| The first section:The interventional effect of traditional medicine that can Tonifying Qi and Activating Blood on natural aging rats’thoracic aorta and cytoskeleton.Objective:To observe the effect of extracts of ginseng, notoginseng, and Chuanxiong Rhizome on the thoracic aorta and skeleton protein of the natural aging rats.Method:The thoracic aorta of natural aging rats as the research object, and 21-month-old rats are used as aging model. The experiment is divided into model group, low dose, middle dose and high dose of herbal treat group and resveratrol group. The intervention group are administered from the 18th month, and last 3 months. The concentration of administration is low dose(0.58g/kg), middle dose(1.16g/kg), and high dose(2.32g/kg) of herbal treat group and resveratrol group(50mg/kg). The model group is given the same volume of pure water, and 3-month-old rats are used as control group. HE staining is used to observe the changes of vascular morphology, and the SA-β-gal is used to identificate the aging degree by calculating blue dye cell ratio. To observe the configuration changes of F-actin by the immunofluorescent staining, and to test the expression of protein of F-actin and SM22a by the western blot.Result:Compared with the control group, the intima of modle group was injury and not smooth, at the same time the middle and outer membrane was hyperplasia obviously and arranged disorder. The elastic plate was twisted, broken, and the structure was not complete. The blue dye cell ratio of SA-β-gal staining increased significantly. The fluorescence intensity and protein of F-actin increasee and the protein of SM22a decreased. The drug intervention reduced the dyed blue cell number effectively, and reduced the fluorescence intensity and protein expression of F-actin, and enhanced the protein expression of SM22a. The drug intervention improved the damage of intima and reduced hyperplasia of the middle and outer membrane.Conclusion:The configuration and protein expression of F-actin and SM22a changed obviously and might be involved in the process of cell aging; The extracts of ginseng, notoginseng, rhizome ligustici wallichii had obvious intervention effect on the F-actin and SM22a, and it might be delay the aging of vessels.The second section:The interventional effect of traditional medicine that can Tonifying Qi and Activating Blood on the replicative senescence vascular smooth muscle cells and cytoskeleton.Objective:To observe the effect of extracts of ginseng, notoginseng, and Chuanxiong Rhizome on the cytoskeleton protein F-action and G-action of the replicative senescence vascular smooth muscle cells.Method:With human aortic smooth muscle cells as the research object, and the replicative senescence 9th generation cells as the senescence models, the experiment was divided into youth group(5th genernation cells),model group(9th generation cells), chinese medicine low dose group(100mg · L-1),middle dose(200mg · L-1),high dose(400mg · L-1) and resveratrol group(10umol/L). The intervention time was 48h. β-Galactosidase specific staining method was used to calculate the ratio of blue dyeing cells. CCK-8 medthod 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-action and G-action and SM22a. The western blot assay was used to determine the expression of F-actin and SM22a protein.Result:Compared with the youth group, the cell size was increased and shape was irregular in model; number of cells in G0/G1 stage was increased and number of cells in S stage was reduced; The ability of CCK-8 to detect the proliferation was decreased, and the number of blue-dyed cells in β-galactosidase staining method was increased, conforming to the characteristics of aging model; F-actin was partially longitudinally arranged in parallel, forming a large number of stress fibers, and the protein expression increased. The staining of G-actin in the central regions of the cells become weak; expended to surrounding area irregularly and part of them become smudged and cavity or showed raincidness flocculent. SM22a staining was funny and bleak, and the fluorescence intensity was weakened significantly, with reduced mRNA and protein expression of SM22a. As compared with the model group, drug intervention effectively reduced the number of blue-dyed cell, reduced the number of cells in G0/G1, increased the number of cells in S stage, Reduce the F-actin protein expression and the formation of stress fibers. Enhance the fluorescence staining intensity of SM22a, and increased protein expressions of SM22a, with statistically significant difference.Conclusion:The replicative senescence vascular smooth muscle cells can be used as aging research models. The morphology, gene and