Abstract
OBJECTIVE: To investigate the effect of dissipating phlegm and blood stasis simultaneously for protecting cardiac microvascular endothelial cells (CMECs) against high glucose-induced injury and the role of AGEs/RAGE axis in the underlying mechanism. METHODS: The primary CMECs were isolated from rat heart by enzymatic digestion and identified by immunofluorescence assay. The CMECs exposed to 33 mmol/L glucose for 48 h were divided into model group (MC), resolving phlegm (RP) group, dissipating blood stasis (DBS) group, dissipating phlegm and blood stasis (RPDBS) group and ALT-711 group. After treatment with 10% drug-containing serum and ALT-711 for 48 h, the content of AGEs in the cells were measured with ELISA. The expressions of RAGE mRNA and protein were measured with real-time quantitative PCR, immunofluorescence assay and Western blotting; The activity of NADPH oxidase and ROS level were measured by cytochrome c reduction and fluorescent probe DHE. RESULTS: High glucose exposure significantly increased the content of AGEs, RAGE expressions at the protein and mRNA levels, NADPH oxidase activity and ROS level in the CMECs (P < 0.01). These changes were significantly mitigated by treatments with RP, DBS, RPDBS and ALT-711 (P < 0.01), among which RPDBS caused the most significant decrements in AGEs content, RAGE expression and NADPH oxidase activity (P < 0.01, P < 0.05). The reduction of ROS level in the RPDBS group was significantly greater than that in RP group (P < 0.01), but similar to that in DBS group (P > 0.05). CONCLUSION: Dissipating phlegm and blood stasis simultaneously can be helpful for prevention and treatment of diabetic myocardial microangiopathy by suppressing the excessive activation of AGEs-RAGE signal axis and oxidative stress, thus protecting CMECs against high glucose-induced damage. Dissipating phlegm and blood stasis simultaneously is better than either of the therapy alone.