RESUMO
In order to reveal the molecular mechanism of the small heat shock proteins (sHSPs) involved in stress resistance and active ingredients accumulation in Salvia miltiorrhiza, a small heat shock protein gene was cloned from Salvia miltiorrhiza by reverse transcription PCR according to the transcriptome data of orange root Salvia miltiorrhiza. The gene is named SmHSP21.8 based on the molecular weight of the protein, and it contains an open reading frame of 585 bp, which encodes 194 amino acids. The results of phylogenetic analysis and amino acid sequence alignment showed that SmHSP21.8 protein belongs to the endoplasmic reticulum (ER) subfamily, and contains a conserved endoplasmic reticulum-specific DPFR-I/V-LE-H/Q-x-P motif at N-terminus. The prokaryotic expression vector pMAL-c2X-SmHSP21.8 was constructed and transformed into E. coli BL21 competent cells. The recombinant protein was successfully expressed after inducted. Temporal and spatial expression analysis showed that SmHSP21.8 gene was the highest expressed in flowers and had significant tissue specificity. The relative expression of the gene was significantly increased in seedlings after induction by 38 ℃, PEG6000, abscisic acid(ABA), and indole-3-acetic acid (IAA), indicating that SmHSP21.8 gene may be involved in abiotic stress such as high temperature and drought, as well as the response to exogenous hormones ABA and IAA. These results lay the foundation for further research on the molecular mechanism of small heat shock proteins involved in adversity stress.
RESUMO
Objective:To explore active components and mechanism of Dipsaci Radix in treating rheumatoid arthritis （RA） based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry （UPLC-QTOF-MS/MS） and Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine （TCMIP）. Method:UPLC-QTOF-MS/MS with electrospray ionization （ESI） was used to qualitatively analyze the chemical components in methanol extract of Dipsaci Radix under positive and negative ion scanning modes. The mobile phase consisted of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-10 min， 0.2%-20%B； 10-20 min， 20%-40%B； 20-25 min， 40%-50%B； 25-33 min， 50%-98%B； 33-35 min， 98%-0.2%B）， and the scanning range was <italic>m</italic>/<italic>z</italic> 50-2 000. Based on TCMIP， candidate target groups of Dipsaci Radix， RA and syndrome with Yin deficiency of liver and kidney were obtained， and correlation analysis on "disease-syndrome-prescription" was used to enrich the main active components and key targets. Cytoscape 3.8.0 and STRING 11.0 database were used to construct protein-protein interaction （PPI） network diagram. Metascape platform was used to analysis gene ontology biological progress and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathways. Result:A total of 81 ingredients were identified by UPLC-QTOF-MS/MS. Based on TCMIP， 283 candidate targets corresponding to 81 ingredients， 7 RA related targets and 215 genes corresponding to syndrome with Yin deficiency of liver and kidney were collected. With further correlation analysis on "disease-syndrome-prescription"， 17 key active ingredients were predicted， mainly including saponins and fatty acids of Dipsaci Radix. It mainly involved 7 hub targets， namely tumor necrosis factor （TNF）， nuclear factor-<italic>κ</italic>B subunit 1 （NF-<italic>κ</italic>B<sub>1</sub>）， hepatocyte nuclear factor 4 alpha （HNF4A）， nuclear receptor subfamily 3 group C member 1 （NR3C1）， peroxisome proliferator activated receptor gamma （PPARG）， nuclear receptor subfamily 1 group H member 4 （NR1H4） and nuclear receptor coactivator 1 （NCOA1）. All of them were related to inflammation， and two of them were related to bile acid pathway. The 7 hub targets and 7 pathways played an important role in RA were screen out， including 4 bile acid related pathways and 3 inflammatory related pathways. Conclusion:UPLC-QTOF-MS/MS combined with TCMIP preliminarily elucidates the regulatory effect of multi-components in Dipsaci Radix on several pathways related to the inflammatory response and bile acid synthesis and metabolism， which lays a foundation for further study on the mechanism of Dipsaci Radix against RA.