Antibiotics Attenuate Methamphetamine-Induced Hepatotoxicity by Regulating Oxidative Stress and TLR4/MyD88/Traf6 Axis

Abstract

Methamphetamine (METH) is a major psychostimulant drug of abuse worldwide, and its neurotoxicity has been studied extensively. In addition to neurotoxicity, METH can also induce hepatotoxicity. The underlying mechanism of intestinal microorganisms in METH-induced hepatotoxicity remains unclear. In this study, mice have received antibiotics intragastrically or PBS once each day for 1 week, followed by METH or saline. The antibiotics attenuated METH-induced hepatotoxicity as evidenced by histopathological observation and biochemical analysis; furthermore, they alleviated METH-induced oxidative stress. The effect of antibiotics on METH-induced hepatotoxicity was investigated using RNA-sequencing (RNA-seq). The RNA-seq results demonstrated that antibiotics could regulate 580 differentially expressed genes (DEGs), of which 319 were upregulated after METH treatment and then downregulated with antibiotic pretreatment and 237 were first downregulated after METH administration and then upregulated after antibiotic pretreatment, in addition to 11 upregulated and 13 downregulated ones simultaneously in METH and antibiotic-pretreated groups. RNA-seq analyses revealed that TLR4 is one of the hub genes. Western blot analysis indicated that antibiotics inhibited the increase of TLR4, MyD88 and Traf6 induced by METH. This research suggests that antibiotics may play an important role in preventing METH-induced liver injury by regulating oxidative stress and TLR4/MyD88/Traf6 axis, though further investigation is required.

Keywords: RNA-seq; RT-qPCR; antibiotics; hepatotoxicity; methamphetamine.

FIGURE 1

Changes in body weight (A) and liver weight (B) of mice compared with the control group. Histopathological assessment of METH liver injury (C). The yellow arrows point to the nucleus and cytoplasm; * indicates the central vein; bar = 20 μm, 20×. METH significantly enhanced serum ALT (D) and AST (E) activities relative to the control group. The increasing level was attenuated in the A + METH (antibiotic + METH) group. *p < 0.05; **p < 0.01; ***p < 0.001; ns, statistically non-significant.

FIGURE 2

Analyses of ROS and SOD activities. The treatment with METH significantly increased ROS (A) and SOD (B) activities, which significantly repressed with antibiotic pretreatment. Antibiotics alone did not change compared to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ns, statistically non-significant.

FIGURE 3

(A) Principal correlation analysis. (B) Venn diagram of DEGs. (C) Volcano plot diagram: the first and third quadrants show the genes both up- and downregulated in both METH and antibiotic-pretreated groups, the second quadrant shows the genes downregulated in the METH group but upregulated in the AM group, and the fourth quadrant shows the genes upregulated in the METH group but downregulated in the AM group. (D) Heatmap diagram of DEG analysis among groups. The blue color represents downregulation, while red represents upregulation. C, control group; M, METH group; AM, antibiotic + METH group.

FIGURE 4

(A) GO analysis of DEGs. The black bars represent biological processes (bp), gray represents cellular components (cc), and white represents molecular functions (mf). (B) KEGG pathway enrichment analysis of DEGs.

FIGURE 5

PPI network (A) and hub genes (B) of all DEGs identified among the three groups. Red balls show the hub genes. PPI, protein–protein interaction.

FIGURE 6

Expression of hub genes in RNA-seq.

FIGURE 7

Consistency of RNA-seq and RT-qPCR results. RT-qPCR results of selected DEGs (Chrna4, Acaca, Nr1d2, and Csrnp1) were consistent with those of RNA-seq. RNA-seq: *p < 0.05, **p < 0.01, ***p < 0.001, significantly different compared to the control group; #p < 0.05, ##p < 0.01, ###p < 0.001, significantly different compared to the METH group. RT-qPCR: *p < 0.05, **p < 0.01, ***p < 0.001, ns, statistically non-significant.

FIGURE 8

(A) Western blots showing the expression of TLR4, MyD88, and Traf6 proteins in the mice liver. (B) The bar chart presents the statistical analysis of TLR4, MyD88, and Traf6 protein expressions. *p < 0.05; **p < 0.01; ***p < 0.001; ns, statistically non-significant.

FIGURE 9

A proposed mechanistic diagram of antibiotic protective action on METH-induced liver hepatotoxicity in this study.