| As one kind of cardiovascular disease, atherosclerosis emerges as a leading cause of death in developed countries, and its morbidity and mortality continue to grow in China in recent years. In the case of adverse drug reaction and the restricting on comprehensive application of anti-atherosclerosis drugs like statin, it is an effective strategy to find compounds with the biological activities to block and delay process of atherosclerosis in natural herbs or traditional medicinal medicine, which plays an important role in the anti-atherosclerosis treatment, and may bring great benefits both on scientific researches and atherosclerosis patients.Vine tea, which was recorded to have the functions of clearing away heat and promoting diuresis, promoting blood circulation as well as removing obstruction in channels (Chinese Material Medica, the Editorial Committee of Chinese Material Medica,1999), has been widely used for centuries in traditional Chinese medicine to treat a number of diseases. Dihydromyricetin, a fiavonoid that can be extracted from vine tea, has a content of more than30%in the tender stems and leaves of (Ampelopsis grossedentata)(Hand-Mazz) W.T.wang. While dihydromyricetin has been reported to have anti-inflammatory, antioxidative, anti-cancer, antimicrobial, and antihypertension as well as hepatoprotective effects, its mechanism of anti-atherosclerosis activities has not been explored.In order to investigate the potential anti-atherosclerosis effects of dihydromyricetin, we studied its endothelial cell protection activities and anti-inflammatory activities, as well as the underlying mechanism. Further, we predicted and analyzed the protein targets of its anti-atherosclerosis effects and other potential pharmacology activities by network pharmacology.Chapter1Dihydromyricetin protects endothelial cells from hydrogen peroxide-induced oxidative stress damageAims:To investigate the cytoprotective effects of dihydromyricetin on hydrogen peroxide (H2O2)-induced oxidative stress damage in human umbilical vein endothelial cells (HUVECs).Methods:The percentage of cell viability was evaluated. We determined the antioxidant properties of dihydromyricetin by measuring the activity of superoxide dismutase (SOD) and Malondialdehyde (MDA). Flow cytometry was used to measure apoptosis in HUVECs that were double stained with Hoechst33342. The generation of intracellular reactive oxygen species (ROS) was measured in2’,7’-dichlorofluorescin diacetate (DCFH-DA)-loaded HUVECs using a fluorescent microscope. In addition, the release of nitric oxide (NO) was analyzed using a commercial kit.Results:HUVECs treated with H2O2had a notable decrease in cell viability that was attenuated when cells were pretreated with dihydromyricetin (37.5-300μM). Dihydromyricetin pretreatment significantly attenuated H2O2-induced apoptosis in HUVECs, inhibited intracellular ROS overproduction.Conclusions: Our study report that dihydromyricetin can protect HUVECs from oxidative stress damage, and may prevent atherosclerosis at the first step of atherosclerotic lesions.Chapter2Dihydromyricetin protects endothelial cells from hydrogen peroxide induced oxidative stress damage by regulating mitochondrial pathwayAims:To explore the possible mechanisms of DMY cytoprotective effects on hydrogen peroxide (H2O2)-induced oxidative stress damage in HUVECs.Methods:HUVECs were grown to confluence, pretreated with dihydromyricetin (37.5-300μM) for2h, and then exposed to H2O2(400μM)) for an additional1h or24h. The expression of apoptosis-related proteins were determined by Western blotting. In addition, the release of nitric oxide (NO) was analyzed using a commercial kit.Results:Pretreatment of cells with dihydromyricetin prior to H2O2exposure resulted in inhibition of p53activation, followed by regulation the expression of Bcl-2and Bax, the release of cytochrome c, the cleavage (activation) of caspase-9and caspase-3, and then the suppression of PARP cleavage in H2O2-induced HUVECs.Conclusions:Our study found that dihydromyricetin can protect HUVECs from oxidative stress damage, an effect that is mediated by the mitochondrial apoptotic pathways.Chapter3The anti-inflammatory effects of dihydromyricetin in vitro and in vivoAims:In this study, we explored the anti-inflammatory activity of dihydromyricetin both in vitro and vivo. Methods:To estimate the serum TNF-a, IL-1β, IL-6and IL-10levels in the LPS-challenged mice, the TNF-a, IL-1β, IL-6and IL-10production in LPS-stimulated Raw264.7macrophages, as well as the effect of DMY on TPA-induced ear oedema.Results: We demonstrated that dihydromyricetin suppressed the levels of pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), interleukin-6(IL-6) as well as increased the level of the anti-inflammatory cytokine interleukin-10(IL-10) in lipopolysaccharide (LPS)-treated mice. Moreover, DMY was found to markedly inhibit the production of nitric oxide (NO), the levels of TNF-a, IL-1β and IL-6, whereas it increased IL-10in lipopolysaccharide (LPS)-induced Raw264.7macrophage cells.Conclusions:We reported that dihydromyricetin could suppression inflammatory reaction both in vitro and vivo, and may prevent atherosclerosis by inhibiting the pro-inflammatory cytokines, as well as increased the anti-inflammatory cytokine released by macrophages in the process of atherosclerosis.Chapter4Suppression of inflammatory responses by dihydromyricetin via inhibiting NF-κB activation and the MAPK signalling pathwayAims:To explore the underlying mechanism of anti-inflammatory effects of dihydromyricetin.Methods:Cells were incubated with various concentrations of dihydromyricetin for2h, and then were stimulated with LPS (1μg/mL) for another1h or24h. Anti-GADPH,β-actin, iNOS, COX-2, TNF-a, phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-IκBa, IκBa, phospho-NF-KB p65were investigated by western blot.Results:Dihydromyricetin reduced the expression of inducible nitric oxide synthase (iNOS), TNF-a and cyclooxygenase-2(COX-2) in macrophage cells. Furthermore, Dihydromyricetin suppressed the phosphorylation of NF-κB and IκBa, as well as the phosphorylation of p38and JNK but not ERK1/2in LPS-stimulated macrophages.Conclusions:The results suggest that dihydromyricetin exerts its topical anti-inflammatory action through suppressing the activation of NF-Iκ via inhibition of the phosphorylation of p38and JNK. Thus, Dihydromyricetin may be a potentially useful therapeutic agent for atherosclerosis. Chapter5Predicting molecular targets for dihydromyricetin using network pharmacologyAims:To predict molecular targets of dihydromyricetin in atherosclerosis, as well as molecular targets in other diseases.Methods:Compounds with a structural similarity of more than95%compared with dihydromyricetin were retrieved by PubChem data base.Results:Based on the retrieved results in PubChem data base and the reported bioactivities of dihydromyricetin, we predicted that Matrix metalloproteinase-9, glutathione S-transferase and heat shock protein beta could be influenced in the process of atherosclerosis. Further, we predicted that the hypoglycemic bioactivities of dihydromyricetin may due to inhibit glycogen phosphorylase, and the icteric hepatitis effects of vine tea may be related with its influence on alkaline phosphatase.Conclusions:Our findings demonstrate that network pharmacology can not only identify potential molecular targets on the anti-atherosclerosis effects of dihydromyricetin, but also predict the molecular targets of dihydromyricetin on other diseases. |
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