Ling Yun, Shi Jiajun, Ma Quanxin, Yang Qinqin, Rong Yili, He Jiangmin, Chen Minli
Animal Experimental Research Center, Academy of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, China.
Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, China.
Front Pharmacol. 2021 Dec 1;12:754527. doi: 10.3389/fphar.2021.754527. eCollection 2021.
Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of (Danshen) and (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (K) and ATP-related potassium channels (K) but not voltage-gated potassium channels (k) and inward rectifying potassium channels (K). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca/calmodulin (CaM)-Ca/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca/CaM-Ca/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.
血管舒张疗法在心血管疾病尤其是高血压和冠心病的治疗中发挥着重要作用。先前的研究发现，由丹参和川芎组成的传统中药复方制剂冠心宁片（GXNT）可增加动脉血流量，但血管舒张是否在此效应中起作用仍不清楚。在此，我们发现，无论有无内皮细胞（ECs），GXNT均能以剂量依赖的方式显著减轻去氧肾上腺素（PE）、去甲肾上腺素（NE）和氯化钾（KCl）诱导的离体兔胸主动脉血管收缩。血管平滑肌细胞（VSMCs）中钙离子水平的变化表明，随着GXNT处理，细胞内钙释放和通过受体依赖性钙通道（ROC）的细胞外钙内流均下降。检测钾通道的实验表明，去内皮血管也受钙激活钾通道（KCa）和ATP敏感性钾通道（KATP）调节，但不受电压门控钾通道（KV）和内向整流钾通道（KIR）调节。对于内皮完整的血管，GXNT处理后血管组织中一氧化氮（NO）和环磷酸鸟苷（cGMP）含量明显增加，用NO合酶抑制剂Nω-硝基-L-精氨酸甲酯（L-NAME）或鸟苷酸环化酶抑制剂亚甲蓝（MB）预处理可显著抑制血管舒张。对NO相关信号通路蛋白表达的评估显示，GXNT以剂量依赖的方式增强磷酸化内皮型NO合酶（eNOS）的表达，但对人脐静脉内皮细胞（HUVECs）中总eNOS、p-Akt、Akt或PI3K水平无影响。除PI3K/AKT信号通路外，钙/钙调蛋白（CaM）-钙/钙调蛋白依赖性蛋白激酶II（CaMKII）信号通路是内皮细胞中参与eNOS激活的主要信号转导通路。进一步的结果表明，GXNT处理的HUVECs中游离钙离子水平降低，同时p-CaMKII表达增加，这意味着钙/钙调蛋白-钙/钙调蛋白依赖性蛋白激酶II级联反应增强。综上所述，这些发现表明，GXNT可能通过激活内皮完整血管环中的内皮CaMKII/eNOS信号通路和去内皮血管中的钙相关离子通道发挥其血管舒张作用。
