ABSTRACT
【Objective】 To explore the effect mechanism of Salvia miltiorrhiza and safflower on combined anti-ischemic stroke and verify relevant action targets in middle cerebral artery occlusion (MCAO) rat model based on network pharmacology. 【Methods】 ①Traditional Chinese Medicine Systems Pharmacology (TCMSP) and GeneCards databases were used to screen the active components, component targets and ischemic stroke targets of Salvia miltiorrhiza and safflower respectively. The above data were imported into STRING database for protein interaction network analysis, and Cytoscape3.8.0 software was used to construct protein interaction network (PPI) and component target interaction network. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis of target genes were performed using David online analysis tool. ② In this experiment, a rat model of ischemic stroke was prepared by using improved MCAO method, and immunohistochemical method and Real-time quantitative polymerase chain reaction (REAL-TIME PCR) to detect the positive expressions of NLRP3 inflammatory body and NF P65 protein in the brain tissue of rats in each group so as to explore the functional mechanism of anti-inflammation reaction against cerebral ischemia injury. 【Results】 ① A total of 87 effective components, corresponding to 253 targets, 1448 targets for ischemic stroke and 161 targets related to drugs and diseases, were screened from the Salvia milticorrhiza and safflower drug pairs. We obtained 730 biological processes, 81 cell components and 128 molecular functions through GO analysis, and 127 signal pathways through KEGG analysis. ②Immunohistochemical method and Real-time PCR determination results showed that compared with control group rats, model group rats had significantly increased tissue NLRP3 inflammatory body and NFkBp65 protein expressions (P<0.01). Compared with those in the model group, NLRP3 inflammatory body and NFkBp65 protein expressions significantly decreased in Dan red compatibility groups and nim horizon groups (P<0.01). 【Conclusion】 Compatibility of effective components in salvia miltiorrhiza, and carthamus tinctorius can further downregulate the release of inflammatory corpuscle NLRP3 through NFkB signaling pathway by blocking inflammatory lesions and thus plays the role of fighting against inflammatory damage.
ABSTRACT
<p><b>BACKGROUND</b>To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two hybrid system.</p><p><b>METHODS</b>Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product ?galactosidase activity was assayed as a measure of transcription activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh?7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin?layer chromatography.</p><p><b>RESULTS</b>The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain present transcriptional activation function in yeast. The transcription activating sequence is localized to the 21 to 47 amino acids of Pre S1 protein Fusion proteins of full length Pre S 1 and GAL 4 DNA binding domain do not show transcriptional activation function in mammalian cells.</p><p><b>CONCLUSION</b>The transcriptional activating sequence of HBVPre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers?from using yeast two hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two?hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.</p>