Alanyl-Glutamine Protects against Lipopolysaccharide-Induced Liver Injury in Mice via Alleviating Oxidative Stress, Inhibiting Inflammation, and Regulating Autophagy

Abstract

Acute liver injury is a worldwide problem with a high rate of morbidity and mortality, and effective pharmacological therapies are still urgently needed. Alanyl-glutamine (Ala-Gln), a dipeptide formed from L-alanine and L-glutamine, is known as a protective compound that is involved in various tissue injuries, but there are limited reports regarding the effects of Ala-Gln in acute liver injury. This present study aimed to investigate the protective effects of Ala-Gln in lipopolysaccharide (LPS)-induced acute liver injury in mice, with a focus on inflammatory responses and oxidative stress. The acute liver injury induced using LPS (50 μg/kg) and D-galactosamine (D-Gal) (400 mg/kg) stimulation in mice was significantly attenuated after Ala-Gln treatment (500 and 1500 mg/kg), as evidenced by reduced plasma alanine transaminase (ALT) (p < 0.01, p < 0.001), aspartate transaminase (AST) (p < 0.05, p < 0.001), and lactate dehydrogenase (LDH) (p < 0.01, p < 0.001) levels, and accompanied by improved histopathological changes. In addition, LPS/D-Gal-induced hepatic apoptosis was also alleviated by Ala-Gln administration, as shown by a greatly decreased ratio of TUNEL-positive hepatocytes, from approximately 10% to 2%, and markedly reduced protein levels of cleaved caspase-3 (p < 0.05, p < 0.001) in liver. Moreover, we found that LPS/D-Gal-triggered oxidative stress was suppressed after Ala-Gln treatment, the effect of which might be dependent on the elevation of SOD and GPX activities, and on GSH levels in liver. Interestingly, we observed that Ala-Gln clearly inhibited LPS/D-Gal exposure-induced macrophage accumulation and the production of proinflammatory factors in the liver. Furthermore, Ala-Gln greatly regulated autophagy in the liver in LPS/D-Gal-treated mice. Using RAW264.7 cells, we confirmed the anti-inflammatory role of Ala-Gln-targeting macrophages.

Keywords: acute liver injury; alanyl-glutamine; apoptosis; inflammation; oxidative stress.

Figure 1

Effects of Ala-Gln on LPS/D-Gal-induced hepatic pathological and biochemical parameters in mice. (A) Liver tissues were stained with HE for histopathological analysis (original magnifications, ×400), and the degree of liver injury was scored. (B–D) plasma ALT, AST, and LDH levels. Data A is presented as medians with interquartile range. Data B–D are expressed as mean ± SD; n = 6–8 in each group. *** p < 0.001 vs. vehicle-treated control group, ## p < 0.01, ### p < 0.001 vs. LPS/D-Gal-treated model group.

Figure 2

Ala-Gln suppressed apoptosis in LPS/D-Gal-treated mice. (A) TUNEL staining (original magnification, ×400) of liver tissues. (B) Quantitation of TUNEL-positive cells in liver. (C,D) Protein expression levels of cleaved-caspase-3 in liver. Data are expressed as mean ± SD, n = 6–8 in each group. *** p < 0.001 vs. vehicle-treated control group, # p < 0.05, ### p < 0.001 vs. LPS/D-Gal-treated model group.

Figure 3

Antioxidant effects of Ala-Gln in liver tissues from mice that received LPS/D-Gal injection. (A) ROS detection via DHE staining (original magnification, ×200) in liver tissues. (B) Hepatic MDA levels. (C) Hepatic SOD activities. (D) Hepatic GPX activities. (E) Hepatic GSH levels. Data are expressed as mean ± SD, n = 6–8 in each group. * p < 0.05, ** p < 0.01 vs. vehicle-treated control group, # p < 0.05, ## p < 0.01 vs. LPS/D-Gal-treated model group.

Figure 4

IHC analysis results showing the levels of F4/80 and CD68 in liver tissues in mice. (A) IHC staining of F4/80 of liver tissues (original magnifications, ×400), and quantitation of F4/80 positive area. (B) IHC staining of CD68 of liver tissues (original magnifications, ×400), and quantitation of CD68 positive area. Data are expressed as mean ± SD, n = 6–8 in each group. *** p < 0.001 vs. vehicle-treated control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS/D-Gal-treated model group.

Figure 5

Impact of Ala-Gln on LPS/D-Gal-induced inflammatory responses in liver and plasma. (A–E) mRNA expression levels of TNF-α, IL-1β, IL-6, RANTES, and MCP-1 in liver tissues. (F–J) Protein levels of TNF-α, IL-1β, IL-6, RANTES, and MCP-1 in plasma. Data are expressed as mean ± SD, n = 6–8 in each group. ** p < 0.01, *** p < 0.001 vs. vehicle-treated control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS/D-Gal-treated model group.

Figure 6

Effect of Ala-Gln on autophagy in liver in LPS/D-Gal-treated mice. (A) Protein expression levels of p62, Beclin-1, ATG-7, and LC3B in liver were assessed using Western blot. (B–E) Quantitation of p62, Beclin-1, ATG-7, and LC3B-II/I expression. Data are expressed as mean ± SD, n = 6–8 in each group. ** p < 0.01, *** p < 0.001 vs. vehicle-treated control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS/D-Gal-treated model group.

Figure 7

Impact of Ala-Gln on LPS-induced inflammatory responses in RAW264.7 cells. (A–C) mRNA expression levels of TNF-α, IL-6, and RANTES in RAW264.7 cells. Data are expressed as mean ± SD. *** p < 0.001 vs. vehicle-treated control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. LPS-treated model group.

Figure 8

Effect of Ala-Gln on H₂O₂-induced oxidative stress in AML-12 cells. Cell viability of AML-12 cells that had received H₂O₂ stimulation (0.3 mM) and Ala-Glu treatment (10 mM). Data are expressed as mean ± SD. *** p < 0.001 vs. vehicle-treated control group, ### p < 0.001 vs. H₂O₂-treated model group.