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Schisantherin A causes endothelium-dependent and -independent vasorelaxation in isolated rat thoracic aorta
Life Sciences 245 (2020) 117357
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Schisantherin A causes endothelium-dependent and -independent vasorelaxation in isolated rat thoracic aorta ⁎
T ⁎
Shuo Yang, ZhiYing Xu, ChengCheng Lin, He Li, JingHui Sun, JianGuang Chen , ChunMei Wang Department of Pharmacology, College of Pharmacy, Beihua University, East Binjiang Road 3999, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Schisantherin A Thoracic aorta Vasorelaxation
Aims: Schisandra is a good choice in Traditional Chinese Medicine for the therapy of cardiovascular diseases, but whether it contains a or some specific component (s) responsible these effects are still unclear. In the present study, we explored whether Schisantherin A (SCA) causes vasorelaxation in isolated rat thoracic aorta. Main methods: We selected SCA, one of the main monomers of lignans from Schisandra, to examine its vasorelaxant effect on the isolated rat thoracic aorta and also exploited several tool inhibitors to probe its underlying mechanisms. Key findings: SCA produced relaxation concentration-dependently on the endothelium-intact (43.56 ± 2.17%) and endothelium-denuded thoracic aorta strips (18.76 ± 3.95%) pre-contracted by phenylephrine (PE). However, after treated with indomethacin or L-NAME, SCA showed only partial vasorelaxant effects. Whereas, this vasorelaxation by SCA was not changed with specific K+-channel inhibitors, i.e. barium chloride (BaCl2), 4aminopyridine (4-AP), tetraethylamine (TEA), and glibenclamide. SCA had no effect on the aorta strips precontracted by PE in neither Ca2+-free nor CaCl2 conditions. But, in the Ca2+ free and high K+ environment, SCA partly abolished the vasocontraction induced by CaCl2. Significance: It was the first report to demonstrate that SCA had endothelium-dependent and -independent vasorelaxant effects on the isolated rat thoracic aorta, and the underlying mechanisms might be involved into its promoting the production of nitric oxide (NO) and prostacyclin (PGI2), and inhibiting the voltage-dependent calcium channels (VDCCs) opening. This study may partially explain the use of Schisandra in cardiovascular diseases and facilitate further drug development as well.
1. Introduction Schisandra chinensis (Turcz.) Baill (Schisandra), a genuine regional herb in the Northeast Mountains of China, was recorded and used as a top-grade herb in an ancient medicine book, Shennong Materia Medica. Nowadays, as a famous traditional Chinese medicine, Schisandra is often used in compound preparations for the treatment of hypertension, coronary heart diseases and heart failure [1–3]. In Korea, Schisandra is also used for the treatment of cardiovascular symptoms caused by menopausal syndrome [4], suggesting it may have effect on the tension of peripheral vessels. It has been proved that its water, methanol and hexane extracts can cause relaxation of rat thoracic aortic rings pre-
contracted by PE [5,6]. Lignans are the main active substances in Schisandra, which include Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A, Schisantherin A (SCA), gomisin A, D, E and J, etc. [7]. Gomicin A and J can inhibit vasoconstriction [4,8], but it is not clear whether the other lignan monomers have an influence on vasotension. Therefore, we selected SCA (Fig. 1) for the further research because of its significant vasorelaxant effect in our previous test. In the present study, by using the isolated rat thoracic aorta strips, we examined not only the vasorelaxation of SCA, but also speculated its underlying mechanism by the aids of several types of tool agents. We hoped this study would provide a new basis for the application of Schisandra in the treatment of cardiovascular diseases associated with vaso-constriction
Abbreviations: SCA, Schisantherin A; ACh, acetylcholine chloride; PE, phenylephrine; L-NAME, Nω-nitro-L-arginine methyl ester; BaCl2, barium chloride; 4-AP, 4aminopyridine; TEA, tetraethylamine; PA, pavaverine; NO, nitric oxide; PGI2, prostacyclin; NOS, nitric oxide synthetase; SR, sarcoplasmic reticulum; p-PI3K, phosphorylated phosphatidylinositol 3 kinase; p-Akt, phosphorylated protein kinase B; p-eNOS, phosphorylated endothelial nitric oxide synthase; SD, standard deviation; ANOVA, analysis of variance; ROCCs, receptor-operated calcium channel; VDCCs, voltage-dependent calcium channel; sGC, soluble guanosine cyclase; AA, arachidonic acid; COX, cyclooxygenase ⁎ Corresponding authors at: Department of Pharmacology, College of Pharmacy, Beihua University, No. 3999 Binjiang East Road, Fengman District, Jilin City 132013, Jilin Province, China. E-mail addresses: [email protected] (J. Chen), [email protected] (C. Wang). https://doi.org/10.1016/j.lfs.2020.117357 Received 25 November 2019; Received in revised form 14 January 2020; Accepted 23 January 2020 Available online 25 January 2020 0024-3205/ © 2020 Published by Elsevier Inc.
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Fig. 1. (A) Fresh Schisandra chinensis (Turcz.) Baill. fruit. (B) The chemical structure of SCA.
their tension to be stable again. The strips with or without endothelium were confirmed by their different responses to the relaxation of ACh. After the strip was pre-contracted with 10−6 M PE, if ACh (10−5 M) could exert its relaxation to > 90%, the strip was judged with intact endothelium, if < 10%, without endothelium. At the end of all experiments, 10−4 M of papaverine was added into the bath in order to get a maximal relaxation as a reference. Then, the relaxation of SCA could be calculated as a percentage relative to that caused by papaverine.
and -relaxation. 2. Materials and methods 2.1. Chemicals and drugs SCA was obtained from Chengdu PufeiDe Biotech Co., Ltd. (Sichuan, China). Acetylcholine chloride (ACh) was bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Phenylephrine (PE), (Nω)nitro-L-arginine methylester (L-NAME), indomethacin, barium chloride (BaCl2), 4-aminopyridine (4-AP), tetraethylamine (TEA), and glibenclamide were all purchased from Sigma (St. Louis, USA). Pavaverine (PA) was supplied by Northeast Pharmaceutical Group Co., Ltd. (Shenyang, China). SCA was dissolved in DMSO, and the final DMSO concentration did not exceed 0.1% (v/v).
2.4. Measurement of direct effect of SCA on the basic tension of isolated rat thoracic aorta To observe the direct effect of SCA on the isolated rat aorta, the cumulative concentrations of SCA (10−7–10−3 M) were applied when the tension of thoracic aorta strips was stable, and the vehicle was applied in the control group.
