Effects and therapeutic mechanism of Yinzhihuang on steatohepatitis in rats induced by a high‐fat, high‐cholesterol diet

We aimed to investigate the therapeutic mechanism of Yinzhihuang (YZH) liquid, a traditional Chinese medicine mainly composed of extracts of four components, on nonalcoholic steatohepatitis (NASH) induced by a high‐fat, high‐cholesterol diet (HFHCD) in rats.


| INTRODUCTION
The incidence of nonalcoholic fatty liver disease (NAFLD), which affects approximately one-quarter of the global population, has been increasing over the past two decades. 1 As an aggressive form of NAFLD, a definitive diagnosis of nonalcoholic steatohepatitis (NASH) is made based on histological evidence of hepatocellular injury and an accumulation of inflammatory cells in addition to hepatic steatosis. 2 Simple steatosis usually runs a benign course in patients, while NASH is more likely to develop into progressive liver fibrosis and eventually cause liver-related complications. This underscores the importance of developing interventions to prevent and treat NASH. 3 Pharmacological therapy is required for patients with NASH or liver fibrosis as well as for those with morbid obesity or musculoskeletal disorders who are unable to exercise sufficiently. 4 Despite its significant burden to the public healthcare system, no medication has yet been approved for the treatment of NASH by the Food and Drug Administration in the United States. Therefore, the need for appropriate treatment options and therapeutic targets is now quite urgent. 5 Due to the complex pathophysiological process of NASH, effective therapy must target several key regulators of cellular and molecular events.
Long-term effects of many monotherapies for systemic diseases have been found to be limited, and adverse events may present during the treatment course. The reasons may include the nature of the diseases, natural evolution of feedback loops and pathway redundancy.
Traditional Chinese herbal medicines are rich in numerous bioactive substances with several biological properties, including anti-inflammatory, anti-infection and immunoregulatory properties. 6 Traditional Chinese herbal medicine has been demonstrated to be effective for treating NAFLD. 7 Yinzhihuang (YZH) oral solution, a traditional Chinese herbal medicine, is mainly composed of extracts of Artemisia capillaris, Gardenia jasminoides and Scutellaria baicalensis, with a small amount of Lonicera japonica. 8 YZH has already been used to remove dampness to eliminate jaundice and detoxify the human body.
In clinical practice, YZH oral solution can be used in the treatment of neonatal jaundice and cholestasis. It can significantly reduce serum bilirubin levels, shorten the time for jaundice recovery, and ultimately achieve a high rate of therapeutic efficacy. 9,10 A clinical study in China reported that 12-week therapy with YZH oral solution could significantly decrease serum levels of alanine transaminase (ALT), aspartate transaminase (AST), total cholesterol (TC), and triglyceride (TG) in patients with NAFLD, without causing any significant adverse reactions. 11 YZH oral solution has also been reported to decrease serum aminotransferase and direct bilirubin levels in rats, together with a protective effect against intrahepatic cholestasis. 10 In addition, it significantly decreased serum levels of ALT, AST, interleukin (IL)-6, and tumor necrosis factor (TNF)-α in mice with immunological liver injuries. 12 Furthermore, our previous study showed that YZH oral solution could ameliorate liver histological features in rats with NASH induced by a methionine and choline-deficient diet (MCD) or a high-fat, highcholesterol diet (HFHCD). 13,14 Though these results suggest that YZH may be effective in the treatment of NAFLD or NASH, its therapeutic mechanism has not been clarified yet.
Complex multicomponent nature may make these traditional Chinese herbal medicines valuable resources due to their synergistic effects. In addition, multiple systems biology platforms have been shown to be the most powerful technologies available to uncover molecular mechanisms and connections between drugs and their multitargeted networks. 15 Based on the rationale and technologies mentioned above, we used transcriptomics and proteomics platforms in the present study to analyze the functional mechanism of YZH oral solution in rats with HFHCD-induced NASH and to investigate the relevant molecular targets and functional pathways from a network perspective.

