Hypertension is a worldwide disease, while the prevalence of hypertension arising and the control rate is less than 20% in China (Armario et al., 2017; Lu et al., 2017). Vascular function plays a vital role in the maintenance of normal blood pressure. Endothelial function, intracellular and extracellular ions in particular balance intracellular Ca²⁺ concentration and calcium sensitization primary control vascular function (Wang et al., 2015; Garcia et al., 2017; Zhou et al., 2017). Previous studies have suggested that Chinese medicine ameliorated endothelial dysfunction, vascular contractility, and vasodilation, which were important in hypertension therapy. Blood-Letting therapy, Qigong Ba Duan Jin and Tai Chi excises provided an alternative approach for hypertension therapy (Sun and Buys, 2015; Xiao et al., 2016; Ma et al., 2018; Xiong et al., 2019). Chinese herb medicine, such as Ilex hainanensis Merr., Longdanxiegan Decoction, also enhanced the effectiveness of anti-hypertension drug (Xiong et al., 2018; Yang et al., 2018). Thus, traditional Chinese medicine plays a vital role in the prevention and treatment of hypertension, especially in China.

Tetrahydropalmatine (THP, Figure 1A) is a natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used in traditional Chinese medicine for treating various pains and cardiovascular disease (Han et al., 2012; Kang et al., 2016). Previous studies have indicated that THP presented multiple pharmacological effects on cardio and neural tissues, such as cardioprotection, neuroprotection, anti-oxidant, anti-apoptosis, and anti-inflammation (Wu et al., 2010; Zhang et al., 2015, 2018). THP improved memory impairment, protected the blood brain barrier and cerebral ischemia-reperfusion injuries in experimental mice models (Mao et al., 2015; Cao et al., 2018; Sun et al., 2018). Chueh et al. found THP could induce hypotension and bradycardia though inhibition of the 5-HT2 and/or D2-receptor in the hypothalamus in rats (Chueh et al., 1995; Fu et al., 1995). THP presented numerous benefits in blood vessels as well. THP reduced the inflammation process of monocyte binding to endothelium by down regulation of ICAM-1 and VCAM-1 in endothelial cells (Yang et al., 2015). THP also inhibited the progression of aortic aneurysms (AAs) though the suppression of matrix metalloproteinase and monocyte chemotactic protein-1 in rat (Wang et al., 2018). Moreover, THP relaxed rabbit atrial strips in a calcium channel blocker like manner (Sun and Li, 1989) and inhibited the kinetic activity of Kv1.5 channels expressed in HEK293 cells (Li et al., 2017). These results suggested that THP presented various pharmacological effects in the vascular system and is potent for anti-hypertension therapy. However, the vascular reactivity of THP and the action mechanism has not yet been fully understood.

In present study, we examined the vascular relaxation effects of THP under different contractors in rat aorta. The roles of endothelium, vascular smooth muscle cell and related signaling pathway and ion channels in the vascular relaxation effect of THP were also evaluated.

The present study examined the vascular reactivity of Tetrahydropalmatine (THP, Figure 1A) in rat aorta. We demonstrated that THP relaxed rat aorta evoked by different contractors including phenylephrine (Phe), KCl, and U46619 (9,11-dideoxy-9α,11α-methanoepoxy Prosta-glandin F2α). Also, the underlying mechanisms were involved in PI3K/Akt/eNOS/NO/cGMP signaling pathway, Ca²⁺ channels and K⁺ channels.

Hypertension is a common cardiovascular disease and the pathogenesis related to the abnormal vascular contractility and vasodilation. Normal endothelium function plays a vital role in the garden of vascular physiological homeostasis and vascular relaxation (Touyz, 2000; Zhou et al., 2017; Maruhashi et al., 2019). In the present study, we found that THP relaxed KCl, Phe, and U46619 induced vascular contraction and the relaxation effect was most effective in Phe-contracted rat aorta (Figure 1B and Table 1). Also, the pre-treatment of β-adrenergic receptor blocker propranolol, angiotensin II receptor blocker losartan and PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced THP-induced vascular relaxation (Figure 1C and Table 1). THP did not affect the vascular contraction induced by angiotensin II (Figure 1D). These results indicated that THP significantly relaxed vascular aorta and the underlying mechanism was not involved in β-adrenergic receptor blockade and RAS inhibition.

Endothelium dependent relaxing factors (EDRF) including nitric oxide (NO) and prostacyclin (PGl₂) play key roles in the maintenance of the normal physical tension and relaxation function of vessel (Vanhoutte, 1989). NO was synthesized by endothelial nitric oxide synthase (eNOS) while cyclooxygenase-2 (COX2) and prostacyclin synthase (PGIS) mediated the production of PGI₂ in endothelial cells (Dorris and Peebles, 2012). We found the vasorelaxation effect of THP was partially inhibited by eNOS inhibitor L-NAME in intact rat aorta regardless of COX2 inhibitor indomethacin (Indo). The inhibition effect of L-NAME + Indo was consistent to mechanical removal of endothelium (-endo) (Figure 2A and Table 1). Cyclic guanosine monophosphate (cGMP) which is generated by guanylyl cyclase mediates the bioavailability of NO and drives NO-dependent vascular relaxation in smooth muscle cells (Furchgott and Vanhoutte, 1989). Pre-treatment of guanylyl cyclase inhibitor ODQ and methyl blue (MB) also reduced the vascular relaxation effect of THP. In addition, L-arginine (L-Arg) which has higher binding ability to eNOS than L-NAME reversed the inhibition effect of L-NAME in THP-induced vascular relaxation. Combination of L-NAME and ODQ significantly inhibited THP-induced vascular relaxation (Figure 2C). PI3K/Akt activates the phosphorylation of eNOS and subsequently generates NO in endothelium. Their inhibitors wortmannin (Wort) and Akt inhibitor IV (Akti IV) slightly suppressed the vascular relaxation effect of THP (Figure 2D and Table 1). In addition, THP induced intracellular NO accumulation in HUVECs (Figure 2E,F). Thus, we could conclude that the underlying mechanism of THP-induced vascular relaxation was endothelium-dependently and involved in the PI3K/Akt/eNOS/NO/cGMP signaling pathway rather than COX2/PIG₂. A previous study also reported that L-THP increased protein expression of phosphatidylinositol 3-kinase (PI3K) and phosphorylation of Akt and eNOS in myocardium (Han et al., 2012). So, PI3K/Akt/eNOS/NO/cGMP signaling pathway seems like an important pharmacological target of THP in cardiovascular disease.