2.2. Animals
2.5. Vasorelaxant effect of SCA on the isolated rat thoracic aorta precontracted with PE
Sixty Male SD rats, weighing 250–300 g, were supplied by Changchun Yisi Laboratory Animal Tech. Co. Ltd. (Jilin, China) [license number: SCXK (JL) 2018–0007, SPF], and all rats were raised in 20 ± 2 °C constant temperature, 50 ± 10% relative humidity, normal light-dark conditions, and free water. Experimental procedures on the present study were approved by the Ethics Committee of Beihua University (Jilin, China).
In order to check the vasorelaxation of SCA on the aortic strips with intact or denuded endothelium, after the strips were contracted with PE (10−6 M), a cumulative concentration of SCA (10−7–10−3 M) was added successively into the bath and the changes in the tension were recorded. The percentage of tension change (diastolic ratio) after using SCA was calculated as (maximal tension by PE − minimal tension by SCA) / (maximal tension by PE − minimal tension by PA) × 100%.
2.3. Preparation of isolated rat thoracic aortic strips The isolated rat thoracic aortic strip was prepared as reported in our previous study [9]. In brief, after anesthetized with 100 mg/kg of urethane, the rat thoracic aorta was quickly removed and put in cold modified Ringer-Locke solution. The fat and connective tissues surrounding the aorta were carefully cut off. Then, the vascular segments, 0.8–1 cm in length, were helically cut into strips about 3 mm in width. So far, these strips had endothelia attached. In order to examine the endothelium-independent vasorelaxant effect of SCA, we also needed the strips with endothelium-denuded, which was prepared by gently rubbing the endothelium away with a small cotton ball. Each strip was vertically fixed in a thermostatic bath (10 mL in capacity) containing modified Ringer-Locke solution, which contained 120 mM of NaCl, 25.0 mM of NaHCO3, 5.4 mM of KCl, 2.2 mM of CaCl2, 1.0 mM of MgCl2, and 5.6 mM of dextrose. The bath solution was maintained at 37 °C, with aerated of 95% O2 and 5% CO2, and pH 7.40. The resting tension was adjusted to optimal 1.5 g for a maximal contraction. Before the formal experiment, the aortic strips should be equilibrated for 30 min, during which the solution was replaced every 10 min. After the tension of the strips was becoming stable, KCl solution (at a final concentration of 30 mM) was added to the bath solution to stimulate the strips 2 times. Then, the strips were flushed and allowed
2.6. Blocking NO and PGI2 production To explore the possible participation of endothelium-derived nitric oxide (NO) and/or prostacyclin (PGI2) pathways, the endothelium-intact strips were firstly incubated with 10−4 M of L-NAME, a nonspecific nitric oxide synthetase (NOS) inhibitor, or 10−5 M of indomethacin, a nonselective cyclooxygenase inhibitor, then, contracted by PE (10−6 M), and finally a cumulative concentration of SCA (10−7–10−3 M) was added to observe its vasorelaxation. 2.7. Blocking four types of potassium channels In order to determine whether SCA-induced relaxation was related to the activation of K+ channels, we selected four types of K+ channel blockers to inhibit the different K+ channels, i.e. BaCl2 (10−3 M) for KIR (inward rectifier K+ channels), 4-AP (10−3 M) for KV (voltage-dependent K+ channels), TEA (10−3 M) for KCa2+ (calcium-activated K+ channels), and glibenclamide (10−6 M) for KATP (ATP-sensitive K+ channels). The endothelium-denuded strips were used in this experiment, a cumulative concentration of SCA (10−7–10−3 M) or the vehicle 2
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Fig. 2. Direct effect of SCA on the basal tension of isolated thoracic aorta of rats. (A) Typical tracings of the responses about SCA on the aorta strips. (B) Direct effects of SCA (10−7–10−3 M) on the thoracic aorta tension. The data are expressed as the means ± SD. of six rats (n = 6).
was added 10 min before the pre-contraction by PE (10−6 M). The cumulative concentrations of SCA (10−7–10−3 M) were added after the vasocontraction of strips was stable.
2.10. Reverse transcription polymerase chain reaction Total RNA was extracted from endothelium-intact thoratic aorta segment after incubating with SCA (10−3 M) for 0, 15 and 30 min at 37 °C by using Trizol reagent (Tiangen). Then the RNA was extracted from the isolated rat thoracic aorta by using Trizol reagent (Tiangen), reverse transcribed, and amplified according to the instructions of HiScript® II One Step RT-PCR Kit. The primer sequences of endothelial NOS (eNOS) and β-actin were as follows: eNOS: Forward: 5′-CGCCTA CCCAAGAAACACCT-3′, Reverse: 5′-GCTGACTCCCTCCCAGTCTA-3; βactin: Forward: 5′-TTACTGCCCTGGCTCCTAG-3′, Reverse: 5′-CGTACT CCTGCTTGCTGATC-3′. The amplification conditions were 50 °C and 30 min for reverse transcription, 94 °C and 3 min for pre-denaturation, 94 °C and 30 s for denaturation, 56 °C and 30 s for annealing of eNOS and β-actin, 72 °C and 30 s for extension with 30 cycles, and 72 °C and 7 min for extension. The gel electrophoresis was performed on 5 μL of PCR products with 100 V for 40 min. The images of the results were observed, photographed and analyzed by Tanon 1600 gel Image system (Shanghai Tianneng Tech. Co., Ltd. China). The image density of eNOS was taken as the relative content of eNOS mRNA.
2.8. Observation of intracellular calcium release Intracellular calcium participates the smooth muscle contraction, PE can activate α-receptor and promote the calcium release from sarcoplasmic reticulum (SR) to bind to troponin-C, then, making exposing myosin-binding site on actin and contracting. This test was designed to check whether SCA was involved into the intracellular calcium release. Endothelium-denuded rat thoracic aorta strips were employed and bathed in Ca2+ free Ringer Locke solution (containing 10−3 M EGTA), and then PE (10−6 M) was added to produce the first transient contraction (T1). Subsequently, the strips were rinsed with Ca2+ Ringer Locke solution 3 times to supplement the intracellular Ca2+ loss and with Ca2+ free Ringer Locke solution (including 10−3 M EGTA) for 2 times in succession. Followed that, SCA (10−3 M) was added and incubated for 10 min and PE (10−6 M) was added again to produce the second transient vasocontraction (T2). T1-T2 was calculated as the tension difference.