protein expressions of SM22a were changed obviously in the process of cell aging, and it may involve in the process of cell aging together with cytoskeleton. The extracts delayed the aging of vascular smooth muscle cells to a certain extent and had obvious intervention effect on SM22a and F-actin, so it might delay the aging of vessels with the cytoskeleton.The third section:The discussion of mechanism on interventional effect of traditional medicine that can Tonifying Qi and Activating Blood on the replicative senescence vascular smooth muscle cells and cytoskeleton.Objective:The discussion of mechanism on extracts of ginseng, notoginseng, and Chuanxiong Rhizome on the expressions of replicative senescence vascular smooth muscle cells and cytoskeleton.Method:With human aortic smooth muscle cells as the research object, Using gene transfection technique, Establish HSP27 gene knockdown aging model. The experiment was divided into six groups, gene unknockdown aging group, gene unknockdown herbal group, gene unknockdown resveratrol group, gene knockdown aging group, gene knockdown herbal group, gene knockdown resveratrol group. Use drug intervention for 48 hours. β-galactsidase specific staining method was used to calculate the ratio of blue dyeing cells. Immunofluorescent staining was used to observe morphological changes of F-action and SM22a. The weatern blot assay was used to determine the protein expression of F-actin and SM22a.Result:Compared with the HSP27 gene unknockdown aging group, the number of HSP27 knockdown gene blue staining cells that stained by SA-β-gal staining increased. The fluorescence intensity and protein expression of F-actin and SM22a was significantly reduced and the differences were statistically significant. The knockdown gene groups have no statistical significance.Conclusion:With knockdown the smooth muscle cells’ HSP27 gene, the phosphorylated HSP27 falls, the F-actin cytoskeletal and SM22a protein expression decreased, the number of blue staining cells that stained by SA-β-gal staining increased. Clear the regulation of HSP27 gene on the cytoskeleton, involved in the regulation of vascular aging process indirectly. HSP27 was no significant difference in each group after the knockdown drug intervention, Ginseng and Radix Notoginseng Chuanxiong extracts’ targets may have a relationship with and HSP27/ phosphorylated and HSP27/F-actin pathway.The fourth section:The interventional effect of traditional medicine that can Tonifying Qi and Activating Blood on the vascular smooth muscle cells induced autophagy.Objective:Observe the extractive of ginseng radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma’s intervention on Replicative aging and starvation-induced autophagy vascular smooth muscle cells.Method:Using replicative senescence smooth muscle cells as object, divided the experiment into four groups, adults group, aging group, herbal group and Resveratrol group, drug intervention for 48 hours. Use transmission electron microscope to observe the formation of autophagy body. Use western blot to detection the expression of autophagy-related protein Beclinl protein and LC3-II.Use hunger to induced autophagy to establish a vascular smooth muscle cells mode afterl a hunger for 2 hours.Result:Compared with the adults group, the aging protein expression of Beclinl and LC3-Ⅱ decreased obviously that have significant difference. Compared with the aging group, the herbal group and resveratrol group protein expression of Beclinl and LC3-Ⅱ increased obviously, there have statistically significant. Compared with normal group, the cells of hunger model group are irregular deformation, and the cell membrane are damaged, the chromatin are In granular form, double membrane structure occurred in the cytoplasm of autophagy and autophagic vacuoles, secondary lysosome increased. protein expression of Beclinl and LC3-Ⅱ increased obviously. Compared with the aging group, the shape of the herbal group and resveratrol group can still. Nuclear membrane are relatively smooth and complete; Cytoplasm produce large amounts of autophagy, autophagic vacuoles and secondary lysosomes, the expression of Beclinl and LC3-Ⅱ increased, which have significant difference.Conclusion:The autophagy levels of replicative senescence smooth muscle cells are reduced. The extractive of ginseng radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma can enhance the autphagy levels of autophagy cells; replicative aging and starvation-induced autophagy vascular smooth muscle cells. The extractive of ginseng radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma may enhance the autphagy levels to delay vascular aging. |
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