Ginsenoside Rb1 Reduces Hyper-Vasoconstriction Induced by High Glucose and Endothelial Dysfunction in Rat Aorta

Acute hyperglycemia induces oxidative damage and inflammation, leading to vascular dysfunction. Ginsenoside Rb1 (Rb1) is a major component of red ginseng with anti-diabetic, anti-oxidant and anti-inflammatory properties. Here, we investigated the beneficial effects and the underlying mechanisms of Rb1 on hypercontraction induced by high glucose (HG) and endothelial dysfunction (ED). The isometric tension of aortic rings was measured by myography. The rings were treated with NG-nitro-L-arginine methyl ester (L-NAME) to induce chemical destruction of the endothelium, and Rb1 was added after HG induction. The agonist-induced vasoconstriction was significantly higher in the aortic rings treated with L-NAME + HG50 than in those treated with HG50 or L-NAME (p = 0.011) alone. Rb1 significantly reduced the hypercontraction in the aortic rings treated with L-NAME + HG50 (p = 0.004). The ATP-sensitive K+ channel (KATP) blocker glibenclamide tended to increase the Rb1-associated reduction in the agonist-induced vasoconstriction in the rings treated with L-NAME + HG50. The effect of Rb1 in the aortic rings treated with L-NAME + HG50 resulted from a decrease in extracellular Ca2+ influx through the receptor-operated Ca2+ channel (ROCC, 10−6–10−4 M CaCl2, p < 0.001; 10−3–2.5 × 10−3 M CaCl2, p = 0.001) and the voltage-gated Ca2+ channel (VGCC, 10−6 M CaCl2, p = 0.003; 10−5–10−2 M CaCl2, p < 0.001), whereas Rb1 did not interfere with Ca2+ release from the sarcoplasmic reticulum. In conclusion, we found that Rb1 reduced hyper-vasoconstriction induced by HG and ED by inhibiting the ROCC and the VGCC, and possibly by activating the KATP in rat aorta. This study provides further evidence that Rb1 could be developed as a therapeutic target for ED in diabetes.


Introduction
Pathological conditions can result in acute hyperglycemia.For example, acute pancreatitis can not only impair glucose metabolism [1] but can also induce endothelial injury and dysregulation of vasomotor tone due to inflammatory reactions [2].High glucose (HG) concentrations have been reported to increase osteogenic protein activity in vascular smooth muscle cells in the presence of elastin-derived peptides and transforming growth factor-beta 1, which can lead to vascular calcifications [3].In addition, HG was found to significantly increase lysine acetylation and the formation of reactive oxygen species (ROS) in vascular smooth muscle cells, processes that may be associated with impaired vascular smooth muscle cell-dependent vasorelaxation in a murine model of type 2 diabetes mellitus [4].Hyperglycemia is a major factor in the development of endothelial dysfunction (ED) through oxidative stress and dysregulation of endothelial nitric oxide synthase [5].A clinical study showed that acute glucose ingestion can increase oxidative stress, possibly degrading vascular endothelial function, even in healthy young men [6].Blood sugar control alone did not effectively prevent cardiovascular complications in patients with type Pharmaceuticals 2023, 16, 1238 2 of 10 2 diabetes and hyperglycemia [7].Consequently, in patients with acute hyperglycemia, efforts should be made to prevent and treat abnormally increased vascular tone under conditions of ED.
Red ginseng (Ginseng Radix Rubra), a plant belonging to the family Araliaceae [8], has been used in traditional Korean medicine due to the pharmacological effects of the ginsenoside saponins contained in its roots, such as anti-oxidant, anti-inflammatory, and anti-diabetic properties [9].It has been reported that there are several types of ginsenosides in red ginseng [10].Ginsenosides can be divided into protopanaxadiol-type compounds, including Rb 1 , Rb 2 , R c , and R d , and protopanaxatriol-type compounds, including Rg 1 , Rg 2 , and Rh 1 , with both of these types possessing a dammarane triterpenoid structure [11].The ginsenosides Rb1 (hereinafter, Rb 1 ) and Rg1 (hereinafter, Rg 1 ) used in the present study are representative components of protopanaxadiol-type and protopanaxatriol-type, respectively [12].The chemical structures of Rb 1 and Rg 1 differ in aspects such as the positions of sugar moieties [13].Both Rb 1 and Rg 1 are known to have antidiabetic effects [14,15].Especially, Rb 1 accounts for the largest proportion of ginsenoside saponins contained in red ginseng [10,16].Rb 1 was shown to stimulate the production of nitric oxide in vascular endothelial cells [17].Rb 1 was also found to down-regulate the Wnt/β-catenin pathway, which is associated with vascular calcification in rat vascular smooth muscle cells [18].In pulmonary arteries, Rb 1 was reported to inhibit store-operated Ca 2+ entry (SOCE) by suppressing stromal interaction molecule activation rather than by altering Ca 2+ release from the sarcoplasmic reticulum (SR), and thereby to decrease endothelin-1-induced vasoconstriction [19].Furthermore, daily intraperitoneal injection of Rb 1 for three weeks was reported to decrease SOCE and vasoconstriction in the pulmonary arteries of rats with pulmonary hypertension [20].However, the effects of Rb 1 on vasoconstriction under hyperglycemia and ED conditions remained unclear.Therefore, in the present study, we investigated the effects of Rb 1 on hyper-vasoconstriction induced by HG and ED in rat aorta, and its underlying mechanisms.