2.11. Western blotting analysis Total proteins were extracted from endothelium-intact thoratic aorta segment after incubating with SCA (10−3 M) for 0, 15 and 30 min at 37 °C by using Trizol reagent (Beytime Biotechnology, Shanghai, China). The protein samples were separated, transferred and then incubated with antibodies (at the indicated dilutions) overnight at 4 °C. Antibodies were used as follows: p-PI3K (phosphorylated phosphatidylinositol 3 kinase) and p-eNOS (phosphorylated endothelial nitric oxide synthase) (1:500 dilution; ABclonal, Wuhan, China), p-Akt (phosphorylated protein kinase) (1:2000 dilution; ABclonal, Wuhan, China) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:20000 dilution; ABclonal, Wuhan, China). After incubation with secondary antibodies, signals were obtained by chemiluminescence, visualized by autoradiography, and quantified Densitometrically (Image J). Values for p-PI3K, p-Akt and p-eNOS were normalized to
2.9. Observation of extracellular calcium influx Endothelium-denuded rat thoracic aorta strips were also employed and incubated in Ca2+-free Ringer Locke solution. Then 10−6 M PE or 60 mM KCl was added to produce a basic contraction, and 10−6–10−2 M of CaCl2 were added in sequence to obtain a concentration–response curve. In order to examine the relation of SCAinduced vasorelaxation to the contractions by PE or KCl, SCA (10−3 M) was pre-incubated for 10 min before adding PE or KCl. The contraction produced by highest concentration of CaCl2 (10−2 M) was taken as 100%, and then, based on which, the inhibitory rate of SCA could be calculated.
3
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GADPH. All were expressed as units relative to the control.
endothelium-independent relaxation by SCA.
2.12. Statistical analysis
3.5. Effect of the sarcoplasmic reticulum (SR) calcium release on vasorelaxation by SCA
SPSS 22.0 software was used for the statistical analysis. All experimental data were expressed by mean ± SD, and the sample means between two groups were compared by Students' t-test. P < 0.05 represents a significant difference in statistics.
In order to investigate whether the vasorelaxatant effect induced by SCA would be related to the inhibition of intracellular release of Ca2+ from the SR, the endothelium-denuded strips were incubated in a Ca2+ free solution and contracted by PE, which promoted calcium release from SR. The pre-treatment with SCA (10−3 M) did not significantly affect PE-mediated vasocontraction (Fig. 6). This result suggested that SCA did not interfere the SR Ca2+ release for its vasodilation.
3. Results 3.1. Direct effect of SCA on the basal tension of isolated thoracic aorta of rats
3.6. Effect of extracellular Ca2+ influx on vasorelaxation by SCA −7
−3
As shown in Fig. 2, SCA (10 –10 M) had no significant effect on the basal tension of normal thoracic aorta strips.
In our present study, we selected two types of calcium channels, i.e. receptor-operated calcium channel (ROCCs) and voltage-dependent calcium channel (VDCCs) to test if their activities were associated with the vasorelaxation by SCA (10−3 M). PE could promoted Ca2+ influx via ROCCs, whereas, high concentration of KCl could promoted Ca2+ influx via VDCCs. Therefore, PE and 60 mM of KCl were employed to contract endothelium-denuded strips in a Ca2+ free solution separately. Our results showed that 10−3 M SCA did not affect the contraction induced by CaCl2 + PE (Fig. 7B), but partially abolished the contraction induced by CaCl2 + KCl (Fig. 7C), suggesting that the relaxation by SCA might be partly related to the blockade of VDCC, but not ROCC.
3.2. Effect of SCA on isolated rat thoracic aortic strips pre-contracted by PE SCA (10−7–10−3 M) induced significant and concentration-dependent relaxation in both endothelium-intact and -denuded strips with PE pre-contraction. As shown in Fig. 3, the Emax were 43.56 ± 2.17% and 18.76 ± 3.95% for SCA in comparison with 3.97 ± 2.53% and 2.89 ± 2.39% for the control in the endothelium-intact and the endothelium-denuded strips respectively. These results indicated that the endothelium might be partly involved in the vasorelaxation of SCA.
3.7. Effect of SCA on eNOS mRNA expression in the isolated rat thoracic aorta
3.3. Effect of blocking NO and PGI2 production on SCA-induced relaxation As showed in Fig. 4A and B, The results showed that L-NAME significantly reduced the vasorelaxation by SCA with Emax of 26.44 ± 5.99%, compared with the control group with Emax of 43.56 ± 2.71% (p < 0.01). Moreover, indomethacin also attenuated the relaxation by SCA with Emax of 32.10 ± 1.08% (p < 0.05). Therefore, the vasodilation by SCA might be related to the functions of both NO and PGI2.
As shown in Fig. 8, after incubation with 10−3 M SCA for 15 and 30 min, eNOS mRNA expression of rat thoracic aorta was significantly up-regulated, and the expression level was more significant at 15 min. 3.8. Effect of SCA on PI3K-Akt-eNOS pathway in the isolated rat thoracic aorta
3.4. Effect of K+ channel blockers on vasorelaxation by SCA
In order to investigate the effect of SCA on the eNOS activity, we further measured the protein expressions of PI3K-Akt-eNOS signal pathway. As shown in Fig. 9, it was found that SCA up-regulated the expression levels of p-PI3K, p-Akt, and p-eNOS after incubation for 15 and 30 min.
In order to elucidate the role of K+ currents on the vasorelaxation induced by SCA, four types of selective K+ channel blockers, BaCl2 (10−3 M), TEA (10−3 M), 4-AP (10−3 M), and glibenclamide (10−6 M) were added into the incubation solution for endothelium-denuded strips separately. The results showed that all these K+ channel blockers did not affect the SCA induced relaxation on the strips (Fig. 5), suggesting that the K+ channel or currents might be not involved in the
4. Discussion In this paper, the effect of SCA on isolated rat thoracic aorta and its
Fig. 3. Effect of SCA on isolated rat thoracic aorta strips pre-contracted by PE. (A) Typical tracings of the responses about SCA on the strips. (B) Cumulative concentration-response curves of SCA on endothelium-intact (+EC) and endothelium-denuded (−EC) aortic strips, pre-contracted with PE (10−6 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group). 4
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Fig. 4. Fig. 5,Fig. 4 Effect of L-NAME or indomethacin on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips pre-incubated with L-NAME or indomethacin. (B) Cumulative concentration-response curves of SCA in endothelium-intact strips pre-contracted with PE in the presence or absence of L-NAME (10−4 M) and indomethacin (10−5 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, *p < 0.05 and **p < 0.01 vs. control group).
