痰瘀同治法对糖尿病大鼠心肌微血管病变AGEs/RAGE轴及氧化应激的影响

目的 观察痰瘀同治对高糖诱导心肌微血管内皮细胞损伤的保护作用并从AGEs/RAGE轴探讨其作用机制。方法 利用酶消化法从大鼠心脏中分离出原代心肌微血管内皮细胞（CMECs），经免疫荧光法鉴定后，培养传代备用。在不同时间段采用不同浓度的葡萄糖对大鼠CMECs进行诱导干预，MTT检测方法发现葡萄糖浓度在33 mmol/L且干预48 h为最佳造模条件；予不同浓度的含药血清，同上法检测得10%含药血清干预48 h为最佳干预方法。将造模成功的CMECs分成5组：模型组（MC）、化痰组（RP）、化瘀组（DBS）、痰瘀同治组（RPDBS）、ALT-711组，予以10%含药血清和ALT-711细胞干预制剂进行干预；另设一组空白对照组（NC）。采用ELISA法检测各组内皮细胞AGEs含量；免疫荧光法、Western blot法和RT-qPCR法检测各组内皮细胞RAGE蛋白及基因表达水平；细胞色素c还原法检测各组内皮细胞还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性水平；二氢乙锭荧光探针法检测各组内皮细胞活性氧自由基（ROS）活性水平。结果 MC组较NC组，细胞内晚期糖基化终末产物（AGEs）含量、RAGE蛋白及基因表达水平、NADPH氧化酶活性水平和ROS活性水平均升高（P<0.01）。与MC组比较，各治疗组的上述指标均降低（P<0.01）。在降低AGEs含量、RAGE蛋白及基因表达水平和NADPH氧化酶活性水平上，RPDBS组较RP组与DBS组显著下降（P<0.01，P<0.05）；在降低ROS水平上，RPDBS组与DBS组差异无统计学意义（P>0.05），较RP组显著下降（P<0.01）。结论 痰瘀同治法可能通过抑制AGEs/RAGE轴及氧化应激对高糖损伤的CMECs起到保护作用，从而防治糖尿病心肌微血管病变，且痰瘀同治法优于单独化痰法和单独化瘀法。

Effect of dissipating phlegm and blood stasis simultaneously on AGEs/RAGE axis and oxidative stress in rats with diabetic myocardial microangiopathy

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.