Effect of Shenmai injection on preventing the development of nitroglycerin-induced tolerance in rats

Long-term nitroglycerin (NTG) therapy causes tolerance to its effects attributing to increased oxidative stress and endothelial dysfunction. Shenmai injection (SMI), which is clinically used to treat cardiovascular diseases, consists of two herbal medicines, Ginseng Rubra and Ophiopogonjaponicas, and is reported to have antioxidant effects. The present study was designed to investigate the potential preventive effects of Shenmai injection on development of nitroglycerin-induced tolerance. The present study involves both in vivo and in vitro experiments to investigate nitroglycerin-induced tolerance. We examined the effect of Shenmai injection on the cardiovascular oxidative stress by measuring the serum levels of malondialdehyde (MDA) and superoxide dismutase (SOD). Endothelial dysfunction was determined by an endothelium-dependent vasorelaxation method in aortic rings and NOS activity. Inhibition of the cGMP/cGK-I signalling pathway was determined from released serum levels of cGMP and the protein expression levels of sGC, cGK-I, PDE1A and P-VASP by western blot. Here, we showed that SMI ameliorated the decrease in AV Peak Vel, the attenuation in the vasodilation response to nitroglycerin and endothelial dysfunction. SMI also reduced the cardiovascular oxidative stress by reducing the release of MDA and increasing the activity of SOD. Shenmai injection further ameliorated inhibition of the cGMP/cGK-I signalling pathway triggered by nitroglycerin-induced tolerance through up-regulating the protein expression of sGC, cGK-I, and P-VASP and down- regulating the proteins expression of PDE1A. In vitro studies showed that Shenmai injection could recover the attenuated vasodilation response to nitroglycerin following incubation (of aortic rings) with nitroglycerin via activating the enzymes of sGC and cGK-I. Therefore, we conclude that Shenmai injection could prevent NTG nitroglycerin-induced tolerance at least in part by decreasing the cardiovascular oxidative stress, meliorating the endothelial dysfunction and ameliorating the inhibition of the cGMP/cGK-I signalling pathway. These findings indicate the potential of Shenmai injection (SMI) as a promising medicine for preventing the development of nitroglycerin-induced tolerance.

长期硝酸甘油（nitroglycerin, NTG）治疗会引发药效耐受，其机制与氧化应激加剧及内皮功能障碍相关。参麦注射液（Shenmai injection, SMI）是临床用于治疗心血管疾病的中药复方制剂，由红参与麦冬两味中药材组成，既往研究证实其具有抗氧化活性。本研究旨在探究参麦注射液对硝酸甘油诱导耐受发生的潜在预防作用。本研究采用体内与体外实验相结合的方案，对硝酸甘油诱导的耐受现象展开研究。我们通过检测血清丙二醛（malondialdehyde, MDA）与超氧化物歧化酶（superoxide dismutase, SOD）水平，评估参麦注射液对心血管氧化应激的调控作用。内皮功能障碍通过主动脉环内皮依赖性血管舒张实验与一氧化氮合酶（NOS）活性检测进行判定。采用蛋白质印迹法（western blot）检测血清环磷酸鸟苷（cGMP）释放水平，以及可溶性鸟苷酸环化酶（sGC）、cGK-I、PDE1A与磷酸化血管扩张刺激磷蛋白（P-VASP）的蛋白表达水平，以此评估cGMP/cGK-I信号通路的抑制状态。本研究结果显示，参麦注射液可改善AV峰值流速下降、硝酸甘油血管舒张反应减弱及内皮功能障碍。参麦注射液还可通过降低MDA释放、提升SOD活性，减轻心血管氧化应激水平。此外，参麦注射液可通过上调sGC、cGK-I与P-VASP的蛋白表达，下调PDE1A的蛋白表达，改善硝酸甘油诱导耐受所触发的cGMP/cGK-I信号通路抑制。体外实验结果表明，参麦注射液可通过激活sGC与cGK-I酶活性，恢复硝酸甘油孵育主动脉环后减弱的硝酸甘油血管舒张反应。综上，本研究认为参麦注射液可至少通过以下途径部分预防硝酸甘油诱导的耐受：减轻心血管氧化应激、改善内皮功能障碍，以及缓解cGMP/cGK-I信号通路的抑制。上述研究结果表明，参麦注射液（SMI）有望成为预防硝酸甘油诱导耐受发生的潜在治疗药物。