by PI3K/Akt/eNOS pathway. PI3K can activate Akt and further phosphorylate eNOS [15,16]. Therefore, we furthermore confirmed SCA upregulated the expression levels of p-PI3K, p-Akt and p-eNOS. These results strongly suggested that the endothelium-dependent vasodilation induced by SCA is related to the regulation of eNOS activity and the promotion of NO production. PGI2 is produced from arachidonic acid (AA) by the catalysis of cyclooxygenase (COX), can bind to the precursor adenosine receptors on smooth muscle, and then activate adenylate cyclase to increase cAMP contents in the smooth muscle cells, resulting in vasodilation [17,18]. We then used indomethacin to inhibit COX and found that it also partly abolished the vasorelaxant effect of SCA, suggesting that the vasorelaxation by SCA was also related to the content of PGI2. Endothelium-independent vasodilation is mainly achieved by antagonizing or activating different ion channels on vascular smooth muscle, such as K+ channel and Ca2+ channel. There are four types of
mechanism were studied. The results showed that SCA had a relaxant effect on the isolated rat thoracic aorta strips in both endothelium-dependent and -independent ways. Endothelium-dependent vasodilation is mainly achieved by endothelium-derived vasodilator factors, such as NO, PGI2 and endothelium-derived hyperpolarizing factor [10,11]. Among them, NO and PGI2 are the main vasorelaxing factors [12]. NO is produced from L-arginine under the catalysis of eNOS and can activate soluble guanosine cyclase (sGC) to transform GTP into cGMP, causing a vasorelaxation [13,14]. L-NAME, an inhibitor of NOS, can inhibit the generation of NO. Therefore, we used L-NAME to clarify whether the relaxation by SCA was through the function of NO. From the result, we could judge that NO was involved in the vasorelaxation by SCA. Moreover, such effect of SCA was proved by the up-regulation of the expression of eNOS mRNA in the rat thoracic aorta after SCA administration. Research showed that the activity of eNOS is mainly regulated
Fig. 5. Effects of K+ channel blockers on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips pre-incubated with K+ channel blockers. (B) The relaxation of aortic strips pre-incubated with BaCl2 (10−3 M), TEA (10−3 M), 4-AP (10−3 M) and glibenclamide (10−6 M). The data are presented as the means ± SD of six rats (n = 6). 5
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Fig. 6. Effect of the SR calcium release on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips stimulated by 10−6 M PE under a Ca2+-free condition. (B) The inhibitory effect of SCA (10−3 M) on endothelium-denuded aortic strips induced contraction by PE (10−6 M). The data are expressed as the means ± SD. of six rats (n = 6).
K+ channels in vascular smooth muscle: KIR, KV, KATP, and KCa2+ [19]. The opening of these K+ channels can make the cell membrane hyperpolarized, reduce the APD and Ca2+ influx, causing vasodilation [20]. In this study, after separate treatment with KIR inhibitor BaCl2, KV inhibitor 4-AP, KCa2+ inhibitor TEA, and KATP inhibitor Glibenclamide, the relaxation by SCA on the endothelium-denuded strips did not change significantly, suggesting SCA exerted its vasorelaxation might not be related to the opening of K+ channels. With regard to Ca2+ channels, they includes two types, ROCC and VDCC, for the maintaining the tension of blood vessels [21]. PE increases the influx of extracellular calcium and then constricts blood vessels by activation of ROCC [22]. Our study found that SCA (10−3 M) had no significant effect on the vasocontraction of CaCl2, indicating that the ROCC might not be involved in the relaxation by SCA. In addition, PE activate the specific receptor of SR to induce Ca2+ releasing, which increase the intracellular calcium and then cause a transient constriction [23]. Treatment with SCA failed to affect on the transient contraction of the strips pre-contracted by PE, suggesting that SCA induced relaxation may not involve the release of calcium from SR. High K+ can promote the opening of VDCC and depolarize cell membranes [24]. The present
Fig. 8. Effects of the SCA (10−3 M) on eNOS mRNA expression of rat thoracic aorta. eNOS mRNA level presented as the ratio of eNOS to β-actin mRNA expression. The data are expressed as the means ± SD. of three rats (n = 3). (Unpaired t-test, *p < 0.05 vs. vehicle).
study showed that, under high K+ condition, SCA could significantly inhibit vasoconstriction caused by CaCl2, suggesting that the vasorelaxation by SCA might be related to the inhibiting the opening of VDCC. 5. Conclusion SCA caused both endothelium-dependent and -independent relaxation of rat thoracic aorta in vitro. NO and PGI2 might be responsible partly for the endothelium-dependent vasorelaxation, while the blockade of VDCC might be responsible partly for the endothelium-independent vasorelaxation (Fig. 10). This study provides an evidence for the clinical application of Schisandra in the treatment of cardiovascular diseases.
Fig. 7. Effect of extracellular Ca2+ influx on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips stimulated by CaCl2 (10−6–10−2 M) under a Ca2+-free. Effects of SCA at 10−3 M on aortic strips incubated in a Ca2+-free solution and contracted with PE 10−6 M (B) or KCl 60 mM (C) before addition of cumulative concentration of CaCl2 (10−6–10−2 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, ***p < 0.001 vs. vehicle). 6
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Fig. 9. Effect of SCA (10–3 M) on PI3K-Akt-eNOS pathway in rat thoracic aorta with intact endothelium. The bar graph represented the relative expression of p-PI3K, p-Akt or p-eNOS protein and GAPDH. The data are expressed as the means ± SD. of three rats (n = 3). (Unpaired t-test, *p < 0.05 vs. vehicle).
Fig. 10. Schematic representation of the mechanisms of vasorelaxant effect of SCA probably involved in both endothelium-dependent and -independent pathway. X represents that SCA does not play the role of vasodilation through this pathway.
References
Declaration of competing interest The authors declare that there is no conflict of interest on the publication of this paper.
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Acknowledgment This work was supported by the Post-graduate Innovation Program of Beihua University, China (No. 2018032).We thank all our colleagues working at the Jilin Schisandra Development and Industrialization Engineering Research Center, Beihua University.