The results in Figure 2A and Table 1 shown denuding aorta endothelium reduced the Emax of THP from 98.97 ± 1.31 to 85.58 ± 3.05% and indicated THP relaxed rat aorta mainly in an endothelium-independent manner. And the regulation of intracellular Ca²⁺ concentration by ion channels especially Ca²⁺ channels dramatically gardens vascular contraction and relaxation. We found THP inhibited the Phe or KCl induced vascular contraction concentration dependently (Figure 3A). And THP reduced CaCl₂ triggered vascular tension of aorta in Ca²⁺-free depolarizing Krebs solution containing KCl or Phe (Figure 3B). Phe and KCl increased intracellular Ca²⁺ concentration by inducing extracellular Ca²⁺ influx and internal Ca²⁺ stores release (Niazmand et al., 2014; Zhou et al., 2017). In order to explore whether THP suppressed intracellular Ca²⁺ concentration, we measured the effect of THP on the cytosol Ca²⁺ amount change triggered by CaCl₂ in Ca²⁺-free Krebs solution containing KCl in smooth muscle cells A7r5 by calcium imaging system. We found THP reduced the cytosol Ca²⁺ concentration in A7r5 cells (Figure 3C,D). L-type channel blocker nifedipine serve as positive control in these experiments. So, we could conclude that THP inhibited the vascular tension by blocking the Ca²⁺ influx. In addition, THP inhibited Phe and KCl-induced rat aorta contraction in the absence of extracellular Ca²⁺ concentration-dependently (Figure 4A,B). It seems that THP blocked the Ca²⁺ released from intracellular Ca²⁺ store and subsequently reduced the Phe and KCl induced transient vascular tension in Ca²⁺ free Krebs solution. Consistently, we found the inhibition of the IP3 and Ryr receptor which mediated the Ca²⁺ release from intracellular Ca²⁺ store attenuated the vascular relaxation effect of THP (Figure 4C and Table 2). Thus, we could conclude that THP reduced the intracellular Ca²⁺ release induced vascular tension by blocking IP3 and Ryr receptors. The Ca²⁺ and Ca²⁺/Calmodulin (CaM) complex activates smooth muscle myosin light chain kinase (MLCK) and induced calcium sensitization during vascular contraction (Martinsen et al., 2014; Zhou et al., 2017). However, THP relaxed PMA (Phorbol 12-myristate 13-acetate) which trigger calcium sensitization by active PKC and ROCK signaling pathway induced vascular contraction at the concentration of 300 μM (Supplementary Figure S1). THP also enhanced vascular relaxation effect of bradykinin and protein expression of bradykinin in endothelial cells (Supplementary Figure S2). So, THP relaxed vascular tension may also relate to the inhibition of intracellular calcium sensitization and the production of bradykinin.

Vascular tension and relaxation were also regulated by K⁺ channels. In this study, we found the voltage-dependent K⁺ channel (Kv) blocker 4-aminopyridine (4-AP), ATP-sensitive K⁺ channel (KATP) blocker glibenclamide (Gly), and inward rectifying K⁺ channel blocker (BaCl₂) attenuated THP induced vascular relaxation in intact rat aorta rather than endothelium denuded rat aorta (Figure 5). Li et al. (2017) also found that THP inhibited the kinetic activity of Kv1.5 channels. Thus, we could conclude that THP relaxed rat aorta partially though K⁺ channels and endothelium also plays a key role.

However, this study has some limitations. Mas-receptor (MS-R) which presents opposite function to angiotensin II receptor type 1 plays a vital role in vasodilation (Katsi et al., 2018) and whether THP could activate MS-R is unknown. THP induced the production of bradykinin while the underlying mechanism needs to be further studied. Moreover, whether THP could lower the blood pressure in vivo and protect the hypertension-related endothelium dysfunction and other complications also needs to be studied in the future.

In summary, THP relaxed vessel contracted by different contractors including Phe, KCl, and U46619. The underlying mechanism was both endothelium-dependent and independent and was involved in the PI3K/Akt/eNOS/NO/cGMP signaling pathway as well as the Ca²⁺ and K⁺ channels. Thus, THP may be a potential treatment for cardiovascular disease like hypertension in clinics.

Z-YZ conducted the experiments related to vascular relaxation effect of THP and wrote the manuscript. W-RZ conducted the experiments about the mechanism of vascular relaxation effect of THP. W-TS conducted the cell culture and related experiments. YX, Z-LM, and L-QZ analyzed the data of these experiments. J-YT and J-GX designed the experiments. X-LC and QY designed the experiments and revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.