Gentiopicroside Produces Endothelium-Independent Vasodilation by Deactivating the PI3K/Akt/Rho-Kinase Pathway in Isolated Rat Thoracic Aorta

Gentiopicroside (GPS), a main active secoiridoid glucoside derived from the roots of perennial herbs in the Gentianaceae family, has antispasmodic and relaxant effects. However, the vasorelaxant effects of GPS on aortic rings and the molecular mechanisms involved in these effects are not yet clear. Therefore, we investigated whether GPS inhibits phenylephrine- (PE-) or KCl-induced contractions in isolated rat thoracic aortic rings. The present study found that GPS produced a dose-dependent relaxation in aortic rings precontracted with PE or KCl and significantly reduced CaCl2-, narciclasine- (Rho-kinase activator-), and phorbol-12,13-diacetate- (PKC activator-) induced vasocontractions. Pretreatment with NG-Nitroarginine methyl ester hydrochloride (L-NAME, NOS inhibitor), methylene blue (sGC inhibitor), indomethacin (COX inhibitor), 4-aminopyridine (KV channel inhibitor), and glibenclamide (KATP channel inhibitor) had no influence on the vasorelaxant effect of GPS, while BaCl2 (Kir channel inhibitor), tetraethylammonium chloride (KCa channel inhibitor), ruthenium red (RYR inhibitor), and heparin (IP3R inhibitor) significantly reduced GPS-induced vasorelaxation. Moreover, GPS pretreatment remarkably inhibited the influx of Ca2+ in vascular smooth muscle cells stimulated using KCl or PE-containing CaCl2 solution. Western blot analysis confirmed that GPS treatment inhibited PE-induced increases in the protein levels of p-Akt, p-myosin light chain (MLC), and p-myosin-binding subunit of myosin phosphatase 1 (MYPT1) in the aortic rings. Additionally, the vasorelaxation activity of GPS was attenuated upon pretreatment with LY294002 (PI3K/Akt inhibitor), Y27632 (Rho-kinase inhibitor), and verapamil (L-type Ca2+ channel inhibitor). These findings demonstrate that GPS exhibits endothelium-independent vasorelaxant effects through inhibition of voltage-dependent, receptor-operated, and inositol triphosphate receptor (IP3R)/ryanodine receptor- (RYR-) mediated Ca2+ channels as well as the PI3K/Akt/Rho-kinase signaling pathway.