Author contributions S.Y. performed experiments, analyzed data and drafted the manuscript. Z. Y. X. performed PCR experiment. C. C. L. performed in vitro vascular experiment. H. L. and J. H. S. contributed to data collection and analysis. J. G. C. polished language. C. M. W. designed the experiments and revised the manuscript. All authors approved final version of manuscript. 7
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Schisantherin A causes endothelium-dependent and -independent vasorelaxation in isolated rat thoracic aorta ⁎
T ⁎
Shuo Yang, ZhiYing Xu, ChengCheng Lin, He Li, JingHui Sun, JianGuang Chen , ChunMei Wang Department of Pharmacology, College of Pharmacy, Beihua University, East Binjiang Road 3999, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Schisantherin A Thoracic aorta Vasorelaxation
Aims: Schisandra is a good choice in Traditional Chinese Medicine for the therapy of cardiovascular diseases, but whether it contains a or some specific component (s) responsible these effects are still unclear. In the present study, we explored whether Schisantherin A (SCA) causes vasorelaxation in isolated rat thoracic aorta. Main methods: We selected SCA, one of the main monomers of lignans from Schisandra, to examine its vasorelaxant effect on the isolated rat thoracic aorta and also exploited several tool inhibitors to probe its underlying mechanisms. Key findings: SCA produced relaxation concentration-dependently on the endothelium-intact (43.56 ± 2.17%) and endothelium-denuded thoracic aorta strips (18.76 ± 3.95%) pre-contracted by phenylephrine (PE). However, after treated with indomethacin or L-NAME, SCA showed only partial vasorelaxant effects. Whereas, this vasorelaxation by SCA was not changed with specific K+-channel inhibitors, i.e. barium chloride (BaCl2), 4aminopyridine (4-AP), tetraethylamine (TEA), and glibenclamide. SCA had no effect on the aorta strips precontracted by PE in neither Ca2+-free nor CaCl2 conditions. But, in the Ca2+ free and high K+ environment, SCA partly abolished the vasocontraction induced by CaCl2. Significance: It was the first report to demonstrate that SCA had endothelium-dependent and -independent vasorelaxant effects on the isolated rat thoracic aorta, and the underlying mechanisms might be involved into its promoting the production of nitric oxide (NO) and prostacyclin (PGI2), and inhibiting the voltage-dependent calcium channels (VDCCs) opening. This study may partially explain the use of Schisandra in cardiovascular diseases and facilitate further drug development as well.
1. Introduction Schisandra chinensis (Turcz.) Baill (Schisandra), a genuine regional herb in the Northeast Mountains of China, was recorded and used as a top-grade herb in an ancient medicine book, Shennong Materia Medica. Nowadays, as a famous traditional Chinese medicine, Schisandra is often used in compound preparations for the treatment of hypertension, coronary heart diseases and heart failure [1–3]. In Korea, Schisandra is also used for the treatment of cardiovascular symptoms caused by menopausal syndrome [4], suggesting it may have effect on the tension of peripheral vessels. It has been proved that its water, methanol and hexane extracts can cause relaxation of rat thoracic aortic rings pre-
contracted by PE [5,6]. Lignans are the main active substances in Schisandra, which include Schisandrin A, Schisandrin B, Schisandrin C, Schisandrol A, Schisantherin A (SCA), gomisin A, D, E and J, etc. [7]. Gomicin A and J can inhibit vasoconstriction [4,8], but it is not clear whether the other lignan monomers have an influence on vasotension. Therefore, we selected SCA (Fig. 1) for the further research because of its significant vasorelaxant effect in our previous test. In the present study, by using the isolated rat thoracic aorta strips, we examined not only the vasorelaxation of SCA, but also speculated its underlying mechanism by the aids of several types of tool agents. We hoped this study would provide a new basis for the application of Schisandra in the treatment of cardiovascular diseases associated with vaso-constriction
Abbreviations: SCA, Schisantherin A; ACh, acetylcholine chloride; PE, phenylephrine; L-NAME, Nω-nitro-L-arginine methyl ester; BaCl2, barium chloride; 4-AP, 4aminopyridine; TEA, tetraethylamine; PA, pavaverine; NO, nitric oxide; PGI2, prostacyclin; NOS, nitric oxide synthetase; SR, sarcoplasmic reticulum; p-PI3K, phosphorylated phosphatidylinositol 3 kinase; p-Akt, phosphorylated protein kinase B; p-eNOS, phosphorylated endothelial nitric oxide synthase; SD, standard deviation; ANOVA, analysis of variance; ROCCs, receptor-operated calcium channel; VDCCs, voltage-dependent calcium channel; sGC, soluble guanosine cyclase; AA, arachidonic acid; COX, cyclooxygenase ⁎ Corresponding authors at: Department of Pharmacology, College of Pharmacy, Beihua University, No. 3999 Binjiang East Road, Fengman District, Jilin City 132013, Jilin Province, China. E-mail addresses: [email protected] (J. Chen), [email protected] (C. Wang). https://doi.org/10.1016/j.lfs.2020.117357 Received 25 November 2019; Received in revised form 14 January 2020; Accepted 23 January 2020 Available online 25 January 2020 0024-3205/ © 2020 Published by Elsevier Inc.
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Fig. 1. (A) Fresh Schisandra chinensis (Turcz.) Baill. fruit. (B) The chemical structure of SCA.
their tension to be stable again. The strips with or without endothelium were confirmed by their different responses to the relaxation of ACh. After the strip was pre-contracted with 10−6 M PE, if ACh (10−5 M) could exert its relaxation to > 90%, the strip was judged with intact endothelium, if < 10%, without endothelium. At the end of all experiments, 10−4 M of papaverine was added into the bath in order to get a maximal relaxation as a reference. Then, the relaxation of SCA could be calculated as a percentage relative to that caused by papaverine.
and -relaxation. 2. Materials and methods 2.1. Chemicals and drugs SCA was obtained from Chengdu PufeiDe Biotech Co., Ltd. (Sichuan, China). Acetylcholine chloride (ACh) was bought from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Phenylephrine (PE), (Nω)nitro-L-arginine methylester (L-NAME), indomethacin, barium chloride (BaCl2), 4-aminopyridine (4-AP), tetraethylamine (TEA), and glibenclamide were all purchased from Sigma (St. Louis, USA). Pavaverine (PA) was supplied by Northeast Pharmaceutical Group Co., Ltd. (Shenyang, China). SCA was dissolved in DMSO, and the final DMSO concentration did not exceed 0.1% (v/v).
2.4. Measurement of direct effect of SCA on the basic tension of isolated rat thoracic aorta To observe the direct effect of SCA on the isolated rat aorta, the cumulative concentrations of SCA (10−7–10−3 M) were applied when the tension of thoracic aorta strips was stable, and the vehicle was applied in the control group.
