[Scr eening of key genes and pathways of brain aging and prediction of potential effective Chinese medicinals: based on bioinformatics].

Microarray data of hippocampal tissue(HC) of the cognitively intact elderly(60-99 years old) were compared with those of the middle-aged and the young(20-59 years old) by bioinformatics techniques to initially screen out differentially expressed genes(DEGs) and then predict potential effective Chinese medicinals for the treatment of brain aging. The gene expression profile(accession: GSE11882) was downloaded from the Gene Expression Omnibus(GEO) and DEGs were screened based on R package. The key DEGs were identified by STRING, Cytoscape and the plug-in, followed by Gene Ontology(GO) term and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Then the key genes and the medical ontology information retrieval platform(Coremine Medical) were mapped against each other to single out the Chinese medicinals for the treatment of brain aging and construct the " Chinese medicinal-active constituent-target" network. Among the resultant 268 DEGs(246 down-regulated and 22 up-regulated), the 15 key genes were mainly involved in biological processes such as leukocyte migration, neutrophil activation, cell chemotaxis, microglia activation and response to external stimulus, and pathways such as inflammatory process, immune response, cytokine-cytokine receptor interaction, PI3 K-Akt signaling pathway, Rap1 signaling pathway, chemokine signaling pathway and Toll-like receptor signaling pathway. The potential effective Chinese medicinals were Notoginseng Radix et Rhizoma, Ginseng Radix et Rhizoma, Salviae Miltiorrhizae Radix et Rhizoma and Astragali Radix. The analysis of DEGs and key genes enhances the understanding of the mechanisms of brain aging. This study provides potential gene targets and ideas for the development of Chinese medicine for brain aging.

[Mechanism of extract of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on SIRT1 autophagy pathway of endothelial cell senescence induced by hydrogen peroxide].

This study aims to explore the effect of extract of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Chuanxiong Rhizoma(hereinafter referred to as GNS) on the SIRT1-autophagy pathway of endothelial cell senescence induced by hydrogen peroxide(H_2O_2). To be specific, vascular endothelial cells were classified into the blank control group(control), model group(model), model + DMSO group(DMSO), resveratrol group(RESV), and GNS low-dose(GNS-L), medium-dose(GNS-M), and high-dose(GNS-H) groups. They were treated with H_2O_2 for senescence induction except the control. After intervention of cells in each group with corresponding drugs for 24 h, cell growth status was observed under an inverted microscope, and the formation of autophagosome under the transmission electron microscope. In addition, the changes of microtubule-associated protein 1 light chain 3ß(LC3 B) were detected by immunofluorescence staining. The autophagy flux was tracked with the autophagy double-labeled adenovirus(mRFP-GFP-LC3) fusion protein. Dansylcadaverine(MDC) staining was employed to determine the autophagic vesicles, and Western blot the expression of sirtuin 1(SIRT1), ubiquitin-binding protein p62, and LC3Ⅱ. After H_2O_2 induction, cells demonstrated slow growth, decreased adhesion ability, raised number of SA-ß-gal-stained blue ones, a certain number of autophagosomes with bilayer membrane and secondary lysosomes in the cytoplasm, and slight rise of autophagy flux level. Compared with the model group, GNS groups showed improved morphology, moderate adhesion ability, complete and smooth membrane, decreased SA-ß-gal-stained blue cells, many autophagosomes, autophagic vesicles, and secondary lysosomes in the cytoplasm, increased autophagolysosomes, autophagy flux level, and fluorescence intensity of LC3 B and MDC, up-regulated expression of SIRT1 and LC3Ⅱ, and down-regulated expression of p62, suggesting the improvement of autophagy level. GNS can delay the senescence of vascular endothelial cells. After the intervention, the autophagy flux and related proteins SIRT1, LC3Ⅱand p62 changed significantly, and the autophagy level increased significantly. However, EX527 weakened the effect of Chinese medicine in delaying vascular senescence. GNS may delay the senescence of vascular endothelial cells through the SIRT1 autophagy pathway.

