RNA-Seq analysis of the protection by Dendrobium nobile alkaloids against carbon tetrachloride hepatotoxicity in mice

Biomed Pharmacother. 2021 May:137:111307. doi: 10.1016/j.biopha.2021.111307. Epub 2021 Feb 6.

Abstract

Objective: Dendrobium nobile is a genuine Chinese medicine. Dendrobium nobile Lindl. alkaloids (DNLA) protects against CCl4-induced acute liver injury. This study used RNA-Seq to explore the mechanisms.

Methods: Mice were pretreated with DNLA (10 and 20 mg/kg, po) for 7 days, and subsequently intoxicated with CCl4 (20 μL/kg, ip for 24 h). Liver RNA was extracted and subjected to RNA-Seq. The bioinformatics, including PCA, GO, KEGG, two-dimensional clustering, Ingenuity Pathways Analysis (IPA), and Illumina BaseSpace Correlation Engine (BSCE) were used to analyze the data. qPCR was performed on selected genes to verify RNA-Seq results.

Results: DNLA protection against CCl4 hepatotoxicity was confirmed by histopathology. PCA revealed the distinct gene expression patterns between the different treatment groups. GO showed that CCl4 induced the activation, adhesion and proliferation of immune cells. KEGG showed CCl4 induced oxidative stress, diseases and compromised adaptive responses. CCl4 induced differentially expressed genes (DEGs) were identified by DESeq2 with Padj < 0.05 and 2D-clustered with other groups. DNLA reverted CCl4-induced DEGs in a dose-dependent manner. qPCR analysis of S100 g, Sprr1, CCL3/7, Saa2/3, IL1rn, Cox7a2 and Rad15 confirmed RNA-Seq results. IPA showed that CCl4 treatment altered some signaling and metabolic pathways, which were ameliorated or returned to normal following DNLA treatment. The CCl4-activated mitochondrial oxidative phosphorylation was illustrated as an example. IPA Upstream Regulator Analysis further revealed the activated or inhibited molecules and chemicals that are responsible for CCl4-induced DEGs, and DNLA attenuated these changes. BSCE analysis verified that CCl4-induced DEGs were highly correlated with the GEO database of CCl4 hepatotoxicity in rodents, and DNLA dose-dependently attenuated such correlation.

Conclusion: RNA-Seq revealed CCl4-induced DEGs, disruption of canonical pathways, activation or inhibition of upstream regulators, which are highly correlated with database for CCl4 hepatotoxicity. All these changes were attenuated or returned to normal by DNLA, demonstrating the mechanisms for DNLA to protect against CCl4 hepatotoxicity.

Keywords: Carbon tetrachloride; GEO database correlation; Hepatotoxicity; Ingenuity pathways analysis; RNA sequencing; qPCR.

MeSH terms

  • Adaptive Immunity / drug effects
  • Alkaloids / chemistry*
  • Alkaloids / therapeutic use*
  • Animals
  • Carbon Tetrachloride Poisoning / drug therapy*
  • Carbon Tetrachloride Poisoning / genetics*
  • Carbon Tetrachloride Poisoning / pathology
  • Cell Adhesion / drug effects
  • Cell Proliferation / drug effects
  • Chemical and Drug Induced Liver Injury / drug therapy*
  • Chemical and Drug Induced Liver Injury / genetics*
  • Chemical and Drug Induced Liver Injury / pathology
  • Computational Biology
  • Databases, Genetic
  • Dendrobium / chemistry*
  • Dose-Response Relationship, Drug
  • Gene Expression Regulation / drug effects
  • Liver / chemistry
  • Liver / pathology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Oxidative Phosphorylation / drug effects
  • Oxidative Stress / drug effects
  • RNA-Seq / methods*

Substances

  • Alkaloids