Effects of HG on Vasoconstriction in Rat Aorta
Analyses of the effects of HG on the agonist-induced vasoconstriction of aortic rings showed that HG concentrations of 25 mM (HG25) or 50 mM (HG50) did not significantly affect the agonist-induced vasoconstriction of rings with intact endothelium (Control, 1.08 ± 0.04 g; HG25, 1.05 ± 0.08 g; HG50, 1.13 ± 0.08 g).Also, there was no significant difference in the agonist-induced vasoconstriction between the rings treated with N G -nitro-L-arginine methyl ester (L-NAME, 1.10 ± 0.06 g) and L-NAME + HG25 (1.18 ± 0.06 g).In contrast, the agonist-induced vasoconstriction was significantly higher in the rings treated with L-NAME + HG50 (1.32 ± 0.06 g) than in the control (p = 0.007) and L-NAMEtreated (p = 0.011) rings.The agonist-induced vasoconstriction of aortic rings was not significantly different between the L-NAME + Mannitol (1.22 ± 0.03 g) group and the control group.These results suggest that vasoconstriction was significantly increased only after simultaneous treatment with HG50 and L-NAME for denudation of the endothelium, and thus was attributable to the HG condition rather than a hyperosmotic effect (Figure 1A).

Effect of K + Channel Blockers on Rb 1 -Treated Hyper-Vasoconstriction Induced by HG and ED in Rat Aorta
To assess the mechanism underlying the effect of Rb 1 10 on agonist-induced vasoconstriction, aortic rings were treated with several K + channel blockers.Tetraethylamine (TEA, 1.09 ± 0.04 g), barium chloride (BaCl 2 , 1.11 ± 0.03 g), and 4-aminopyridine (4-AP, 1.09 ± 0.04 g) did not significantly alter the agonist-induced vasoconstriction in the aortic rings treated with L-NAME + HG50 + Rb 1 10 (1.11 ± 0.05 g), whereas the agonist-induced vasoconstriction tended to be higher in the rings treated with L-NAME + HG50 + Rb 1 10 + Glibenclamide (Gliben, 1.21 ± 0.03 g) than with L-NAME + HG50 + Rb 1 10 (1.11 ± 0.05 g; p = 0.062).These results suggested that activation of the ATP-sensitive K + channel (K ATP ) may be involved in the inhibitory effect of Rb 1 on hyper-vasoconstriction induced by HG and ED (Figure 2A).

