Abstract
Scutellarein (SCU) is an herbal flavonoid, showing hepatoprotective potentials. The study was aimed to investigate whether the hepatoprotective effect of SCU is dependent on the integrity of gut microbiota. Mice received repeated intraperitoneal injections of CCl4, followed with or without SCU treatment (15, 30, and 60 mg/kg). Gut microbial community of mice was disrupted by administrating a cocktail of antibiotics (ampicillin, neomycin sulfate, metronidazole, and vancomycin) in drinking water. The results showed SCU plus antibiotics aggravated CCl4-induced chronic liver injury, as demonstrated by liver function analysis, histological analysis, and TUNEL assay. SCU activated CYP2E1 expression and worsened CYP2E1-mediated lipid peroxidation and oxidative stress as coadministered with antibiotics. Moreover, when gut microbiota was disrupted by antibiotics, SCU activated IκBα/NF-κB pathway and promoted the release of subsequent proinflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Remarkably, the 16 S rRNA sequencing demonstrated that SCU greatly decreased the relative abundance of Bifidobacterium and Lactobacillus and increased the relative abundance of Enterococcus in gut microbiota-dysbiosis mice. Spearman correlation analysis showed that Lactobacillus was positively correlated with SOD and negatively correlated with AST. Collectively, the hepatoprotective effect of SCU is reversed under antibiotics intervention, which may partly involve the activation of CYP2E1 and IκBα/NF-κB pathway and diminishment of Lactobacillus.
Highlights
DNA was measured by NanoDrop 2000 ultraviolet spectrophotometer. e bacterial 16 S rRNA gene V3–V4 region was amplified by PCR using the forward primer (338F: 5′ACTCCTACGGGAGGCAGCA-3′) and the reverse primer (806R: 5′-GGACTACHVGGGTWTCTAAT -3′). e gut microbiota composition was analyzed on an IlluminaMiSeq platform. e alpha diversity including Chao1 and Shannon index was calculated using OUT in QIIME (Denver, USA)
Effects of SCU plus Antibiotics on Hepatic Injury and Apoptosis. e effects of SCU in gut microbiota-dysbiosis mice were first assessed by serum biochemical parameters (ALT, AST, TBIL, and ALB) and 60 mg/kg SCU without antibiotics intervention (MH group) acts as a positive control
Histological observation and TUNEL assay showed that SCU plus antibiotics treatment had higher histological score and hepatocyte apoptosis areas (Figures 1(e) and 1(f )), which was consistent with the results of serum parameters
Summary
Mice were euthanized and blood samples, stools, and liver tissues were collected 12 h after the final injection of CCl4. Embedded tissues were cut into 4 μm thick sections and stained with hematoxylin and eosin (H&E) for the histological analyses. Hepatocyte apoptosis in the liver tissue was detected by DeadEndTM Fluorometric TUNEL System according to the manufacture’s protocol (Promega, USA). Immunohistochemistry for CYP2E1 was performed with the paraffin-embeded liver sections. Total RNA of 100 mg snap-frozen liver samples was extracted by Trizol reagent ( ermo Fisher Scientific, NY, USA). Total RNA was reverse-transcribed into complementary DNA using RevertAid First Strand cDNA Synthesis Kit according to the manufacture’s instructions ( ermo Fisher Scientific, NY, USA). Total protein of 100 mg snap-frozen liver tissues was extracted by RIPA lysis buffer and protein concentration was determined by BCA assay kit (Solarbio, Beijing, China) based on the manufacturer’s protocols. Fresh feces samples were collected and the total DNA was isolated using QIAamp DNA Stool Kit (Qiagen, Valencia, USA). e purity and concentration of
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