2.2. Animals
2.5. Vasorelaxant effect of SCA on the isolated rat thoracic aorta precontracted with PE
Sixty Male SD rats, weighing 250–300 g, were supplied by Changchun Yisi Laboratory Animal Tech. Co. Ltd. (Jilin, China) [license number: SCXK (JL) 2018–0007, SPF], and all rats were raised in 20 ± 2 °C constant temperature, 50 ± 10% relative humidity, normal light-dark conditions, and free water. Experimental procedures on the present study were approved by the Ethics Committee of Beihua University (Jilin, China).
In order to check the vasorelaxation of SCA on the aortic strips with intact or denuded endothelium, after the strips were contracted with PE (10−6 M), a cumulative concentration of SCA (10−7–10−3 M) was added successively into the bath and the changes in the tension were recorded. The percentage of tension change (diastolic ratio) after using SCA was calculated as (maximal tension by PE − minimal tension by SCA) / (maximal tension by PE − minimal tension by PA) × 100%.
2.3. Preparation of isolated rat thoracic aortic strips The isolated rat thoracic aortic strip was prepared as reported in our previous study [9]. In brief, after anesthetized with 100 mg/kg of urethane, the rat thoracic aorta was quickly removed and put in cold modified Ringer-Locke solution. The fat and connective tissues surrounding the aorta were carefully cut off. Then, the vascular segments, 0.8–1 cm in length, were helically cut into strips about 3 mm in width. So far, these strips had endothelia attached. In order to examine the endothelium-independent vasorelaxant effect of SCA, we also needed the strips with endothelium-denuded, which was prepared by gently rubbing the endothelium away with a small cotton ball. Each strip was vertically fixed in a thermostatic bath (10 mL in capacity) containing modified Ringer-Locke solution, which contained 120 mM of NaCl, 25.0 mM of NaHCO3, 5.4 mM of KCl, 2.2 mM of CaCl2, 1.0 mM of MgCl2, and 5.6 mM of dextrose. The bath solution was maintained at 37 °C, with aerated of 95% O2 and 5% CO2, and pH 7.40. The resting tension was adjusted to optimal 1.5 g for a maximal contraction. Before the formal experiment, the aortic strips should be equilibrated for 30 min, during which the solution was replaced every 10 min. After the tension of the strips was becoming stable, KCl solution (at a final concentration of 30 mM) was added to the bath solution to stimulate the strips 2 times. Then, the strips were flushed and allowed
2.6. Blocking NO and PGI2 production To explore the possible participation of endothelium-derived nitric oxide (NO) and/or prostacyclin (PGI2) pathways, the endothelium-intact strips were firstly incubated with 10−4 M of L-NAME, a nonspecific nitric oxide synthetase (NOS) inhibitor, or 10−5 M of indomethacin, a nonselective cyclooxygenase inhibitor, then, contracted by PE (10−6 M), and finally a cumulative concentration of SCA (10−7–10−3 M) was added to observe its vasorelaxation. 2.7. Blocking four types of potassium channels In order to determine whether SCA-induced relaxation was related to the activation of K+ channels, we selected four types of K+ channel blockers to inhibit the different K+ channels, i.e. BaCl2 (10−3 M) for KIR (inward rectifier K+ channels), 4-AP (10−3 M) for KV (voltage-dependent K+ channels), TEA (10−3 M) for KCa2+ (calcium-activated K+ channels), and glibenclamide (10−6 M) for KATP (ATP-sensitive K+ channels). The endothelium-denuded strips were used in this experiment, a cumulative concentration of SCA (10−7–10−3 M) or the vehicle 2
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Fig. 2. Direct effect of SCA on the basal tension of isolated thoracic aorta of rats. (A) Typical tracings of the responses about SCA on the aorta strips. (B) Direct effects of SCA (10−7–10−3 M) on the thoracic aorta tension. The data are expressed as the means ± SD. of six rats (n = 6).
was added 10 min before the pre-contraction by PE (10−6 M). The cumulative concentrations of SCA (10−7–10−3 M) were added after the vasocontraction of strips was stable.
2.10. Reverse transcription polymerase chain reaction Total RNA was extracted from endothelium-intact thoratic aorta segment after incubating with SCA (10−3 M) for 0, 15 and 30 min at 37 °C by using Trizol reagent (Tiangen). Then the RNA was extracted from the isolated rat thoracic aorta by using Trizol reagent (Tiangen), reverse transcribed, and amplified according to the instructions of HiScript® II One Step RT-PCR Kit. The primer sequences of endothelial NOS (eNOS) and β-actin were as follows: eNOS: Forward: 5′-CGCCTA CCCAAGAAACACCT-3′, Reverse: 5′-GCTGACTCCCTCCCAGTCTA-3; βactin: Forward: 5′-TTACTGCCCTGGCTCCTAG-3′, Reverse: 5′-CGTACT CCTGCTTGCTGATC-3′. The amplification conditions were 50 °C and 30 min for reverse transcription, 94 °C and 3 min for pre-denaturation, 94 °C and 30 s for denaturation, 56 °C and 30 s for annealing of eNOS and β-actin, 72 °C and 30 s for extension with 30 cycles, and 72 °C and 7 min for extension. The gel electrophoresis was performed on 5 μL of PCR products with 100 V for 40 min. The images of the results were observed, photographed and analyzed by Tanon 1600 gel Image system (Shanghai Tianneng Tech. Co., Ltd. China). The image density of eNOS was taken as the relative content of eNOS mRNA.
2.8. Observation of intracellular calcium release Intracellular calcium participates the smooth muscle contraction, PE can activate α-receptor and promote the calcium release from sarcoplasmic reticulum (SR) to bind to troponin-C, then, making exposing myosin-binding site on actin and contracting. This test was designed to check whether SCA was involved into the intracellular calcium release. Endothelium-denuded rat thoracic aorta strips were employed and bathed in Ca2+ free Ringer Locke solution (containing 10−3 M EGTA), and then PE (10−6 M) was added to produce the first transient contraction (T1). Subsequently, the strips were rinsed with Ca2+ Ringer Locke solution 3 times to supplement the intracellular Ca2+ loss and with Ca2+ free Ringer Locke solution (including 10−3 M EGTA) for 2 times in succession. Followed that, SCA (10−3 M) was added and incubated for 10 min and PE (10−6 M) was added again to produce the second transient vasocontraction (T2). T1-T2 was calculated as the tension difference.