| Preparation and analysis of YZH liquid
The YZH liquid was purchased from China Resources Sanjiu Medical & Pharmaceutical (Beijing, China). The identification and quantification of the marker compounds in the YZH liquid were performed according to the methods described in the Updated Pharmacopeia of the People's Republic of China. 16 Briefly, 12 g of Herba Artemisiae Capillariae, 6.4 g of Fructus Gardeniae and 8 g of L. japonica were mixed and dissolved by adding 300 mL of double-distilled water. The pH value was adjusted to 6.5 by adding 10% sodium hydroxide solution and the solution was filtered and reserved. S. baicalensis (40 g) was then dissolved in 300 mL of double-distilled water and the pH value was adjusted to 6.5-7.0 with a 10% sodium hydroxide solution. After filtration, the filtrate was mixed with the reserved solution. The compound was thoroughly mixed with 0.5 g of citric acid, 100 g of sucrose, 50 g of honey, 2 g of aspartame and 3 g of sodium benzoate. The mixture was refrigerated at 4-8 C for 24 hours and the pH value was adjusted to neutral. An appropriate amount of double-distilled water was added to this solution to adjust the total amount to 1000 mL.
Finally, the solution was left to stand, filtered and sterilized to finally obtain the YZH liquid.
Marker compounds were analyzed using high-performance liquid chromatography (Agilent 1260 Infinity; Agilent Technologies, Palo Alto, CA, USA) with a Venusil MP C18 column (250 mm × 4.6 mm, 5 μm; Agela Technologies, Wilmington, DE, USA), as described in a previous study. 17 The reference substances for five marker compounds, including chlorogenic acid, geniposide, hyperoside, baicalin and luteolin, were purchased from the National Institutes for Food and Drug Control (Beijing, China). Measurements were all made at a flow rate of 1 mL/min and a detection wavelength of 327 nm. The column temperature was 35 C and the injection volume was 10 μL.
The five characteristic peaks were identified by comparison with each reference substance ( Figure S1).

| Animal experiments
In total 30 male Sprague-Dawley rats (6 weeks old) obtained from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China) were kept (5 rats per cage) under controlled temperature (24 C ± 2 C) and humidity (50% ± 5%) with free access to food and water. The animal experiments were performed with reference to the National Research Council's Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee of Society for Human Resource Management (no. SHRM-IACUC-001).
Following a week of acclimation, the rats were randomly divided into three groups using random number table. The control group was given a normal diet composing of 64.5% carbohydrates, 13.3% fat, and

| High-throughput RNA sequencing and bioinformatics analysis
Total RNA was extracted according to the manufacturer's protocol (TaKaRa, Shiga, Japan) from the livers of 12 rats (four rats per group).
The RNA sequencing experimental process is shown in Figure 2A. The necessity of a resequencing step was determined by quality control of the primary sequencing data. After quality control, the raw reads were filtered into clean reads that were aligned to the reference sequences.
The distribution of reads on the reference genes and the mapping ratio were calculated based on the alignment data. Then, gene expression analysis and deep analysis based on the gene expression data were included into the downstream analyses. Genes expressed differentially were screened according to the default criteria: a log2-fold change ≥1.0 and a divergence probability ≥0.8. Furthermore, we performed a KEGG pathway enrichment analysis on the differentially expressed genes.

