GC-MS Profile of Hua-Feng-Dan and RNA-Seq Analysis of Induced Adaptive Responses in the Liver.

Jia-Jia Liu,Rui-Xia Wu,Jie Liu,Feng Zhang,Yan Liang,Jing-Shan Shi,Ya Zhang,Shang-Fu Xu,Ying-Lian Song,Zhang Wang

doi:10.3389/fphar.2022.730318

Abstract

Background: Hua-Feng-Dan is a patent Chinese medicine for stroke recovery and various diseases. This study used GC-MS to profile its ingredients and RNA-Seq to analyze the induced adaptive response in the liver. Methods: Hua-Feng-Dan was subjected to steam distillation and solvent extraction, followed by GC-MS analysis. Mice were orally administered Hua-Feng-Dan and its “Guide drug” Yaomu for 7 days. Liver pathology was examined, and total RNA isolated for RNA-Seq, followed by bioinformatic analysis and quantitative real-time PCR (qPCR). Results: Forty-four volatile and fifty liposoluble components in Hua-Feng-Dan were profiled and analyzed by the NIST library and their concentrations quantified. The major components (>1%) in volatile (5) and liposoluble (10) were highlighted. Hua-Feng-Dan and Yaomu at hepatoprotective doses did not produce liver toxicity as evidenced by histopathology and serum enzyme activities. GO Enrichment revealed that Hua-Feng-Dan affected lipid homeostasis, protein folding, and cell adhesion. KEGG showed activated cholesterol metabolism, bile secretion, and PPAR signaling pathways. Differentially expressed genes (DEGs) were identified by DESeq2 with p < 0.05 compared to controls. Hua-Feng-Dan produced more DEGs than Yaomu. qPCR on selected genes largely verified RNA-Seq results. Ingenuity Pathways Analysis of the upstream regulator revealed activation of MAPK and adaptive responses by Hua-Feng-Dan, and Yaomu was less effective. Hua-Feng-Dan-induced DEGs were highly correlated with the Gene Expression Omnibus database of chemical-induced adaptive transcriptome changes in the liver. Conclusion: GC-MS primarily profiled volatile and liposoluble components in Hua-Feng-Dan. Hua-Feng-Dan at the hepatoprotective dose did not produce liver pathological changes but induced metabolic and signaling pathway activations. The effects of Hua-Feng-Dan on liver transcriptome changes point toward induced adaptive responses to program the liver to produce hepatoprotective effects.

Highlights

Hua-Feng-Dan has over 300 years of history in the treatment of stroke and various diseases in China and is still used today alone or in combination with other medications (Liu et al, 2018)
Gene Ontology (GO) Enrichment revealed that Hua-Feng-Dan affected lipid homeostasis, protein folding, and cell adhesion
Hua-Feng-Dan-induced Differentially expressed genes (DEGs) were highly correlated with the Gene Expression Omnibus database of chemical-induced adaptive transcriptome changes in the liver

Summary

Introduction

Hua-Feng-Dan has over 300 years of history in the treatment of stroke and various diseases in China and is still used today alone or in combination with other medications (Liu et al, 2018). We have demonstrated the protective effects of Hua-Feng-Dan against LPS plus MPTP-induced Parkinson’s disease (PD) mouse model (Hu et al, 2020), and LPS plus rotenone-induced PD rat model (Chen et al, 2020), and identified that cinnabar and realgar are two active ingredients in the recipe both in LPS-induced neuroinflammation in rat midbrain neuron–glia cocultures (Zhang et al, 2012a) and in LPS plus rotenone-induced dopaminergic neuron loss in rats (Chen et al, 2020). Hua-Feng-Dan is a patent Chinese medicine for stroke recovery and various diseases. This study used GC-MS to profile its ingredients and RNA-Seq to analyze the induced adaptive response in the liver

Methods

Results

Discussion

Conclusion