[Effect of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts on intestinal flora of vascular aging mice induced by high glucose and high lipid].

The aim of this paper was to observe the changes of intestinal flora in vascular aging mice, in order to explore the relationship between vascular aging and intestinal flora and the effects of extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on intestinal flora of vascular aging mice. A model of vascular aging in mice was induced through intrape-ritoneal injection with streptozotocin(STZ) combined with high-fat diet. Biochemical detection was performed on serum cholesterol(CHO), triglyceride(TG), high-density liptein cholesterol(HDL-C), low-density liptein cholesterol(LDL-C) and blood glucose(GLU). HE staining was used to detect mice thoracic aorta morphology, and the expressions of cyclin-dependent kinase inhibitor 2 A(p16) and cyclin-dependent kinase inhibitor 1 A(p21) protein in mice thoracic aorta were detected by Western blot. The 16 S rDNA gene of mice intestinal flora was detected by Illumina MiSeq high-throughput sequencing technology to explore the changes of intestinal flora in each group. The results demonstrated that the GLU level in low-dose and high-dose TCM groups decreased, but with unobvious changes in blood lipid indexes. Metformin could significantly decrease the levels of GLU(P<0.01), CHO and LDL-C in mice(P<0.05). Intravascular injury was not obvious in each drug group, and the expressions of p16 and p21 protein were significantly decreased(P<0.05). The intestinal flora of each group was mainly composed of Firmicutes(F) and Bacteroidetes(B) at the level of the phylum, but the B/F ratio was different from that of the youth group and the blank control group. The B/F ratio of the model group was significantly lower(P<0.01), and compared with the model group, the B/F ratio of the high-dose group and the metformin group was signi-ficantly higher(P<0.05). There were dominant and differential floras in the intestine of each group of mice. The results showed that extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma could improve the intestinal flora structure and create a good intestinal environment by increasing the B/F ratio, which provides a new possible pathway for lowering blood glucose and blood lipids and delaying vascular aging.

[Effect of microRNA-34a/SIRT1/p53 signal pathway on notoginsenoside R1 delaying vascular endothelial cell senescence].

This study aimed to investigate the effect of notoginsenoside R1 in delaying H2O2-induced vascular endothelial cell senescence through microRNA-34a/SIRT1/p53 signal pathway. In this study， human umbilical vein endothelial cells(HUVECs) were selected as the study object; the aging model induced by hydrogen peroxide(H2O2) was established， with resveratrol as the positive drug. HUVECs were randomly divided into four groups， youth group， senescence model group， notoginsenoside R1 group and resveratrol group. Notoginsenoside R1 group and resveratrol group were modeled with 100 µmoL·L⁻¹ H2O2 for 4 h after 24 h treatment with notoginsenoside R1(30 µmoL·L⁻¹) and resveratrol(10 µmoL·L⁻¹) respectively. At the end， each group was cultured with complete medium for 24 h. The degree of cellular senescence was detected by senescence-associated ß-galactosidase(SA-ß-Gal) staining kit， the cell viability was detected by cell counting kit-8， the cell cycle distribution was analyzed by flow cytometry， and the cellular SOD activity was detected by WST-1 method in each group. The expressions of SIRT1， p53， p21 and p16 proteins in HUVECs were detected by Western blot. In addition， the mRNA expressions of miRNA-34a， SIRT1 and p53 in HUVECs were assayed by Real-time PCR. These results indicated that notoginsenoside R1 significantly reduced the positive staining rate of senescent cells， enhanced the cell proliferation capacity and intracellular SOD activity， decreased the proportion of cells in G0/G1 phase， and increased the percentage of cells in S phase simultaneously compared with the senescence model group. Moreover， notoginsenoside R1 decreased the mRNA expressions of miRNA-34a and p53 and the protein expression of p53， p21 and p16.At the same time， notoginsenoside R1 increased the protein and mRNA expressions of SIRT1. The differences in these results between the senescence model group and the notoginsenoside R1 group were statistically significant(P<0.05). However， there was not statistically significant difference in these results between the notoginsenoside R1 group and the resveratrol group. In conclusion， the senescence of endothelial cells induced by H2O2 can be used as a model for studying aging. Notoginsenoside R1 has an obvious anti-aging effect on vascular endothelial cells in this study. The possible mechanism is that notoginsenoside R1 can delay the senescence process of vascular endothelial cells induced by H2O2 by regulating microRNA-34a/SIRT1/p53 signal pathway.