Introduction
Hypertension, which is associated with vasoconstriction and vascular remodeling, is a serious threat to global public health by causing cardiovascular diseases, such as atherosclerosis, myocardial infarction, and vascular hypertrophy [1][2][3][4]. The incidence and mortality rates of hypertension are rapidly increasing worldwide; hypertension causes approximately 9 million deaths each year, and the total number of hyperten-sive patients in the world is expected to reach 1.5 billion by 2025 [5,6]. Therefore, lowering blood pressure and relaxing blood vessels can greatly alleviate the risk of cardiovascular disease development caused by elevated blood pressure.
Accumulating evidence [7][8][9] suggests that the hypercontractility of vascular smooth muscle (VSM) is closely related to raised blood pressure, while intracellular calcium concentration ([Ca 2+ ] in ) is the primary regulator of tension in VSM. Ca 2+ is a critical factor in excitation-contraction coupling in VSM, an increase in [Ca 2+ ] in results in vasoconstriction and a decrease in [Ca 2+ ] in results in vasodilation [10]. It is well known that the regulation of vascular tone is mainly triggered by releasing vasodilator factors [nitric oxide (NO), prostacyclin (PGI 2 )], changing the resting membrane potential (K + channels), the influx of extracellular Ca 2+ through receptor-operated calcium channel (ROCC) and voltagedependent calcium channel (VDCC, including L-type Ca 2+ channels), and the release of intracellular Ca 2+ from sarcoplasmic reticulum [11,12]. Additionally, many signaling pathways have been reported to play an essential role in vasoconstriction processes [13]. For example, PI3K/Akt upon activation can induce the VSM constriction by coupling membrane receptors to L-type Ca 2+ channels [14,15]. PKC and Rho-kinase Ca 2+ -sensitizing pathways leading to myosin phosphatase inhibition are critically involved in α1-adrenoceptor-mediated VSM contraction [16]. Although many vasodilators are commercially available such as nitroglycerin (NO donor drug), verapamil (Ca 2+ channel antagonist), and fasudil (ROCK inhibitor), they are limited by their adverse effects and patient compliance [17,18]. Therefore, the development of natural vasodilator compounds has far-reaching research significance and high relevance for the discovery of new treatment of cardiovascular diseases.
Gentiopicroside (GPS, C 16 H 20 O 9 , Figure 1(a)) is a secoiridoid glucoside that is isolated from the roots of perennial herbs in the Gentianaceae family, such as Gentiana straminea Maxim., Gentiana macrophylla Pall., Gentiana manshurica Kitag., Gentiana dahurica Fisch., and Gentiana scabra Bge., which are used widely as medicinal herbs in China for the treatment of rheumatoid arthritis, hemiplegia, arthralgia, stroke, and hypertension [19]. GPS has been proven to display potential protective effects against osteoarthritis, hepatitis, diabetic renal fibrosis, osteoclastogenesis, and alcoholic hepatosteatosis [20]. Kesavan et al. [21] reported that Gentiana lutea root extracts consisting of GPS significantly inhibit the proliferation of VSMCs induced by platelet-derived growth factor-BB, which may have a cardiovascular protective effect in the prevention and treatment of atherosclerosis. Given that GPS can inhibit the spontaneous contractions of smooth muscle induced by histamine, KCl, and BaCl 2 in isolated pig ileum [22], we hypothesized that GPS may inhibit vascular contraction by blocking ion channels or the corresponding signal transduction pathways.
To the best of our knowledge, the effects of GPS on aortic rings and the molecular mechanisms involved in these effects have not yet been clarified. Therefore, this study aims to investigate the effects of GPS on the vasoconstriction of aortic rings induced by PE and KCl. We also explored the underlying mechanisms of the GPS-promoted vasodilatation effects by studying Ca 2+ and K + channels and the PI3K/Akt/Rhokinase signaling pathway. These data could provide a novel insight into the molecular mechanisms underlying the vasodilatory effects of GPS.

Preparation of Rat Thoracic Aortic
Rings. Fifty-one specific pathogen-free grade healthy male Sprague-Dawley rats (4-6 months old and weighing an average 250 g) were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine (no: SCXK-2013-0020). All procedures in this study were approved by the Ethics Committee for the Use of Experimental Animals of Guangzhou University of Chinese Medicine (Permit no: 20190513056). As described previously [23], after the SD rats were euthanized, the thoracic aorta was carefully dissected and placed into ice-cold modified Krebs solution. The isolated aortas were cleaned of adipose and connective tissue and cut into 3-4 mm long rings, which were then mounted with two stainless steel hooks into an organ bath containing Krebs solution (gassed with 95% O 2 and 5% CO 2 at 37°C) at an initial force of 1 g tension. The alternation of isometric tension was recorded with a force-displacement transducer connected to a ML870 Power Lab Biological Signal Collection System (AD Instruments, Castle Hill, NSW, Australia). The endothelium of the aortic ring was removed carefully by rotating a manipulator inside the ring lumen, and its absence was verified by examining the capacity of 10 μM acetylcholine to induce less than 10% relaxation of rings precontracted with 1 μM PE. The endothelium was considered intact when the relaxation was more than 80% in response to acetylcholine. Only the aortic rings that met this standard were used for the subsequent experiments.

Action of GPS on Baseline Tension.
After the aortic rings, with or without endothelium, were stabilized at primary 1 g tension, cumulative concentrations of GPS (0, 5, 10, 20, 40, 80, 160, and 320 μΜ) were added to the organ chambers. Changes in vascular tension were recorded, and a cumulative concentration-response curve for GPS was obtained.

