Tongxinluo On Cardiac Microvascular Endothelial Ischemia And Reperfusion Injury Protection Mechanism

Induction of autophagy by Tongxinluo via the MEK/ERK pathway protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injuryBackgrounds and Objectives:In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL) is a Traditional Chinese Medicine formulation that is widely used by more than6million patients with cardio-cerebral vascular diseases in China for its vascular-protective effect. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury.Methods:CMECs were exposed to different treatments for30min and subjected to hypoxia/reoxygenation (H/R) each for2h. The TXL solution at different concentrations, ginsenoside Rgl, paeoniflorin,3-methyladenine (3-MA), rapamycin and PD98059were used to further investigate the role and the modulatory mechanism of autophagy in CMECs.Results:The results indicated that H/R significantly induced autophagy, as identified by an increased number of monodansylcadaverine (MDC)-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain3ratio, but not Beclin-1expression. Autophagy inhibition using3-methyladenine (3-MA) was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis rates in a dose-dependent manner, reaching its largest effect at800μg/mL, which decreased apoptosis from21.04±1.11%to9.92±0.49%.3-MA attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared to TXL alone. TXL upregulated MEK and ERK phosphorylation; however, PD98059abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared to TXL alone.Conclusions:These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy via activation of the MEK/ERK pathway. Traditional Chinese Medicine Tongxinluo modulates the secretion of cytokines in vitro by cardiac microvascular endothelial cells in ischemia/reperfusion injury.Backgrounds and Objectives:Cardiac microvascular endothelial cells (CMECs) regulate the function of cardiomyocytes and blood cells in ischemia/reperfusion injury via autocrine and paracrine methods. Tongxinluo (TXL) is a Traditional Chinese Medicine compound which is constituted of Radix ginseng and other11kinds of natural products. It is proved to improve the function of endothelium and be protective from ischemia/reperfusion injury. Thus, we designed the study to find alterations in paracrine function of CMECs under the hypoxia/reoxygenation (H/R) situation and its modulation by TXL.Methods:CMECs were exposed to different concentrations of TXL for30min and then subjected to H/R for12h/2h. Apoptotic rates of the cells were measured to determine the optimal concentration. Protein antibody arrays were used to find the alterations of cytokines in conditioned medium (CM) secreted by CMECs. Gene Ontology (GO) project was adopted to describe the functions of changed cytokines.Results:TXL inhibited apoptosis of CMECs dose-dependently under H/R and reached its peak effect at the dose of800μg/mL, which reduce cell apoptosis from35.15±0.78%to14.64±2.23%. Thirty-three types of cytokines were significantly changed by H/R (19factors decreased and14increased), and TXL at800μg/ml changed121types of cytokines compared to the H/R group (93factors decreased and28increased). The cytokines with significant alterations were involved in cell differentiation and proliferation, regulation of signalling pathway, and transportation. Among these cytokines,10kinds of cytokines were increased by H/R but were decreased by TXL, such as MCP-2;5were decreased by H/R but increased by TXL, such as Follistatin;8were attenuated by H/R and further decreased by TXL, such as bFGF; IGFBP-1were upregulated by H/R and further increased by TXL.Conclusions:TXL inhibited apoptosis of CMECs and modulated the paracrine function of the cells in ischemia/reperfusion injury.