Structural changes of gut microbiota in a rat non-alcoholic fatty liver disease model treated with a Chinese herbal formula

Abstract

Accumulating evidence indicates that disruption of the gut microbiota by a high-fat diet (HFD) may play a pivotal role in the progression of metabolic disorders such as non-alcoholic fatty liver disease (NAFLD). In this study, the structural changes of gut microbiota were analyzed in an HFD-induced NAFLD rat model during treatment with an ancient Chinese herbal formula (CHF) used in clinical practice – Qushi Huayu Fang. CHF treatment significantly reduced body weight, alleviated hepatic steatosis, and decreased the content of triglycerides and free fatty acids in the livers of the rats. Gut microbiota of treated and control rats were profiled with polymerase chain reaction-denaturing gradient gel electrophoresis and bar-coded pyrosequencing of the V3 region of 16S rRNA genes. Both analyses indicated that the CHF-treated group harbored significantly different gut microbiota from that of model rats. Partial least squares discriminant analysis and taxonomy-based analysis were further employed to identify key phylotypes responding to HFD and CHF treatment. Most notably, the genera Escherichia/Shigella, containing opportunistic pathogens, were significantly enriched in HFD-fed rats compared to controls fed normal chow (P < 0.05) but they decreased to control levels after CHF treatment. Collinsella, a genus with short chain fatty acid producers, was significantly elevated in CHF-treated rats compared to HFD-fed rats (P < 0.05). The results revealed that the bacterial profiles of HFD-induced rats could be modulated by the CHF. Elucidation of these differences in microbiota composition provided a basis for further understanding the pharmacological mechanism of the CHF.

Introduction

Non-alcoholic fatty liver disease (NAFLD), a manifestation of the metabolic syndrome in the liver, is closely associated with insulin resistance, obesity, and diabetes mellitus [27]. Although it is a major threat to public health, the pathogenesis of NAFLD is not yet well understood, nor are there effective therapies [28].

Emerging evidence in the last two decades indicates a possible role of gut microbiota in the etiology of NAFLD [1]. Small intestinal bacterial overgrowth is commonly found in NAFLD patients and results in increased intestinal permeability, endogenous ethanol production, and choline deficiency [3], [11], [44]. In addition, the gut microbiota promotes absorption of monosaccharides and short chain fatty acids by fermentation, and thus increases de novo hepatic lipogenesis and enhances fat storage by regulating lipoprotein lipase activity [12]. NAFLD is typically associated with low-grade, chronic inflammation [13], and a major cell wall component of Gram-negative bacteria, lipopolysaccharide (LPS), is known to be potent in promoting inflammation [5]. When binding to CD14 and toll-like receptor 4 on the surface of immune cells, a low concentration of LPS from gut microbiota can trigger a series of inflammatory processes [31], [45], which in turn contributes to the pathogenesis of NAFLD. Consequently, the gut microbiota has become a novel target for potential therapies [14], with probiotics, prebiotics, and antibiotics being tested for treatment or prevention of NAFLD [4], [19], [22].

Traditional medicine has long been used in China, and most of the formulas are orally administered. Recent research has revealed that many ingredients in these herbs could only be absorbed and they exert their biological effects with the help of the host's gut microbiota [8]. Furthermore, intriguing but limited data also show that the efficacy of some traditional herbs may be related to modulation of gut microbiota [48]. One formula, Qushi Huayu Fang, has a long history of use in clinical practice to alleviate NAFLD. However, to date, no systematic and credible explanations have been proposed for its mechanism. Whether Qushi Huayu Fang can modulate the host's gut ecosystem while alleviating NAFLD symptoms and whether gut bacteria are a potential target for the herbal formula are still unanswered questions.

Nowadays, advanced technologies have facilitated the exploration of gut microbiota, especially with the development of high-throughput sequencing technology. Researchers are able to look in detail at the structure of gut microbiota, and study how external factors, such as age and drug use among others, influence gut microbiota. In this study, gut microbial composition during Qushi Huayu Fang treatment in a high-fat diet (HFD)-induced NAFLD rat model was monitored by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and bar-coded pyrosequencing. Using a microbiome-wide association strategy [34], [41], [47], the modulation of this Chinese herbal formula (CHF) in the gut microbiome was validated, and key phylotypes closely related to NAFLD development and CHF treatment were expected to be identified.