Effect of GPS on Aortic Rings Precontracted with PE and
KCl. After equilibration for 60 min, PE (1 μM) or KCl (60 mM) was used to induce a steady contraction in the aortic ring with or without endothelium. This was followed by the addition of cumulative concentrations of GPS (0, 5, 10, 20, 40, 80, 160, and 320 μΜ) to the ring for 20 min to verify its vasorelaxant activity. The vasodilation rate (%) was calculated as follows: Relaxation ð%Þ = ðmaximal tension by PE or KCl − tension after incubation with corresponding compoundsÞ/ðmaximal tension by PE or KCl − basal tension before precontraction with PE or KClÞ × 100%.
2.9. Western Blot Analysis. Isolated aortic rings without endothelium were transferred to DMEM and incubated at 37°C with 5% CO 2 . PE was added to the medium for 30 min, followed by GPS (160 μΜ) for 20 min. Aortic rings were snap frozen with liquid nitrogen; then, the protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer mixed with 1% phenylmethanesulfonyl fluoride (PMSF). According to the standard WB procedure, the membranes were incubated with primary antibodies against GAPDH, Akt, p-Akt, MLC, p-MLC, MYPT1, and p-MYPT1 (1 : 1000 dilution, Cell Signaling Technologies, Beverly, MA, USA) overnight at 4°C, followed by incubation with the corresponding secondary antibody for 1 h. The protein bands were visualized using an ECL reagent (EMD Millipore) and analyzed using the Tanon 5200 image acquisition system (Tanon Science and Technology Co., Ltd., Shanghai, China).
2.10. Statistical Analysis. All data are expressed as mean ± SEM. Data were plotted using the GraphPad Prism software (version 6.0; GraphPad Software, Inc.), with sigmoidal curve fitting performed by nonlinear regression using the Prism software. The maximal relaxation or contraction response was presented as E max , and the half-maximal effective concentration was presented as EC 50 . Data were analyzed by one-way and two-way analysis of variance (ANOVA), followed by Bonferroni's as a posttest, using the SPSS software (version 23.0; IBM Corp.). Differences with p < 0:05 were considered statistically significant.