Discussion
The present study found that the agonist-induced vasoconstriction of aortic rings with intact endothelium was not affected by the HG concentration.In the presence of ED, however, the agonist-induced vasoconstriction was significantly increased by the addition of 50 mM glucose.This concentration is relevant to that seen in the hyperosmolar hyperglycemic state [21] leading to acute pancreatitis [22].This effect was independent of vascular contracting factors from the endothelium, such as endothelin-1 [23], indicating that HG has a direct effect on vascular smooth muscle cells.Furthermore, a mannitol-based hyperosmotic control did not show a vasoconstriction increase comparable to that seen in the HG50 group, suggesting that the HG was responsible for enhancing the agonist-induced vasoconstriction of vascular smooth muscle cells.The hyperpolarization of mitochondria in mouse vascular smooth muscle cells treated with HG was found to inhibit myosin light chain phosphatase, resulting in vascular smooth muscle contraction [24].The exposure of murine aortic vascular smooth muscle cells to 30 mM glucose for 48 h was found to increase intracellular Ca 2+ levels through SOCE [25].Because acute pancreatitis is frequently accompanied by ED [2] and hyperglycemia [26], intracellular Ca 2+ concentrations may be increased in the vascular smooth muscle cells of patients with acute pancreatitis, resulting in vascular hypercontraction.
The ability of Rb 1 and Rg 1 to reduce hyper-vasoconstriction induced by HG and ED was also evaluated.Although neither Rb 1 nor Rg 1 altered vasoconstriction in the aortic rings treated with L-NAME and L-NAME+HG25, Rb 1 significantly reduced hypervasoconstriction induced by 50 mM glucose and ED, whereas Rg 1 was ineffective.Rb 1 was more effective than Rg 1 in mitigating vascular smooth muscle dysfunction in the presence of angiotensin 2-induced abdominal aortic aneurysm [27] and inhibiting vascular inflammatory action in the coronary artery endothelium [28].These different effects of Rb 1 and Rg 1 may reflect differences in their chemical configurations, such as the positions of sugar moieties and aglycone structures [13].
The effects of Rb 1 on hyper-vasoconstriction induced by HG and ED were not altered by the K + channel inhibitors, TEA, BaCl 2 , and 4-AP.In contrast, the K ATP blocker Gliben tended to suppress the Rb 1 inhibition of hyper-vasoconstriction induced by HG and ED.HG has been found to inhibit K ATP activity in vascular smooth muscle, an inhibition mediated by increased superoxide production in the human omental artery [29].Furthermore, the K ATP is inhibited when intracellular ATP is decreased, followed by the opening of the VGCC, resulting in increased cytosolic Ca 2+ levels [30,31].Thus, Rb 1 may inhibit extracellular Ca 2+ influx through the activation of the K ATP , thereby reducing hyper-vasoconstriction induced by HG and ED.
Rb 1 has been found to reduce intracellular Ca 2+ concentrations in the myocardial H9C2 cell hypoxia model, through a mechanism involving the downregulation of the calcium/calmodulin-dependent protein kinase II and the ryanodine receptor 2 [32].Furthermore, Rb 1 selectively suppressed the L-type VGCC in cultured rat hippocampus neurons [33].To determine whether the effects of Rb 1 on hyper-vasoconstriction induced by HG and ED were associated with Ca 2+ flow, the effects of Rb 1 on vasoconstriction induced by sarcoplasmic SR Ca 2+ release or extracellular Ca 2+ influx were measured.The ability of Rb 1 to ameliorate the increased vasoconstriction induced by treatment with L-NAME and 50 mM glucose was found to be independent of SR Ca 2+ release.In contrast, Rb 1 was found to reduce hyper-vasoconstriction induced by HG and ED through inhibiting extracellular Ca 2+ influx via the ROCC and the VGCC, regarded as the main Ca 2+ channels for controlling vascular tension in vascular smooth muscle [34,35].
Pretreatment with Rb 1 was found to inhibit Ca 2+ increase through SOCE in pulmonary arterial smooth muscle cells [20].Moreover, Rb 1 significantly inhibited Ca 2+ influx through SOCE only in vascular smooth muscle cells, but not in vascular endothelial cells, exposed to 30 mM glucose for 48 h [25].In agreement with these findings, the present study found that Rb 1 did not affect the agonist-induced vasoconstriction in aortas with HG and intact endothelium.This lack of effect may be due to endothelium-derived contracting factors, including angiotensin II [36], thromboxane [37], and endothelin-1 [38], released by HG stimulation.
Rb 1 is expected to have therapeutic potential in diseases accompanied by hyperglycemia and ED.The present study indicates that Rb 1 , which possesses anti-diabetic properties [39], reduced hyper-vasoconstriction induced by HG and ED through the ROCCand VGCC-mediated inhibition of extracellular Ca 2+ influx and/or possibly through the K ATP channel activation.
These findings indicate the importance of maintaining healthy endothelium to prevent hypercontraction of vascular smooth muscles, especially in patients with hyperglycemia.If endothelial vessels are damaged, however, it is crucial to manage hypercontraction of vascular smooth muscle in hyperglycemic conditions.The finding that Rb 1 is effective as a preventive and therapeutic intervention for hyper-vasoconstriction induced by HG and ED in rat aorta provides a potential rationale for clinical trials aimed at evaluating the efficacy of Rb 1 in patients with both hyperglycemia and ED.