2.11. Western blotting analysis Total proteins were extracted from endothelium-intact thoratic aorta segment after incubating with SCA (10−3 M) for 0, 15 and 30 min at 37 °C by using Trizol reagent (Beytime Biotechnology, Shanghai, China). The protein samples were separated, transferred and then incubated with antibodies (at the indicated dilutions) overnight at 4 °C. Antibodies were used as follows: p-PI3K (phosphorylated phosphatidylinositol 3 kinase) and p-eNOS (phosphorylated endothelial nitric oxide synthase) (1:500 dilution; ABclonal, Wuhan, China), p-Akt (phosphorylated protein kinase) (1:2000 dilution; ABclonal, Wuhan, China) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:20000 dilution; ABclonal, Wuhan, China). After incubation with secondary antibodies, signals were obtained by chemiluminescence, visualized by autoradiography, and quantified Densitometrically (Image J). Values for p-PI3K, p-Akt and p-eNOS were normalized to
2.9. Observation of extracellular calcium influx Endothelium-denuded rat thoracic aorta strips were also employed and incubated in Ca2+-free Ringer Locke solution. Then 10−6 M PE or 60 mM KCl was added to produce a basic contraction, and 10−6–10−2 M of CaCl2 were added in sequence to obtain a concentration–response curve. In order to examine the relation of SCAinduced vasorelaxation to the contractions by PE or KCl, SCA (10−3 M) was pre-incubated for 10 min before adding PE or KCl. The contraction produced by highest concentration of CaCl2 (10−2 M) was taken as 100%, and then, based on which, the inhibitory rate of SCA could be calculated.
3
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GADPH. All were expressed as units relative to the control.
endothelium-independent relaxation by SCA.
2.12. Statistical analysis
3.5. Effect of the sarcoplasmic reticulum (SR) calcium release on vasorelaxation by SCA
SPSS 22.0 software was used for the statistical analysis. All experimental data were expressed by mean ± SD, and the sample means between two groups were compared by Students' t-test. P < 0.05 represents a significant difference in statistics.
In order to investigate whether the vasorelaxatant effect induced by SCA would be related to the inhibition of intracellular release of Ca2+ from the SR, the endothelium-denuded strips were incubated in a Ca2+ free solution and contracted by PE, which promoted calcium release from SR. The pre-treatment with SCA (10−3 M) did not significantly affect PE-mediated vasocontraction (Fig. 6). This result suggested that SCA did not interfere the SR Ca2+ release for its vasodilation.
3. Results 3.1. Direct effect of SCA on the basal tension of isolated thoracic aorta of rats
3.6. Effect of extracellular Ca2+ influx on vasorelaxation by SCA −7
−3
As shown in Fig. 2, SCA (10 –10 M) had no significant effect on the basal tension of normal thoracic aorta strips.
In our present study, we selected two types of calcium channels, i.e. receptor-operated calcium channel (ROCCs) and voltage-dependent calcium channel (VDCCs) to test if their activities were associated with the vasorelaxation by SCA (10−3 M). PE could promoted Ca2+ influx via ROCCs, whereas, high concentration of KCl could promoted Ca2+ influx via VDCCs. Therefore, PE and 60 mM of KCl were employed to contract endothelium-denuded strips in a Ca2+ free solution separately. Our results showed that 10−3 M SCA did not affect the contraction induced by CaCl2 + PE (Fig. 7B), but partially abolished the contraction induced by CaCl2 + KCl (Fig. 7C), suggesting that the relaxation by SCA might be partly related to the blockade of VDCC, but not ROCC.
3.2. Effect of SCA on isolated rat thoracic aortic strips pre-contracted by PE SCA (10−7–10−3 M) induced significant and concentration-dependent relaxation in both endothelium-intact and -denuded strips with PE pre-contraction. As shown in Fig. 3, the Emax were 43.56 ± 2.17% and 18.76 ± 3.95% for SCA in comparison with 3.97 ± 2.53% and 2.89 ± 2.39% for the control in the endothelium-intact and the endothelium-denuded strips respectively. These results indicated that the endothelium might be partly involved in the vasorelaxation of SCA.
3.7. Effect of SCA on eNOS mRNA expression in the isolated rat thoracic aorta
3.3. Effect of blocking NO and PGI2 production on SCA-induced relaxation As showed in Fig. 4A and B, The results showed that L-NAME significantly reduced the vasorelaxation by SCA with Emax of 26.44 ± 5.99%, compared with the control group with Emax of 43.56 ± 2.71% (p < 0.01). Moreover, indomethacin also attenuated the relaxation by SCA with Emax of 32.10 ± 1.08% (p < 0.05). Therefore, the vasodilation by SCA might be related to the functions of both NO and PGI2.
As shown in Fig. 8, after incubation with 10−3 M SCA for 15 and 30 min, eNOS mRNA expression of rat thoracic aorta was significantly up-regulated, and the expression level was more significant at 15 min. 3.8. Effect of SCA on PI3K-Akt-eNOS pathway in the isolated rat thoracic aorta
3.4. Effect of K+ channel blockers on vasorelaxation by SCA
In order to investigate the effect of SCA on the eNOS activity, we further measured the protein expressions of PI3K-Akt-eNOS signal pathway. As shown in Fig. 9, it was found that SCA up-regulated the expression levels of p-PI3K, p-Akt, and p-eNOS after incubation for 15 and 30 min.
In order to elucidate the role of K+ currents on the vasorelaxation induced by SCA, four types of selective K+ channel blockers, BaCl2 (10−3 M), TEA (10−3 M), 4-AP (10−3 M), and glibenclamide (10−6 M) were added into the incubation solution for endothelium-denuded strips separately. The results showed that all these K+ channel blockers did not affect the SCA induced relaxation on the strips (Fig. 5), suggesting that the K+ channel or currents might be not involved in the
4. Discussion In this paper, the effect of SCA on isolated rat thoracic aorta and its
Fig. 3. Effect of SCA on isolated rat thoracic aorta strips pre-contracted by PE. (A) Typical tracings of the responses about SCA on the strips. (B) Cumulative concentration-response curves of SCA on endothelium-intact (+EC) and endothelium-denuded (−EC) aortic strips, pre-contracted with PE (10−6 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group). 4
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Fig. 4. Fig. 5,Fig. 4 Effect of L-NAME or indomethacin on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips pre-incubated with L-NAME or indomethacin. (B) Cumulative concentration-response curves of SCA in endothelium-intact strips pre-contracted with PE in the presence or absence of L-NAME (10−4 M) and indomethacin (10−5 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, *p < 0.05 and **p < 0.01 vs. control group).
