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Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

ABSTRACT: Treatment with Panax notoginseng saponin (PNS) can prevent neurological damage in middle cerebral artery occlusion model rats to promote recovery after a stroke. However, the exact molecular mechanisms are unknown and require further study. In the present study, mRNA sequencing was employed to investigate differential gene expression between model and sham groups, and between model and PNS?treated groups. Enrichment of gene data was performed using Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes database. Hub genes were identified and networks were constructed using Cytoscape that were further verified by reverse transcription?quantitative PCR. A total of 1,104 genes of interest were found, which included 690 upregulated and 414 downregulated genes that were identified when the model was compared with the sham group. Additionally, 817 genes of interest, which included 390 upregulated and 427 downregulated genes, were identified when the PNS?treated group was compared with the model group. There were 303 overlapping genes of interest between the analysis of model to sham groups, and the analysis of model to PNS?treated groups. The top 10 genes from the 303 aberrantly expressed genes of interest included ubiquitin conjugating enzyme E2 variant 2, small ubiquitin?related modifier 1, small RNA binding exonuclease protection factor La, Finkel?Biskis?Reilly murine sarcoma virus (FBR?MuSV) ubiquitously expressed, centrosomal protein 290 kDa, DNA?directed RNA polymerase II subunit K, cullin?4B, matrin?3 and vascular endothelial growth factor receptor 2. In conclusion, these genes may be important in the underlying mechanism of PNS treatment in ischemic stroke. Additionally, the present data provided novel insight into the pathogenesis of ischemic stroke.

SUBMITTER: Lin J

PROVIDER: S-EPMC7646891 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

Lin Jun J Liang Ping P Huang Qing Q Jian Chongdong C Huang Jianmin J Tang Xionglin X Li Xuebin X Liao Yanling Y Huang Xiaohua X Huang Wenhua W Su Li L Meng Lanqing L

Molecular medicine reports 20200929 6

Treatment with Panax notoginseng saponin (PNS) can prevent neurological damage in middle cerebral artery occlusion model rats to promote recovery after a stroke. However, the exact molecular mechanisms are unknown and require further study. In the present study, mRNA sequencing was employed to investigate differential gene expression between model and sham groups, and between model and PNS‑treated groups. Enrichment of gene data was performed using Gene Ontology analysis and the Kyoto Encycloped ...[more]

PMID: 33173991

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Project description:BackgroundPanax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown.ResultsUsing the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif.ConclusionThis study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species.

Project description:BACKGROUND:Panax notoginseng is commonly used for the treatment of cardiovascular diseases in China. The present study investigates the effects of three different saponin fractions (ie total saponins, PNS; protopanaxadiol-type saponin, PDS; and protopanaxatriol-type saponin, PTS) and two major individual ingredients (ie ginsenoside Rg1 and Rb1) from P. notoginseng on the endothelial inflammatory response in vitro and in vivo. METHODS:Recombinant human tumor necrosis factor-? (TNF-?) was added to the culture medium of human coronary artery endothelial cells (HCAECs) to induce an inflammatory response. A cell adhesion assay was used to determine the effect of the P. notoginseng saponin fractions on endothelial-monocyte interaction. The cell adhesion molecule (CAMs) expression, including ICAM-1 and VCAM-1, in the protein level on the surface of endothelial cells were measured by cellular ELISA. CAMs expression in mRNA level was also assayed by qRT-PCR in the HCAECs and the aorta of rat fed with high cholesterol diet (HCD). Western blotting was used to detect effect of the saponin fractions on CAMs protein expression in HCAECs. In addition, nuclear translocation of p65, a surrogate marker for NF-?B activation, was measured by immunostaining. RESULTS:Three saponin fractions and two individual ginsenosides exhibited the inhibitory effects on monocyte adhesion on TNF-?-activated HCAECs and expression of ICAM-1 and VCAM-1 at both mRNA and protein levels in vitro. The saponin fractions exhibited a similar trend of the inhibitory effects on the mRNA expression of CAMs in the aorta of HCD-fed rat in vivo. These inhibitory effect of saponin fractions maybe attribute partially to the suppression of the TNF-?-induced NF-?B activation. CONCLUSION:Our data demonstrate that saponin fractions (ie PNS, PDS and PTS) and major individual ginsenosides (ie Rg1 and Rb1) have potential anti-atherogenic effects. Among the tested saponin fractions, PDS is the most potent saponin fraction against TNF-?-induced monocyte adhesion as well as the expression of adhesion molecules in vitro and in vivo.

Dataset Information

Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

Publications

Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

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