Abstract
Hemorrhage stroke is a severe vascular disease of the brain with a high mortality rate in humans. Salvia miltiorrhiza Bunge (Danshen) is a well-known Chinese Materia Medica for treating cerebral vascular and cardiovascular diseases in traditional Chinese medicine. Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, which is the main active ingredient of Danshen. In our previous study, we established a zebrafish model of cerebral hemorrhage and found that STS dramatically decreased both the hemorrhage rate and hemorrhage area, although the underlying mechanism was not fully elucidated. We conducted a transcriptome analysis of the protective effect of STS against atorvastatin (Ator)-induced cerebral hemorrhage in zebrafish using RNA-seq technology. RNA-seq revealed 207 DEGs between the Ator-treated group and control group; the expression levels of 53 DEGs between the Ator-treated group and control group were reversed between the STS + Ator-treated group and Ator-treated group. GO enrichment analysis indicated that these 53 DEGs encode proteins with roles in hemoglobin complexes, oxygen carrier activity and oxygen binding, etc. KEGG analysis suggested that these 53 DEGs were most enriched in three items, namely, porphyrin and chlorophyll metabolism, ferroptosis, and the HIF-1 signaling pathway. The PPI network analysis identified 12 hub genes, and we further verified that Ator elevated the mRNA expression levels of hemoglobin (hbae1.3, hbae3, hbae5, hbbe2, and hbbe3), carbonic anhydrase (cahz), HIF-1 (hif1al2) and Na+/H+ exchanger (slc4a1a and slc9a1) genes, while STS significantly suppressed these genes. In addition, we found that pharmacological inhibition of PI3K/Akt, MAPKs, and mTOR signaling pathways by specific inhibitors partially attenuated the protective effect of STS against Ator-induced cerebral hemorrhage in zebrafish, regardless of mTOR inhibition. We concluded that hemoglobin, carbonic anhydrase, Na+/H+ exchanger and HIF-1 genes might be potential biomarkers of Ator-induced cerebral hemorrhage in zebrafish, as well as pharmacological targets of STS. Moreover, HIF-1 and its regulators, i.e., the PI3K/Akt and MAPK signaling pathways, were involved in the protective effect of STS against Ator-induced cerebral hemorrhage. This study also provided evidence of biomarkers involved in hemorrhage stroke and improved understanding of the effects of HMG-COA reductase inhibition on vascular permeability and cerebral hemorrhage.
Highlights
Hemorrhage stroke, which is one of the most severe vascular diseases of the brain, is the second most common type of stroke after ischemic stroke and has a high mortality (Leclerc et al., Abbreviations: Akt, protein kinase B; Ator, atorvastatin; cell adhesion molecules (CAMs), cell adhesion molecule; CFDA, China State Food and Drug Administration; cGMP, cyclic guanosine monophosphate; Danshen, salvia miltiorrhiza Bunge; DMSO, dimethylsulfoxide; differentially expressed genes (DEGs), differentially expressed gene; FDR, false discovery rate; GO, Gene Ontology; HIF-1, hypoxia inducible factor-1; HMG-COA, 3-hydroxy3-methyl glutaryl coenzyme A; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PKG, cGMP-dependent protein kinase; PPI, protein-protein interaction; RNA-seq, RNA sequencing; Tan IIA, tanshinone IIA; Sodium tanshinone IIA sulfonate (STS), sodium tanshinone IIA sulfonate.2015)
78.05% sequences matched with annotated genes in the databases and 25,858 genes were detected in total; 207 DEGs were subsequently identified between the Ator-treated group and control group according to the criteria of fold change ≥ 2 and p value < 0.05
The expression levels of 53 DEGs, among which five genes were upregulated and 48 genes were downregulated, in the Atortreated group vs. control group analysis were reversed in the Ator + STS-treated group vs. Ator-treated group analysis (Figure 3B). These genes were subjected to GO and KEGG analysis, the results of which indicated that the mechanisms underlying the protective effect of STS against Ator-induced zebrafish cerebral hemorrhage were involved in hemoglobin complex, oxygen carrier activity, oxygen binding, iron ion binding, CAMs and tight junctions, etc., and their related signaling pathways such as the ferroptosis and HIF-1 signaling pathways, etc. (Figures 3C, D)
Summary
Hemorrhage stroke, which is one of the most severe vascular diseases of the brain, is the second most common type of stroke after ischemic stroke and has a high mortality Potential biomarkers of Ator-induced cerebral hemorrhage, and the underlying mechanisms of the hemorrhage stroke-protective effect of STS, were not well elucidated. In the present study, we investigated the potential biomarkers and underlying mechanisms of STS against Atorinduced cerebral hemorrhage in zebrafish via transcriptome profile analysis and RNA-seq technology
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