Essential hypertension (EH), a systemic disease caused by genetic and environmental factors, is one of the important risk factors of cardio-cerebrovascular diseases, and can significantly increase the incidence and mortality of acute myocardial infarction, cerebral infarction, peripheral vascular disease and other diseases worldwide (Vallée A et al., 2019). According to the national sample survey of China (The Writing Committee of the Report on Cardiovascular Health and Diseases in China, 2022), the crude prevalence rate of hypertension in Chinese people ≥15 years old is on the rise, and it is estimated that 435 million population have high normal blood pressure (BP). Furthermore, the results of the China Hypertension Survey (The Writing Committee of the Report on Cardiovascular Health and Diseases in China, 2022) indicated that about 1.4 billion people suffer from hypertension worldwide and there are 245 million people suffering from hypertension in China and the trend shows an increasing. Thus, how to effectively prevent and control EH to reduce the morbidity and mortality of cardiovascular events has become a major social problem.

Endothelial dysfunction (ED) is an important feature of the initial stage and early presentation of hypertension in the past and today (Feliu and Hasson, 2014; Hirsch, 2003). Strongly supportive evidence in favor of endothelial cells (ECs) regulating normal vasodilation function and BP level, and it is considered to be the initiating factor and carrier of the chain of “endothelial dysfunction-hypertension-cardiovascular events" (Hirsch, 2003). Exposure of the hypertension risk factor results in ED and is associated with the balance of BP level and endothelial function. Vascular ED is associated with a reduction in nitric oxide (NO) and prostacyclin (PGI2) bioavailability (Ozkor and Quyyumi, 2011), an increase in generation of potential vasoconstrictor substances such as angiotensin II (Ang II) and endothelin-1 (Boegehold and Matthew, 2002). In the physiological processes, some agonists, such as acetylcholine (Ach) and bradykinin (BK), which increase the activities of endothelial nitric oxide synthase (eNOS) and cyclooxygenase (COX), promote NO and PGI2-mediated vasodilation. The endothelin-dependent vasodilator substances jointly regulate normal vasodilation function and BP level.

MicroRNAs (miRNAs), a kind of endogenous non-coding RNAs of 18–22 nucleotides in length, negatively regulate target genes expression at post-transcriptional level Many supportive evidences have demonstrated that high BP and endothelial dysfunction can be regulated by miRNAs and that the vascular specific miRNAs is regarded as the key regulators of BP (Zhang et al., 2015). MicroRNA 24 (miR-24) belongs to the microRNA-23-27-24 with the 22 bp length, which is expressed in multiple tissues such as the kidneys, lungs and heart, especially in vascular tissues (Zhou et al., 2011). MiR-24 is involved in the regulation of multiple signaling pathways, and is closely related to the occurrence and development of cardiovascular diseases. It had been proved that miR-24 could significantly up-regulated in hypertensive diseases and positively correlated with the severity and complications of hypertension, which had been considered as an important marker in hypertension (Chen and Ou, 2016; Lars Maegdefessel et al., 2015). It had been well known that vascular endothelial cells (VECs) dysfunction is the initiating factors of hypertension. miR-24 had been proved widely involved in inhibition the proliferation, differentiation and apoptosis of VECs (Chen and Ou, 2016). Studies had shown that miR-24 could affect the proliferation, differentiation and apoptosis of VECs through the down-regulating of eNOS (Zhang et al., 2015). Additionally, it has been well found that the PI3K/AKT signaling pathway is also closely related to eNOS and ED (Abeyrathna and Su, 2015; Hashimoto et al., 2006). Meanwhile, others demonstrated the up-regulation of miR-24 suppressed activation of PI3K/AKT signaling pathway and attenuated vascular remodeling in diabetic rats (Cai et al., 2019).

Radix et adix et Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Honghua in Chinese) were all recorded in a famous China medical classic book named Compendium of Materia Medica. They had been used in China for over thousand years to treat cardiovascular disease based on the theory of “activating blood circulation and removing blood stasis”. Danhong Formula (DHF) was consisting of extract of Danshen and Honghua, which is prepared from Salvia miltiorrhiza roots (Danshen in Chinese) and Carthamus tinctorius flowers (Honghua in Chinese) at a quantity ratio of 3:1. DHF injection had been approved by the State Food and Drug Administration of China (Permission Number Z20026866) to treat cardio-cerebrovascular diseases, such as anti-angina (Chen et al., 2022), heart failure, ect (Jun et al., 2021; Zi et al., 2016) according to the traditional Chinese Medicine (TCM) theory of “promoting blood circulation, removing blood stasis and dredging collaterality” in clinic. DHF is a polycomponent of herbal medicine (He et al., 2019), containing several main water-soluble bioactive compounds, such as Danshensu, salvianolic acid, hydroxysafflor yellow A, protocatechuic aldehyde, rosmarinic acid (Li et al., 2017, Li et al., 2017, 2018). Studies have found that DHF has a variety of pharmacological effects (A et al., 2019), including improving microcirculation, anti-inflammatory (B et al., 2013). Our previous studies have found that DHF improved ED and decreased high BP by up-regulating the Kallikrein-kinin system (KKS) in SHR (Yang et al., 2017). Others studies found that DHF also could promote endothelial function by up-regulating the expression of AKT, eNOS through activating of PI3K/AKT signaling pathway in atherosclerosis (Zhou et al., 2019). Since DHF has multi-effect with multi-target in endothelial function, and miR-24 was demonstrated regulation of PI3K/AKT signaling pathway, whether DHF alleviated ED and reduced BP in hypertension via miR-24-PI3K/AKT/eNOS axis is still unclear.

Therefore, in this work we analyzed the ability of DHF to attenuate high BP in SHR and alleviate endothelial dysfunction in isolated aortic rings. Moreover, the expression of miR-24-3p and PI3K- AKT -eNOS in aortic were detected by PT-PCR or immunohistochemistry analysis. Further, the present study also investigated the effect of DHF on expression of PI3K-Akt-eNOS in miR-24-3p over-expression HUVECs model. Our present work found that DHF could down-regulate the expression of miR- 24-3p and activate the expression of PI3K/AKT/eNOS. This work firstly provides a direction for antihypertensive mechanism of DHF from microRNA aspect and will promote its clinical applications.