[Effects of ginseng, notoginseng, and Chuanxiong Rhizoma extracts on cytoskeleton protein of replicative senescence vascular smooth muscle cells].

To observe the effect of extracts of ginseng, notoginseng, and Chuanxiong Rhizome on the cytoskeleton protein F-actin and G-actin of the replicative senescence vascular smooth muscle cells, with human aortic smooth muscle cells (HASMC) as the research object, and the replicative senescence 9th generation cells as the senescence models, the experiment was divided into youth group (5th generation cells), model group (9th generation cells), Chinese medicine low dose group (100 mg•L⁻¹), middle dose group (200 mg•L⁻¹), and high dose group (400 mg•L⁻¹) and resveratrol group (10 µmol•L⁻¹). The intervention time was 48 h. ß-Galactosidase specific staining method was used to calculate the ratio of blue dyeing cells. CCK-8 method was used to detect the cells proliferation. The flow cytometry was used to analyze the cell cycle. Immunofluorescent staining was used to observe morphological changes of F-actin and G-actin. The western blot assay was used to determine the expression of F-actin protein. Compared with the model group, the Chinese medicine groups and resveratrol group significantly reduced the number of blue dyeing cells, improved the ability of cells proliferation, reduced the number of cells in G0/G1 phase, increased the number of cells in S phase, and reduced the protein expression of F-actin and the formation of stress fibers, with obvious intervention effect and statistically significant difference. Therefore, the replicative senescence vascular smooth muscle cells can be used as the models for senescence research, with significant changes in morphology and protein expression of cytoskeleton protein F-actin and G-actin in the process of cells aging. The extracts of ginseng, notoginseng, and Chuanxiong Rhizome have obvious intervention effect on F-actin and G-actin, and it might be indirectly associated with delaying the aging of blood vessels.

Ginseng-Sanqi-Chuanxiong (GSC) Extracts Ameliorate Diabetes-Induced Endothelial Cell Senescence through Regulating Mitophagy via the AMPK Pathway.

Vascular endothelial senescence induced by high glucose and palmitate (HG/PA) contributes to endothelial dysfunction, which leads to diabetic cardiovascular complications. Reduction of endothelial senescence may attenuate these pathogenic processes. This study is aimed at determining whether Ginseng-Sanqi-Chuanxiong (GSC) extracts, traditional Chinese medicine, can ameliorate human aortic endothelial cell (HAEC) senescence under HG/PA-stressed conditions and further explore the underlying mechanism. We found that GSC extracts significantly increased antisenescent activity by reducing the HG/PA-induced mitochondrial ROS (mtROS) levels in senescent HAECs. GSC extracts also induced cellular mitophagy formation, which mediated the effect of GSC extracts on mtROS reduction. Apart from this, the data showed that GSC extracts stimulated mitophagy via the AMPK pathway, and upon inhibition of AMPK by pharmacological and genetic inhibitors, GSC extract-mediated mitophagy was abolished which further led to reverse the antisenescence effect. Taken together, these data suggest that GSC extracts prevent HG/PA-induced endothelial senescence and mtROS production by mitophagy regulation via the AMPK pathway. Thus, the induction of mitophagy by GSC extracts may provide a novel therapeutic candidate for cardiovascular protection in metabolic syndrome.