Absorption Of Delphinidin-3-glucoside In Vascular Endothelial Cells And The Its Protective Effects Against The Endothelial Injury

Cardiovascular and cerebrovascular diseases caused by atherosclerosis (AS) havebecome a class of major diseases which have serious threat to human life. AS is amulti-factor and multi-stage complex pathological process, accordding to Ross,“oxidativedamage and the inflammatory response doctrine” is the most important theory to explainthe pathogenesis of AS. The theory demonstrates that oxidized low-density lipoprotein(oxLDL) inducing vascular endothelial cell injury is the initiative step of AS.. oxLDL caninduce oxidative stress, endothelium-dependent vasodilation function decline, thrombosisand inflammation, subsequently, leading to AS. In recent decades, more and more studiesshow that AS are closely related to unhealthy lifestyles, particularly unreasonable dietaryhabits. World Health Organization demonstrated that nearly50%of AS can be prevented byimproving diet and other lifestyle, suggesting tips on how to effectively prevent AS throughchanging diet pathway has important theoretical and practical significance. Epidemiologicalsurveys show that vegetables, fruits and other plant food intake can significantly reduce theincidence of AS and coronary heart disease, suggesting that plant foods may contain somepreventable ingredients of AS. Further studies show that flavonoids are an important classof biologically preventable ingredients of AS in plant foods. Flavonoids are naturalpolyphenols, containing various classes. Accordding to its structure, it was divided into sixcategories: flavones, flavonols, flavanones, flavanols, isoflavones and anthocyanins, amongwhich anthocyanins are natural pigments, widely found in all kinds of colored fruits andvegetables. Intervention study in humans showes that daily intake of600mg anthocyaninscan significantly increase total plasma antioxidant and reduce vascular inflammatorymediators, thereby decreasing the incidence of AS and coronary heart disease. However, theanti-AS-related mechanisms of anthocyanins are not yet fully elucidated. On the one hand, metabolism and bioavailability studies have found that anthocyanins have lowbioavailability due to their rapid clearance rate and short half-life in vivo, which forming aclear contrast to their significant cardiovascular protective effect, suggesting that there maybe unique effective uptake and tissue targeting distribution of anthocyanins in endothelialcells or vascular wall tissue. On the other hand, recent studies have found that vascularendothelial cell autophagy plays an important role in the development of AS. Moderateautophagy can protect endothelial cells or plaque cells against oxidative stress damagethrough degradation of intracellular oxidative damage contents, especially polarizedmitochondria, thereby, inhibiting AS. Whether Anthocyanins are able to prevent endothelialcells from oxidative damage through inducing autophagy, as well as the related mechanismsneed further studies.We previously investigated21anthocyanin compounds with pharmacodynamicscreening test and screened out delphinidin-3-glucoside (Dp), which was most effective oninhibiting vascular endothelial oxidative injury (). Based on the previous work, the presentstudy aimed to investigate the uptake of Dp and the molecular mechanisms involved in theinhibitive effects on endothelial oxidative damage. First, the transmembrane transport anddistribution of Dp in vascular endothelial cells were studied with animal model and theprimary cultured human umbilical vein endothelial cells (HUVECs),throughhigh-performance liquid chromatography (HLPC) and confocal laser scanning microscopyassays. We found that sodium-dependent glucose transporter (SGLT1) plays an importantrole in Dp transmembrane transport. Second, The effect of Dp on endothelial proliferation,apoptosis, ROS genearation and mitochondrial oxidative damage were observed withoxLDL-induced endothelial cellsin vitro. Finally, we used electron microscopy, confocallaser scanning microscopy and western blot assays to explore the effects of Dp on cellautophagy in endothelial cells and the role of AMPK/SIRT1/autophagy-mediated signalingpathways.The results are summarized as follows:(1) Depending on autofluorescence characteristics of Dp, our study showed thatHUVECs effectively absorbed Dp through active and passive elements and Dp existed as the prototype form in cells; intracellular Dp are mainly distributed in cytoplasm andmembrane of endothelial cells; After intravenous injection of Dp in mice, it was found thatDp was permanently available in blood vessels. Immunofluorescence cytochemistry foundthat SGLT1was expressed in endothelial cell membrane, knockdowm SGLT1resulting insignificant decrease in Dp uptake, suggesting that the absorption of Dp is at least partlydependent on SGLT1activity in entothelial cells(2) Flow cytometry detection found that Dp can significantly inhibit apoptosisinduced by oxLDL in HUVECs; confocal laser scanning microscopy revealed that oxLDLcan cause mitochondrial membrane potential (△ψ) reduction and mitochondrialpermeability transition pore (mPTP) remain open, and Dp can concentration-dependentlyinhibite the△ψ reducing and mPTP remain open causing by oxLDL, suggesting that Dpcan inhibit oxLDL-induced mitochondrial dysfunction; then we examined the intracellularROS and O2and found that oxLDL can significantly increase the levels of ROS and O2,Dp can cause dose-dependent inhibition of oxLDL induced increase in ROS and O2. Theseresults suggested that Dp may reduce mitochondrial damage through inhibiting the increaseof free radicals induced by oxLDL. Finally, via western-blot, we found that oxLDL canincrease pro-apoptotic factor AIF, Cyt C, Bax expression and decrease the anti-apoptoticfactor Bcl-2expression, while increasing apoptotic protein Caspase-3expression;Furthermore, Dp can inhibit the expression of the above apoptotic proteins induced byoxLDL, suggesting that Dp can inhibit oxLDL-induced mitochondrial apoptosis pathway.(3) Dp pretreatment can dose-dependently increase autophagy under oxLDLstimulation in endothelial cells, thereby enhancing protective activity against oxidativestress response; confocal double staining detection found that Dp can induce mitophagy inendothelial cells; blocking autophagy could significantly inhibit the protective effect of Dpon oxLDL-caused apoptosis in HUVECs, suggesting that Dp inhibit oxLD-caused oxidativedamage by inducing autophagy in endothelial cells; western-blot experiments found that Dpcan significantly increase pAMPK and SIRT1expression levels; suppression of pAMPKand SIRT1expression can significantly reduce the Dp-induced autophagy, suggesting thatDp induced autophagy through AMPK-SIRT1pathway in endothelial cells. Conclusion:In summary, Dp could be effectively absorbed by vascular endothelial cells and waspermanently available in blood vessels in vivo. Furthermore, Dp could attenuate endothelialoxidative injury through the activation of AMPK/SIRT1/autophagy-related signaling pathway.