Effects Of Conjugates Of Danshensu And Cysteine ​​Derivatives On Antioxidation And Anti - Apoptotic Effects Of HUVECs And SH-SY5Ys

Background and purpose:Danshensu, an active component of Danshen（the dried root of salvia miltiorrhiza）, has been well-known for a lot of biological activities, such as inhibiting platelet aggregation, dilating coronary arteries, myocardial cell apoptosis and anti-inflammatory property. These activities are at least partially related to its anti-oxidation and anti-apoptosis. So Danshensu has been considered as a good anti-oxidant and anti-apoptosis drug candidate in our laboratory for further studies. However, several issues have to be overcome on the therapeutic development of Danshensu. For example, it is difficult to isolate and purify Danshensu from natural resources due to the chemical unstability of phenolic hydroxyl groups, low content in Danshen and a variety of structural analogs as impurities. Several synthetic strategies have been explored to produce （±）-Danshensu. Moreover, being a hydrophilic molecule and thus poorly soluble in lipidic matrices, Danshensu uneasily enters into cells by crossing the cell membranes. Therefore, we spontaneously considered that structural modification of Danshensu may be a potential way to improve its pharmacological efficacy. Combination of two mutually complementary biological active entities into one by conjugation has been widely used in drug design. So we designed3compouds.In this study we attempt to evaluate the anti-apoptosis activity of compounds1,2and3on SH-SY5Ys and HUVECs stimulated by H₂O₂.Experimental approach:On SH-SY5Ys. Some cells were pretreated with50μM compounds1,2,3and5mM NAC for4h prior to incubation with200μM H₂O₂for an additional12h. Untreated cells were used as control. On HUVEC. Some cells were pretreated with5μM,50μM and100μM compound3and5mM NAC for4h prior to incubation with200μM H₂O₂for an additional12h. Untreated cells were used as control.Key results:Compounds1,2and3at50μM significantly increased cell viability, reduced LDH release, decreased MDA content, increased SOD and GSH content and increased the cellular Gpx activity in H2O2-induced SH-SY5Ys. The results above suggested that there were no statistical differences in anti-apoptotic activity of compounds1,2and3. Compound3was much easier to get. so we focused on compound3.In studies of HUVEC. Compound3at50uM and100μM significantly increased cell viability, reduced LDH release, decreased MDA content and increased GSH content. To assess the anti-apoptotic potential of compound3, JC-1staining and PI and FITC double staining detected by flow cytometry were performed to observe the changes in ratio of survival rate/Apoptosis rateCompound3at50μM,100μM significantly increased ratio of survival rate/Apoptosis rate, indicateing compound3could decrease the probability of apoptosis. At the molecular level, Systemic levels of anti-apoptotic protein bcl-2and protein pro-caspase-3,procaspase-9were significantly higher in cells pretreated with compound3at50μM,100μM, pro-apoptotic protein bax was lower in compound3at50μM,100μM treated group compared with the group stimulated by H₂O₂alone. Conclusions and implications:In summary, we have designed and successfully synthesized new derivatives of Danshensu, compounds1,2and3, Pharmacological evaluation has shown its potent anti-apoptotic activity and we assume the mechanism is in partial related to blocking oxidative stress and apoptotic pathways. The results reported here demonstrate that these Danshensu derivatives merit attention as anti-apoptosis agents.