| Isobaric tags for relative and absolute quantification (iTRAQ) proteomics and bioinformatics analysis
Protein was extracted from the remaining liver tissues of these 12 rats using a lysis buffer and two magnetic beads (diameter 5 mm). The main procedures of the iTRAQ quantitative proteomics experiment are shown in Figure 2B

| Establishment of a network-based regulatory model
The generation of a network-based regulatory model was based on the Cytoscape web application (Cytoscape Consortium, San Diego, CA, USA) with information obtained from several levels of functional analysis: the fold-change analysis of genes or proteins, the proteinprotein interaction analysis, the KEGG pathway enrichment analysis and the biological process enrichment analysis. The protein-protein interaction analysis was accomplished by selecting genes and proteins expressed differentially via the widely used STRING biological database (https://string-db.org) and web resource. The default confidence cut-off value was set as 400; solid lines were used between genes and proteins when interactions with scores were over 400, while F I G U R E 1 Flowchart of grouping of the control, high-fat, highcholesterol diet (HFHCD) + saline, and HFHCD + Yinzhihuang (YZH) rats. The saline and YZH liquid were given to the rats by gavage once daily beginning at the end of the 8th week at doses of 15 mL/kg. HFHCD, high-fat, high-cholesterol diet; SD, Sprague-Dawley rats, YZH, yinzhihuang dashed lines were used in interactions with scores lower than 400.
Genes/proteins are represented with circular nodes, while KEGG pathways are indicated with rectangles. The pathways are colored in a gradient from yellow, indicating a larger P value, to blue, indicating a smaller P value. Genes/proteins are colored red (upregulated) and green (downregulated) in fold-change analysis.

| Statistical analysis
The data are presented as the mean ± standard error. One-way ANOVA followed by a Newman-Keuls post-test was used to compare differences in continuous variables among groups with SPSS version 21.0 software (IBM, Armonk, NY, USA). Graphs were generated using GraphPad Prism Software version 6 (San Diego, CA, USA). Differences were considered statistically significant at P < 0.05.

| Effects of YZH liquid on HFHCD-induced NASH rats
At the end of the 16th week, all rats in the HFHCD + saline group developed NASH with significant hepatocellular steatosis, ballooning and lobular inflammation. Compared with the control group, the HFHCD + saline group had a higher body weight, liver and fat index (P < 0.01), while the HFHCD + YZH group had a significantly lower body weight, liver index and fat index than the HFHCD + saline group (P < 0.01; Figure 3A). In addition, intake of YZH liquid improved hepatic histological features, including steatosis, ballooning and lobular inflammation ( Figure 3B). Furthermore, serum ALT, AST and TC levels of the HFHCD + YZH group significantly decreased compared with those of the HFHCD + saline group (P < 0.05; Figure 3C), although they did not significantly differ compared with those of the control group.

| Differentially expressed genes in liver tissues after YZH liquid administration
Using the cut-off criteria mentioned above, we identified 308 differentially expressed genes, including 261 upregulated genes and 47 downregulated genes, in the livers of HFHCD + saline rats compared with those of control rats. While compared with the HFHCD + saline rats, liver tissues of HFHCD + YZH rats exhibited 89 differentially expressed genes, with 16 upregulated and 73 downregulated genes ( Figure 4A). Altogether 56 genes were found to be upregulated in the HFHCD + saline group while downregulated after treated with YZH liquid; however, eight genes were downregulated in HFHCD + saline F I G U R E 2 Main procedures of the RNA-sequencing experiment and isobaric tags for relative and absolute quantification. A, the RNA sequencing experimental process; B, the main procedures of the iTRAQ quantitative proteomics experiment but upregulated in HFHCD + YZH ( Figure 4B). The expression patterns of these 64 hepatic genes were found to have been reversed after the administration of YZH liquid in the NASH rat models. A heat map of the gene expression analysis showing the hierarchical clustering of these differentially expressed genes after YZH administration in the different groups is shown in Figure 4C.
The KEGG pathway enrichment analysis included 64 selected genes which were expressed differentially. These genes were significantly enriched in 10 signaling pathways, including the chemokine signaling pathway, the Toll-like receptor signaling pathway, the TNF signaling pathway, the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, biosynthesis of unsaturated fatty acids, the nuclear factor-κB signaling pathway, fatty acid degradation, fatty acid metabolism, glycolysis and gluconeogenesis and bile secretion ( Table 1).