Animals
Three-week-old male Sprague Dawley rats (weighing 65 ± 15 g) were purchased from Young Bio (Seongnam, Korea) and housed at a temperature of 21-23 • C and under a 12 h photoperiod.The rats were given free access to tap water and a standard chow diet comprised of 60% carbohydrate, 27% protein, and 13% fat (percentages of total kcal).After acclimatization, the rats were assigned to distinct groups (N = 4-11).The experimental protocol in this study was approved by the Institutional Committee for Animal Research Ethics of Korea University (KUIACUC-2021-0088).

Preparation of Aortic Rings
Rats were anesthetized with isoflurane, and their thoracic aortas were dissected.The aortic tissue was immersed in Krebs solution (118.3 mM NaCl, 25 mM NaHCO 3 , 1.22 mM KH 2 PO 4 , 11.1 mM glucose, 4.78 mM KCl, 1.2 mM MgCl 2 , 2.5 mM CaCl 2 ) and the connective tissue was removed carefully.Each aorta was cut into rings 2-3 mm in length and placed in 37 • C chambers, which were continually aerated with a mixture of 5% CO 2 and 95% O 2 .The aortic rings were connected to a DMT 620M (Danish Myo Technology, Aarhus, Denmark) with tungsten wires, and then stabilized at 1.0 g tension for 80 min.The aortic rings were pretreated with 10 −4 M L-NAME to denudate the endothelium chemically and thus to assess only the vasoconstrictive responses of vascular smooth muscle.For the HG condition, the total glucose concentration in each chamber was adjusted to 25 mM [40] or 50 mM [41] for 30 min.Mannitol was used to prepare a high osmolarity control corresponding to HG50.The aortic rings were pretreated with 10 −6 or 10 −5 M Rb 1 or Rg 1 , or with 10 −5 M NAC, (an antioxidant) [42], and contraction was induced by adding 10 −5 M PE.When the maximum vasoconstriction plateau was reached, 10 −5 M acetylcholine was added to confirm endothelial denudation.

Involvement of K + Channel in Vasoconstriction
To investigate whether the inhibitory effect of Rb 1 on hyper-vasoconstriction under HG and ED conditions was associated with the activation of the K + channel, the aortic rings were pretreated with K + channel blockers.Briefly, the aortic rings with chemically denuded epithelium were treated with 10 −3 M TEA to block the Ca 2+ -activated K + channel (K Ca2+ ), 10 −6 M Gliben to block the K ATP , 10 −3 M BaCl 2 to block the inward rectifier K + channel (K IR ), or 10 −3 M 4-AP to block the voltage-dependent K + channel (K V ) [20].The rings were subsequently treated with 10 −5 M Rb 1 prior to agonist-induced vasoconstriction.

Measurement of SR Ca 2+ Release-Induced Vasoconstriction
To determine whether Rb 1 was involved in PE-induced SR Ca 2+ release, which causes the exposure of myosin-binding sites on actin and vascular smooth muscle contraction [35], the aortic rings were immersed in Ca 2+ -free Krebs solution supplemented with 10 −4 M L-NAME, followed by the addition of 10 −5 M PE to induce vasoconstriction due to SR Ca 2+ release.The aortas were rinsed with Krebs solution three times to restore lost intracellular Ca 2+ , followed by rinsing with Ca 2+ -free Krebs solution two times.The rings were subsequently pretreated with 10 −5 M Rb 1 for 10 min, followed by the addition of 10 −5 M PE.PE-induced vasoconstriction was calculated to estimate the amount of Ca 2+ release from the SR [35].

