Mechanism of Yiqi Huoxue Huatan recipe in the treatment of coronary atherosclerotic disease through network pharmacology and experiments.

In recent years, with population aging and economic development, morbidity and mortality of atherosclerotic cardiovascular disease associated with atherosclerosis (AS) have gradually increased. In this study, a combination of network pharmacology and experimental verification was used to systematically explore the action mechanism of Yiqi Huoxue Huatan Recipe (YHHR) in the treatment of coronary atherosclerotic heart disease (CAD). We searched and screened the active ingredients of Coptis chinensis, Astragalus membranaceus, Salvia miltiorrhiza, and Hirudo. We also searched multiple databases for related target genes corresponding to the compounds and CAD. STRING was used to construct the protein-protein interaction (PPI) network of genes. Metascape was used to perform gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for common targets to analyze the main pathways, and finally, the molecular docking and main possible pathways were verified by experimental studies. Firstly, a total of 1480 predicted target points were obtained through the Swiss Target Prediction database. After screening, merging, and deleting duplicate values, a total of 768 targets were obtained. Secondly, "Coronary atherosclerotic heart disease" was searched in databases such as the OMIM, GeneCards, and TTD. 1844 disease-related targets were obtained. Among PPI network diagram of YHHR-CAD, SRC had the highest degree value, followed by AKT1, TP53, hsp90aa1 and mapk3. The KEGG pathway bubble diagram was drawn using Chiplot, the Signal pathways such as NF kappa B signaling pathway, Lipid and AS, and Apelin signaling pathway are closely related to the occurrence of CAD. The PCR and Western blot methods were used to detect the expression of NF-κB p65. When compared with that in the model group, the expression of NF-κB p65mRNA decreased in the low-concentration YHHR group, with P < .05, while the expression of NF-κB p65mRNA decreased significantly in the high-concentration YHHR group, with P < .01. On the other hand, when compared with that in the model group, the expression of NF-κB p65 decreased in the low-concentration YHHR group, but was not statistically significant, while the expression of NF-κB p65 was significant in the high-concentration YHHR group, and has statistical significance with P < .05. YHHR has been shown to resist inflammation and AS through the SRC/NF-κB signaling pathway.

MicroRNA profiling analysis of developing berries for 'Kyoho' and its early-ripening mutant during berry ripening.

BACKGROUND: 'Fengzao' is an early-ripening bud mutant of 'Kyoho', which matures nearly 30 days earlier than 'Kyoho'. To gain a better understanding of the regulatory role of miRNAs in early-ripening of grape berry, high-throughput sequencing approach and quantitative RT-PCR validation were employed to identify miRNAs at the genome-wide level and profile the expression patterns of the miRNAs during berry development in 'Kyho' and 'Fengzao', respectively. RESULTS: Nine independent small RNA libraries were constructed and sequenced in two varieties from key berry development stages. A total of 108 known miRNAs and 61 novel miRNAs were identified. Among that, 159 miRNAs identified in 'Fengzao' all completely expressed in 'Kyoho' and there were 10 miRNAs specifically expressed in 'Kyoho'. The expression profiles of known and novel miRNAs were quite similar between two varieties. As the major differentially expressed miRNAs, novel_144, vvi-miR3626-3p and vvi-miR3626-5p only expressed in 'Kyoho', vvi-miR399b and vvi-miR399e were down-regulated in 'Fengzao', while vvi-miR477b-3p up-regulated in 'Fengzao'. According to the expression analysis and previous reports, miR169-NF-Y subunit, miR398-CSD, miR3626-RNA helicase, miR399- phosphate transporter and miR477-GRAS transcription factor were selected as the candidates for further investigations of miRNA regulation role in the early-ripening of grape. The qRT-PCR analyses validated the contrasting expression patterns for these miRNAs and their target genes. CONCLUSIONS: The miRNAome of the grape berry development of 'Kyoho', and its early-ripening bud mutant, 'Fengzao' were compared by high-throughput sequencing. The expression pattern of several key miRNAs and their target genes during grape berry development and ripening stages was examined. Our results provide valuable basis towards understanding the regulatory mechanisms of early-ripening of grape berry.