| Network-based regulatory model of YZH liquid on NASH
The 64 differentially expressed genes and 73 differentially expressed proteins after YZH administration were selected to establish a geneprotein interaction network ( Figure 6). The results indicate that there are 33 genes or proteins involved in the proposed regulatory model.     24 Fads expression has been found to be negatively associated with hepatic fat content, and FADS can suppress the accumulation of hepatic lipids. 25 In addition, YZH liquid decreased the expression levels of Pla2 family members, which are upstream checkpoints regulating fatty acid uptake that are upregulated in patients with NASH. 26,27 The knockout of Pla2 has been proven to achieve a 56.5% decrease in fatty acid influx in hepatocytes. 26 The administration of YZH liquid also increases the expression of alcohol dehydrogenase, the impairment of which has been noticed in the liver tissue of NASH patients. 28 Overall, YZH liquid reduces hepatic lipid accumulation in HFHCD-fed NASH rats by limiting lipogenesis and accelerating β-oxidation. F I G U R E 6 Network-based regulatory model of Yinzhihuang (YZH) liquid for nonalcoholic steatohepatitis. The 64 differentially expressed genes and 73 differentially expressed proteins after YZH liquid administration were selected to establish a gene-protein interaction network. The circular nodes indicated genes or proteins, while the rectangles indicated KEGG pathways. The pathways are colored with a gradient from yellow to blue, with yellow color indicates a larger P value, while blue color indicates a smaller P value. In the case of fold-change analysis, the genes/ proteins are colored red (upregulated) and green (downregulated), respectively. Abbreviations: NOD, nucleotide-binding oligomerization domain; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor Hepatocyte injury is the key feature that differentiates NASH from isolated steatosis. 32 Injured hepatocytes release factors promoting the accumulation of immune cells that produce hepatotoxic substances and induce further injury and inflammation. 33,34 In our study, the genes involved in the Toll-like receptor signaling pathway, the NF-κB signaling pathway, the NOD-like receptor signaling pathway, and TNF signaling pathway were significantly increased in the NASH rat models. These genes, including Lbp, Stat1, Ripk2, Irf1, Gbp2 and several chemokines, were suppressed after YZH liquid therapy.

F I G U R E 3 Effects of yinzhihuang (YZH) liquid on high-fat, high-cholesterol diet (HFHCD)-induced nonalcoholic steatohepatitis (NASH). A, Effects of YZH liquid on body weight, liver index and fat index in NASH rats. B, Effects of YZH liquid on hepatic histological features of NASH
Chemokines are chemoattractants for leukocyte trafficking, growth and activation at sites of injury and inflammation, and in the livers of patients with NASH, the expression of chemokines and their receptors was increased based on accumulated data. 35 Studies have demonstrated the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration and amelioration of steatosis and inflammation development by the inhibition of Ccl2 or Cxcl10, which links hepatocyte lipotoxicity to macrophage-associated liver inflammation in NASH as a potent chemotactic ligand. 36,37 There is evidence that the endotoxemia marker Lbp is increased in patients with NASH and this correlates with lobular inflammation, 38  There were some limitations to our study. First, TG levels were measured only in the serum, while its accumulation in hepatocytes is actually the main component of hepatic steatosis. Second, the therapeutic mechanism of YZH liquid was evaluated in NASH rat models. The efficacy and functional mechanism of this herbal medicine need further evaluation in clinical studies. Third, the specific regulatory relationships between genes and proteins in this network-based regulatory model have not been fully elucidated in our study. Therefore, the verification of RNA sequencing in several selected pathways is needed and functional experiments need to be specifically designed to support our claims.

| CONCLUSION
In conclusion, YZH liquid was effective in reducing body weight and serum aminotransferase levels and alleviating pathological liver injury in HFHCD-fed NASH models. The network-based regulatory model of YZH liquid on NASH shows that YZH liquid can reduce lipid deposi-