by PI3K/Akt/eNOS pathway. PI3K can activate Akt and further phosphorylate eNOS [15,16]. Therefore, we furthermore confirmed SCA upregulated the expression levels of p-PI3K, p-Akt and p-eNOS. These results strongly suggested that the endothelium-dependent vasodilation induced by SCA is related to the regulation of eNOS activity and the promotion of NO production. PGI2 is produced from arachidonic acid (AA) by the catalysis of cyclooxygenase (COX), can bind to the precursor adenosine receptors on smooth muscle, and then activate adenylate cyclase to increase cAMP contents in the smooth muscle cells, resulting in vasodilation [17,18]. We then used indomethacin to inhibit COX and found that it also partly abolished the vasorelaxant effect of SCA, suggesting that the vasorelaxation by SCA was also related to the content of PGI2. Endothelium-independent vasodilation is mainly achieved by antagonizing or activating different ion channels on vascular smooth muscle, such as K+ channel and Ca2+ channel. There are four types of
mechanism were studied. The results showed that SCA had a relaxant effect on the isolated rat thoracic aorta strips in both endothelium-dependent and -independent ways. Endothelium-dependent vasodilation is mainly achieved by endothelium-derived vasodilator factors, such as NO, PGI2 and endothelium-derived hyperpolarizing factor [10,11]. Among them, NO and PGI2 are the main vasorelaxing factors [12]. NO is produced from L-arginine under the catalysis of eNOS and can activate soluble guanosine cyclase (sGC) to transform GTP into cGMP, causing a vasorelaxation [13,14]. L-NAME, an inhibitor of NOS, can inhibit the generation of NO. Therefore, we used L-NAME to clarify whether the relaxation by SCA was through the function of NO. From the result, we could judge that NO was involved in the vasorelaxation by SCA. Moreover, such effect of SCA was proved by the up-regulation of the expression of eNOS mRNA in the rat thoracic aorta after SCA administration. Research showed that the activity of eNOS is mainly regulated
Fig. 5. Effects of K+ channel blockers on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips pre-incubated with K+ channel blockers. (B) The relaxation of aortic strips pre-incubated with BaCl2 (10−3 M), TEA (10−3 M), 4-AP (10−3 M) and glibenclamide (10−6 M). The data are presented as the means ± SD of six rats (n = 6). 5
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Fig. 6. Effect of the SR calcium release on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips stimulated by 10−6 M PE under a Ca2+-free condition. (B) The inhibitory effect of SCA (10−3 M) on endothelium-denuded aortic strips induced contraction by PE (10−6 M). The data are expressed as the means ± SD. of six rats (n = 6).
K+ channels in vascular smooth muscle: KIR, KV, KATP, and KCa2+ [19]. The opening of these K+ channels can make the cell membrane hyperpolarized, reduce the APD and Ca2+ influx, causing vasodilation [20]. In this study, after separate treatment with KIR inhibitor BaCl2, KV inhibitor 4-AP, KCa2+ inhibitor TEA, and KATP inhibitor Glibenclamide, the relaxation by SCA on the endothelium-denuded strips did not change significantly, suggesting SCA exerted its vasorelaxation might not be related to the opening of K+ channels. With regard to Ca2+ channels, they includes two types, ROCC and VDCC, for the maintaining the tension of blood vessels [21]. PE increases the influx of extracellular calcium and then constricts blood vessels by activation of ROCC [22]. Our study found that SCA (10−3 M) had no significant effect on the vasocontraction of CaCl2, indicating that the ROCC might not be involved in the relaxation by SCA. In addition, PE activate the specific receptor of SR to induce Ca2+ releasing, which increase the intracellular calcium and then cause a transient constriction [23]. Treatment with SCA failed to affect on the transient contraction of the strips pre-contracted by PE, suggesting that SCA induced relaxation may not involve the release of calcium from SR. High K+ can promote the opening of VDCC and depolarize cell membranes [24]. The present
Fig. 8. Effects of the SCA (10−3 M) on eNOS mRNA expression of rat thoracic aorta. eNOS mRNA level presented as the ratio of eNOS to β-actin mRNA expression. The data are expressed as the means ± SD. of three rats (n = 3). (Unpaired t-test, *p < 0.05 vs. vehicle).
study showed that, under high K+ condition, SCA could significantly inhibit vasoconstriction caused by CaCl2, suggesting that the vasorelaxation by SCA might be related to the inhibiting the opening of VDCC. 5. Conclusion SCA caused both endothelium-dependent and -independent relaxation of rat thoracic aorta in vitro. NO and PGI2 might be responsible partly for the endothelium-dependent vasorelaxation, while the blockade of VDCC might be responsible partly for the endothelium-independent vasorelaxation (Fig. 10). This study provides an evidence for the clinical application of Schisandra in the treatment of cardiovascular diseases.
Fig. 7. Effect of extracellular Ca2+ influx on SCA induced vasorelaxation. (A) Typical tracings of the responses about SCA on the strips stimulated by CaCl2 (10−6–10−2 M) under a Ca2+-free. Effects of SCA at 10−3 M on aortic strips incubated in a Ca2+-free solution and contracted with PE 10−6 M (B) or KCl 60 mM (C) before addition of cumulative concentration of CaCl2 (10−6–10−2 M). The data are expressed as the means ± SD. of six rats (n = 6). (Unpaired t-test, ***p < 0.001 vs. vehicle). 6
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Fig. 9. Effect of SCA (10–3 M) on PI3K-Akt-eNOS pathway in rat thoracic aorta with intact endothelium. The bar graph represented the relative expression of p-PI3K, p-Akt or p-eNOS protein and GAPDH. The data are expressed as the means ± SD. of three rats (n = 3). (Unpaired t-test, *p < 0.05 vs. vehicle).
Fig. 10. Schematic representation of the mechanisms of vasorelaxant effect of SCA probably involved in both endothelium-dependent and -independent pathway. X represents that SCA does not play the role of vasodilation through this pathway.
References
Declaration of competing interest The authors declare that there is no conflict of interest on the publication of this paper.
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Acknowledgment This work was supported by the Post-graduate Innovation Program of Beihua University, China (No. 2018032).We thank all our colleagues working at the Jilin Schisandra Development and Industrialization Engineering Research Center, Beihua University.
Author contributions S.Y. performed experiments, analyzed data and drafted the manuscript. Z. Y. X. performed PCR experiment. C. C. L. performed in vitro vascular experiment. H. L. and J. H. S. contributed to data collection and analysis. J. G. C. polished language. C. M. W. designed the experiments and revised the manuscript. All authors approved final version of manuscript. 7
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