| Aging is a worldwide problem for humanity. It is predicted that the incidence of cardiovascular diseases increase as the population ages despite effective treatments for several established cardiovascular risk factors. This apparent incongruity results from the fact that age is associated with both morphological and functional changes in the vasculature. "Vascular age" rather than chronological age is the best predictor of cardiovascular events and mortality. Therefore, the research of vascular aging is significant. The pathological changes of vascular aging are related to intima, media and adventitia. The biological changes of endothelial cells play an important role in the progression of vascular aging. Cytoskeleton is fibrous protein system in cytoplasm. Its changes are related to the permeability, systolic and diastolic function of blood vessels, apoptosis and signal transduction in endothelial cells. Therefore, this study mainly analyzes the pathology change of the cytoskeleton in endothelial aging, researches the effect and mechanism that Chinese herbal medicines for nourishing qi and resolving blood stasis delay endothelial senescence.The first part:The establishment of replicative senescence model in microvascular endothelial cell and the observation of the cytoskeleton in replicative senescence modelObjective:To observe the characteristics of the aging and F/G-actin changes in replicative senescence model of human cardiac microvascular endothelial cells (HCMEC).Method:Combined with the aging β-galactosidase staining aging, detecting cell proliferation by CCK-8, and analyzing cell cycle by flow cytometry, we established replicative senescence model of HCMEC. Laser scanning confocal microscope was used to observe the changes of F/G-actin.Results:Compared with 5 passage cells, the morphology of 8 passage cells was mainly characterized by cellular enlargement and flattening with a concomitant increase in the size of the nucleus and nucleoli, and an increase in the number of cytoplasmic granules. In addition to the morphological changes, the proportion of aging staining accounted for about 80%, the ability of cell proliferation reduced, the proportion of G0/G1 phase increased, the proportion of S phase and G2/M phase decreased, and the ability to split decreased. In this condition,8 passage HCMECs can be used as replicative senescence model. F-actin was mainly distributed in the edge of endothelial cell, which displayed typical cobblestone contour; G-actin was mainly distributed in the central region of endothelial cells, which was shown a dense rounded oval in 5 passage HCMECs. The F-actin around the cell periphery became irregular, tended to disappear, jagged fracture and stress fiber gradually formed. The G-actin around nucleus of cell stretched to the cell periphery, an increase in the number of punctate staining in cytoplasmic domain. This shew that the complete structure of F-actin and G-actin disrupted, the proportion of the conversion from G-actin to F-actin increased, the mean optical density of the F/G-actin ratio decreased.Conclusion:Eight passage HCMECs can be used for the research of replicative senescence. The form and structure of F-actin and G-actin damaged, the proportion of the conversion from G-actin to F-actin increased, and stress fiber gradually formed in replicative senescence model.The second part:The intervention effects of Chinese herbal medicines for nourishing qi and resolving blood stasis on cytoskeletal proteins in replicative senescence model of microvascular endothelial cellObjective:To observe the effects of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong on aging, F-actin and vascular endothelial cadherin (VE-cadherin) in replicative senescence model of microvascular endothelial cell.Method:The replicative senescence model was established in vitro. The cultured HCMECs in vitro were divided into 6 groups, i.e.5 passage cells (youth group),8 passage cells (old group), resveratrol group (10 umol/L), herbal treated low dose group (50mg/L), herbal treated Intermediate dose group (100mg/L), and herbal treated high dose group (200mg/L). The intervention time of drug was 48h. The aging was identified by senescence β-galactosidase staining kit. The cell proliferation was detected by CCK-8. The cell cycle was analyzed by flow cytometry. Supernatant NO levels in cell was detected by nitrate reductase. The morphological changes of the F-actin, G-actin and VE-cadherin were observed by Laser scanning confocal microscope. The protein and gene expression of F-actin, VE-cadherin and heat shock protein 27(HSP27) were observed by real-time PCR and western blot.Results:Compared with the old group, the extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could decrease the proportion of aging stained, promote cell proliferation, reduce the proportion of G0/G1 phase, increase the proportion of S and G2/M phase, and increase the secretion of NO in cell supernatants. In addition, the extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong maintained F-actin distribution in the cell periphery, reduced the formation of stress fibers, maintained complete and clear structure of VE-cadherin, increased mean optical density value of F/G-actin and VE-cadherin. The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong also down-regulated the protein expression of F-actin, up-regulated the protein expression of VE-cadherin and HSP27.Conclusion:The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong effectively delayed microvascular endothelial cell aging, maintained the functions of endothelial cells. The mechanism may be related to the down-regulation of the protein expression of F-actin, up-regulation of the protein expression of VE-cadherin and HSP27.The thrid part:The mechanisms of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong on F-actin and VE-cadherin in replicative senescence model of microvascular endothelial cellObjective:To observe the mechanisms of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong on actin and VE-cadherin in replicative senescence model of microvascular endothelial cell.Method:By using lentiviral interference, we established the replicative senescence model of HCMECs after HSP27 gene silenced. They were divided into mock group and unknockdown groups:old group, resveratrol group (10 umol/L), herbal treated low dose group (50mg/L), herbal treated Intermediate dose group (100mg/L), and herbal treated high dose group (200mg/L). They also included gene knockdown cells:HSP27shRNA group (old group), HSP27shRNA+resveratrol group, HSP27shRNA+herbal treated low dose group, HSP27shRNA+herbal treated Intermediate dose group, and HSP27shRNA+ herbal treated high dose group. The intervention time of drug was 48h. The aging was identified by senescence P-galactosidase staining kit. The cell proliferation was detected by CCK-8. The cell cycle was analyzed by flow cytometry. Supernatant NO levels in cell was detected by nitrate reductase. The morphological changes of the F-actin, G-actin and VE-cadherin were observed by Laser scanning confocal microscope. The protein and gene expression of F-actin, VE-cadherin and heat shock protein 27(HSP27) were observed by real-time PCR and western blot.Results:Compared with mock groups and control groups, the proportion of aging stained decreased, cell proliferation promote in cells belong to HSP27shRNA+ herbal treated groups. The F-actin around the cell periphery became irregular, jagged fracture gradually occurred and tended to dissipate. Some dynamic actin stress fiber filaments appeared. The G-actin surrounding area of cell stretched to the cell periphery, punctate staining had scattered in cytoplasmic domain. The mean optical density value of F/G-actin and VE-cadherin decreased. The protein expressions of F-actin and VE-cadherin down-regulated in HSP27shRNA+herbal treated groups.Conclusion:The mechanism that the extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong delay microvascular endothelial cell aging was the down-regulation of the protein expression of F-actin and the up-regulation of the protein expression of VE-cadherin by HSP27.In summary,8 passage HCMECs can be used for the research of replicative senescence. The form and structure of F-actin and G-actin damaged, the proportion of the conversion from G-actin to F-actin increased, and stress fiber gradually formed in replicative senescence model. The extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong could decrease the proportion of aging stained, promote cell proliferation and mitosis, increase the secretion of NO in cell supernatants, maintain F-actin distribution in the cell periphery, reduce the formation of stress fibers, maintain complete and clear structure of VE-cadherin, and increase mean optical density value of F/G-actin and VE-cadherin. Besides, the extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong downregulated the protein expression of F-actin, and upregulated the protein expression of VE-cadherin. By HSP27shRNA, the mechanism that the extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chanxiong delayed microvascular endothelial cell aging was closely related to down-regulation of the protein expression of F-actin and up-regulation of the protein expression of VE-cadherin by HSP27. |
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