Discussion
Hypertension, angina pectoris, and acute coronary syndrome are usually accompanied by the pathological characters of decreased vasodilation ability and enhanced vasoconstriction that may lead to vessel occlusion and insufficient blood supply to important organs and tissues, which serve as the main major risk factors for cardiovascular diseases [27]. Therefore, improving the vasodilation function of patients is considered an effective method for the prevention and treatment of cardiovascular diseases [28]. To date, rat thoracic aortic rings are used by many researchers as classical models to investigate the vasorelaxation effects of drugs. The present study is the first to investigate the vasodilatory effect of GPS in 5 BioMed Research International isolated rat thoracic aorta and to investigate the mechanism of action involved.
Vasodilation can be divided into two types: endotheliumdependent and endothelium-independent. The former is mainly related to the production of endothelium-derived relaxing factor NO and prostaglandins in the thoracic aorta, while the latter is related to a reduction in [Ca 2+ ] in levels caused by drugs directly acting on VSM [29]. Contraction of VSM depends on the influx of extracellular Ca 2+ through VDCC and ROCC in the cell membrane and the release of intracellular Ca 2+ through stimulation of IP 3 R-and RYRmediated Ca 2+ channels in the sarcoplasmic reticulum [30]. KCl-induced contractions mainly result from membrane depolarization and openness of VDCC, while PE, an α-adrenoreceptor agonist, leads to an aortic contraction in response to extracellular Ca 2+ influx through ROCC [31]. Previous studies [32,33] have demonstrated that 4.2 mM of gentiopicroside exhibited no significant cytotoxicity to chondrocytes, and the methanol extract of Swertia corymbosa (family: Gentianaceae) did not produce any mortality and delayed toxicity orally up to 2000 mg/kg (approximately 81.45 mg/kg of GPS) when the animals were monitored for a further 14 days. The data in the present study indicates that GPS dosedependently produced vasorelaxation effects on aortic rings with endothelium that were precontracted with PE or KCl; this effect of GPS was not significantly different in aortic rings without endothelium. Furthermore, we found that the GPS-induced vasorelaxation effect was unaffected upon pre-treatment with NOS inhibitor (L-NAME), COX inhibitor (indomethacin), and sGC inhibitor (methylene blue) in aortic rings with endothelium. These results evidently indicated that the vasodilation effects of GPS on thoracic aortic rings were endothelium-independent, independent of endothelium-derived relaxing factors such as NO and PGI 2 , and that GPS is likely to directly act on the VSM by inhibition of VDCC and ROCC.
K + channels play a critical role in regulating vascular tone by K + efflux, causing cell membrane hyperpolarization, and inhibiting extracellular Ca 2+ influx leading to vasodilation [34]. The four types of K + channels including K ATP , K ir , K Ca , and K V on the VSM can be blocked using Gli, BaCl 2 , TEA, and 4-AP, respectively. These results showed that the effects of GPS in PE-precontracted rings were attenuated upon treatment with BaCl 2 and TEA but not 4-AP and Gli, which manifested in the vasodilatory effects related to K + channels. The relationship between Ca 2+ channels and the vasodilation effects of GPS was further explored in this study. The data showed that GPS dose-dependently attenuated the contraction of high K + depolarized aortic rings induced by gradually increasing CaCl 2 (0.1-10 mM) input in Ca 2+ -free solution; in addition, GPS obviously decreased the [Ca 2+ ] in fluorescence intensity induced by KCl and CaCl 2 in A7r5 cells, which indicated that GPS inhibited extracellular calcium influx, resulting in vasodilation. PE-induced contractions in Ca 2+ -free solution are ascribed to intracellular Ca 2+ release via activation of PKC/Rho-kinase-and IP 3 R/RYR-  Figure 7: Effects of GPS on the PI3K/Akt/Rho-kinase pathway. (a) Protein expression levels of p-Akt, Akt, p-MYPT1, MYPT1, p-MLC, and MLC in aortic rings without endothelium were analyzed using western blotting. Data are presented as SEM (n = 3). * p < 0:05 compared to the control group; # p < 0:05 compared to the PE group. (b) Endothelium-denuded aortic rings were preincubated with verapamil, LY294002, and Y27632, and then, the rings were treated with GPS (20, 80, and 160 μΜ) after precontracted with PE. Data are presented as mean ± SEM (n = 6). * p < 0:05 compared to the control group. mediated Ca 2+ sensitization [16,31,35]. Our results indicated that GPS treatment significantly decreased Rhokinase activator-(narciclasine-) and PKC activator-(phorbol-12,13-diacetate-) mediated contraction, and the vasoconstriction of GPS was attenuated upon pretreatment with RYR inhibitor (ruthenium red) and IP 3 R inhibitor (heparin) in aortic rings without Ca 2+ . These results indicated that GPS inhibited the upregulation of [Ca 2+ ] in via blockade of both extracellular Ca 2+ influx in the cell membrane and intracellular Ca 2+ release through IP 3 R/RYR-mediated Ca 2+ channels in the sarcoplasmic reticulum by inhibition of VDCC and ROCC, and this inhibitory effect may be closely related to Rho-kinase-and PKC-induced phosphorylation of MLC [11,36].
It is well documented that activation of the PI3K/Akt pathway significantly enhances the contraction of VSM through stimulation of the L-type Ca 2+ channel and activation of Rho-kinase [37,38]. As shown in Figure 8, Ca 2+ binds to calmodulin in the cytoplasm, activates MLC kinase, phosphorylate MLC, and finally causes vasoconstriction [11,39]. On the other hand, Rho-kinase can inhibit MLC phosphatase via phosphorylation of MYPT1, resulting in MLC constantly being in the phosphorylated state but not in the dephosphorylated state, which eventually leads to vasoconstriction [40][41][42]. Our results indicated that treatment with GPS significantly decreased the PE-upregulated levels of Akt, MLC, and MYPT1 phosphorylation. L-type Ca 2+ channel inhibitor (verapamil), PI3K/Akt inhibitor (LY294002), and Rhokinase inhibitor (Y27632) were used to confirm that the vasodilatory effect of GPS on aortic rings involves regulation of the PI3K/Akt/Rho-kinase signaling pathway, as evidenced by the fact that vasodilatory effects of GPS were significantly attenuated compared to the control group upon pretreatment with these three inhibitors. Therefore, we suggested that GPS inhibited PI3K/Akt/Rho-kinase signaling pathway leading to vasodilation of aortic rings.

Conclusions
In summary, the present study demonstrates for the first time that GPS has evident vasodilation effects that are endothelium-independent, and the underlying mechanisms of action for these effects involve the activation of K + channels and inhibition of Ca 2+ channels by suppressing the activation of the PI3K/Akt/Rho-kinase signaling pathway.

Data Availability
The research article data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors declare that there is no conflict of interest.