peroxisome proliferator-activated receptor-gamma coactivator-1α, both of which mediate mitochondrial homeostasis. SIRT1 silencing suppressed the BP-induced changes in liver mitochondria, blunting its efficacy.

Conclusions: The ingredients of BP ameliorate hepatocyte mitochondrial dysfunction in AFLD mice.

Keywords: Alcoholic fatty liver disease; Blueberry; Mitochondrial dysfunction; Probiotics; SIRT1.

Fig. 1

SIRT1 deficiency abrogates the effect of BP in AFLD hepatocyte ultrastructure. A Hepatocyte ultrastructure in mice of different groups (Scale bar: 10 μm). The arrow indicates mitochondria; B Hepatic necrosis area and mitochondrial swelling. CG, control group; MG, AFLD model group; SI, AFLD mice treated with SIRT1 siRNA; BJ, AFLD mice treated with blueberry; BJSI, AFLD mice treated with blueberry and SIRT1 siRNA; BJB, AFLD mice treated with BP; BJBSI, AFLD mice with BP and SIRT1 siRNA. Different lowercase letters (a, b, c, and d) represent significant differences (*P < 0.05, Student’s t-test, n = 8)

Fig. 2

SIRT1 deficiency limits the effect of BP on mitochondrial function of AFLD mice. A Functional indexes of hepatic mitochondria were measured using a biochemical marker kit. B Hepatic mitochondrial respiratory function is expressed as state 4 and 3 respiration rates, RCR, and the ADP/O ratio. C Hepatic mitochondrial synthesis of ATP, ADP, AMP, and EC. Lowercase letters (a, b, c, and d) represent significant differences (*P < 0.05, Student’s t-test, n = 8)

Fig. 3

Knockdown of SIRT1 attenuated the decreasing mitochondrial oxidative stress in AFLD mice with BP treatment. A, B Biomarkers of oxidative stress in liver and hepatic mitochondria. C ROS was detected by immunofluorescence assay. Representative photographs of samples from each experimental group (Scale bar: 50 μm). Lowercase letters (a, b, c, and d) represent significant differences (*P < 0.05, Student’s t-test, n = 8)

Fig. 4

BP restored the decreased level of SIRT1 in AFLD mice. A Expression of SIRT1 mRNA in mouse livers was detected by RT-qPCR. Data are mean ± SD. B Immunohistochemical analysis was performed to evaluate the expression of SIRT1 in mouse livers of different groups (Scale bar: 80 μm). C SIRT1 expression was measured by western blotting. Data are the result of triplicate experiments. Lowercase letters (a, b, c, and d) represent significant differences (*P < 0.05, Student’s t-test, n = 8)

Fig. 5

BP increased PGC-1α expression via SIRT1. A RT-qPCR of PGC-1α mRNA in mouse liver. PGC-1α expression is expressed relative to GAPDH. B Immunohistochemical staining was used to evaluate SIRT1 expression (Scale bar: 80 μm). C Western blot analysis of hepatic PGC-1α. Date are shown as mean ± SD, n = 8. Lowercase letters (a, b, c, and d) represent significant differences (*P < 0.05, Student’s t-test, n = 8)