Measurement of Extracellular Ca 2+ Influx-Induced Vasoconstriction
Two types of Ca 2+ channels, ROCC and VGCC, were selected to determine whether the effects of Rb 1 included extracellular Ca 2+ influx, thereby affecting vasoconstriction under HG and ED conditions.The aortic rings were immersed in Ca 2+ -free Krebs solution containing L-NAME, followed by the addition of 10 −5 M PE (for Ca 2+ influx via the ROCC) or 60 mM KCl (for Ca 2+ influx via the VGCC) to induce vasoconstriction.The rings were subsequently treated with 10 −6 -10 −2 M CaCl 2 to obtain concentration-response curves.After rinsing with Ca 2+ -free Krebs solution, to evaluate the inhibitory effect of Rb 1 on hyper-vasoconstriction induced by HG and ED via the ROCCs or VGCCs, the aortic rings were treated with 10 −5 M Rb 1 for 10 min prior to the addition of 10 −5 M PE or 60 mM KCl and subsequent 10 −6 -10 −2 M CaCl 2 .The percent PE-or KCl-induced vasoconstriction was calculated based on the maximal contractile response to 10 −2 M CaCl 2 [35].

Statistical Analysis
All data were expressed as mean ± SEM and analyzed using SPSS statistics version 26 software (IBM, IL, USA).The data for extracellular Ca 2+ influx-and SR Ca 2+ releaseinduced vasoconstriction were compared by paired t-tests.The other data were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test for post hoc comparison.p < 0.05 was deemed statistically significant.

Conclusions
In this study, we have demonstrated that Rb 1 effectively reduced hyper-vasoconstriction induced by HG and ED (p = 0.004) via the inhibition of Ca 2+ influx through the ROCC (10 −6 -10 −4 M CaCl 2 , p < 0.001; 10 −3 -2.5 × 10 −3 M CaCl 2 , p = 0.001) and the VGCC (10 −6 M CaCl 2 , p = 0.003; 10 −5 -10 −2 M CaCl 2 , p < 0.001) and partially through the activation of the K ATP in rat aorta for the first time (Figure 3).Our research provides further evidence into clinical applicability that Rb 1 could be recommended as a therapeutic agent for ED in diabetes and provides a guideline for clinical studies evaluating the importance of managing vascular function in patients with hyperglycemia and ED, including in patients with acute pancreatitis.
In this study, we have demonstrated that Rb1 effectively reduced hyper-vasoconstriction induced by HG and ED (p = 0.004) via the inhibition of Ca 2+ influx through the ROCC (10 −6 -10 −4 M CaCl2, p < 0.001; 10 −3 -2.5 × 10 −3 M CaCl2, p = 0.001) and the VGCC (10 −6 M CaCl2, p = 0.003; 10 −5 -10 −2 M CaCl2, p < 0.001) and partially through the activation of the KATP in rat aorta for the first time (Figure 3).Our research provides further evidence into clinical applicability that Rb1 could be recommended as a therapeutic agent for ED in diabetes and provides a guideline for clinical studies evaluating the importance of managing vascular function in patients with hyperglycemia and ED, including in patients with acute pancreatitis.Informed Consent Statement: Not applicable.

Figure 3 .
Figure 3. Proposed mechanisms of action of Rb1 in reducing hyper-vasoconstriction induced by high glucose and endothelial dysfunction in rat aorta.Abbreviations: IP3R, inositol 1,4,5-trisphosphate receptor; KATP, ATP-sensitive K + channel; KCa2+, Ca 2+ -activated K + channel; KIR, inward rectifier K + channel; KV, voltage-dependent K + channel; Rb1, ginsenoside Rb1; ROCC, receptor-operated calcium channel; SR, sarcoplasmic reticulum; VGCC, voltage-gated calcium channel.Author Contributions: conceptualization, G.H.S.; methodology, G.H.S.; formal analysis, J.P., Y.K.S., U.K., and G.H.S.; investigation, J.P., Y.K.S., and U.K.; writing-original draft preparation, J.P., Y.K.S., and G.H.S.; writing-review and editing, G.H.S.; visualization, J.P., Y.K.S., and G.H.S.; supervision, G.H.S.; project administration, G.H.S.; funding acquisition, G.H.S.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C2004118) and the Institute of Nursing Research, Korea University Grant.This manuscript is a revision of J.P.'s master's thesis from Korea University.Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Research and Ethics Committee of Korea University (KUIACUC-2021-0088).Informed Consent Statement: Not applicable.Data Availability Statement: Data are available on request from the corresponding author on reasonable request.Conflicts of Interest:The authors declare no conflicts of interest.