The
American
Journal
of
Chinese
Medicine
(AJCM-D-13-00115.R1)
Treatment of Stress Urinary Incontinence by Ginsenoside Rh2
Yung-Hsiang Chen,*, Yu-Ning Lin,* Wen-Chi Chen,*, Wen-Tsong Hsieh* and Huey-Yi Chen*,
*Graduate Institute of Integrated Medicine, College of Chinese Medicine, Department of Pharmacology, China Medical University, Taichung 40402, Taiwan
Departments of Medical Research, Urology, and Obstetrics and Gynecology, China Medical
University Hospital, Taichung 40402, Taiwan
Correspondence to: Dr. Huey-Yi Chen, Graduate Institute of Integrated Medicine,
China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-22053366#3512, Fax: +886-4-22037690, E-mail: d888208@ms45.hinet.net
Abstract:
Stress urinary incontinence (SUI) is a common disorder in middle-aged women and the elderly. Although surgical treatment of SUI has progressed, there are no effective pharmacological therapies without side effect. We studied the effect of ginsenoside Rh2 against SUI. Here, we studied the effect of ginsenoside Rh2 on contractile force of the urethra and blood vessels in an ex vivo organ bath assay. We further investigated the mechanisms and effects of Rh2 in cell culture and animal models. Ginsenoside Rh2 dose-dependently reduced lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. In the vaginal distension (VD)-induced SUI mouse model, ginsenoside Rh2 significantly reversed VD-induced SUI physical signs and reduced blood pressure. The modulation of several SUI-related proteins, includingmyosin, survival motor neuron (SMN) protein, -adrenergic receptor 1a (AdR1a), and superoxide dismutase 3 (SOD3), may play some crucial roles in the therapeutic approaches against SUI. In conclusion, the ginsenoside Rh2 may offer therapeutic potential against SUI.
Keywords: Chinese herbal medicine; Ginsenoside Rh2; Leak point pressure;
Introduction
Stress urinary incontinence (SUI) is an increasingly more prevalent disorder given the steep worldwide increases in elderly populations. It is also a common medical condition affecting middle-aged women (Hilton et al., 2012; Herderschee et al., 2013; Lensen et al., 2013). Approximately 40% of women aged 20–45 years old suffer from SUI that significantly affects their quality of life (van der Vaart et al., 2002). Although progress has been made in the surgical treatment of SUI, there are no effective pharmacological therapies. Therefore, drug discovery and development against SUI is urgently needed. Some therapies for SUI cause urethral contractions,
including -adrenergic agonists such as ephedrine, phenylephrine (PE), midodrine, norfenefrine, phenylpropanol-amine, and imipramine, but their efficacies are
inconclusive. Treatment of women with SUI using -adrenergic stimulating drugs was withdrawn because of unwanted side effects, including insomnia, restlessness, elevated blood pressure, arrhythmia, chest pain, and headache (Alhasso et al., 2005; Canda et al., 2008).
Because conventional therapy is not completely efficacious and older individuals may be unwilling to undergo surgical treatment, alternative treatments may be potentially used as adjunctive therapies for SUI (Cherniack, 2006). Some have reported the use of traditional Chinese herbal medicines for SUI, including several herbs and polyherbal formulae, but convincing evidence regarding the benefits of these treatments is lacking. Recently, integrative medicine has been used for treating health conditions in middle-aged women, and several herbal therapies have been tested to determine their ability to ameliorate the physical symptoms of SUI. In an uncontrolled trial, subjects were given a Chinese herbal formula; 78% experienced decreased frequencies of incontinent episodes (Murakami, 1988). More recently, a
traditional Chinese herbal mixture, Ba-Wei-Die-Huang-Wan, was found to inhibit detrusor overactivity (Imamura et al., 2013). These data suggest the potential application of Chinese herbal medicine for lower urinary tract symptoms (Lee et al., 2006; Wu et al., 2008; Yang et al., 2012).
Comprehending that the urethral function and pharmacology may lead to the development of promising new agents (Chen et al., 2010), which could be useful in the management of SUI in women (Canda et al., 2008). Ginseng is any one of 11 species of slow-growing perennial plants with fleshy roots belonging to the genus
Panax of the family Araliaceae. The root is most often available in dried form, either
whole or sliced. Although not as highly prized, Ginseng leaf is also used as a medicine; the root is most often available in dried form (Jiang et al., 2013). Folk medicine attributes various benefits to the oral use of ginseng roots, including roles as an aphrodisiac; stimulant; and as a treatment for type II diabetes, sexual dysfunction, and urinary incontinence (Shergis et al., 2013). The major effective components of ginseng are ginsenosides. Among them, ginsenoside Rh2 possesses a dammarane skeleton that has been proven to have a remarkable potentiality of various biological effects (Guo et al., 2012). Ginsenoside Rh2 belongs to the protopanaxadiol family and has drawn some attention because of its antitumor and immunomodulation effects (Wang et al., 2006). In our unpublished pilot study, several candidate Chinese herbal medicines were screened, and the botanical agents for SUI were proposed for further development. This study established an ex vivo organ bath assay, an in vitro cell culture, and an in vivo animal SUI models to evaluate the effects of botanical agents against SUI and to explore the possible mechanisms. Among these botanical herbs,
Panax ginseng and its major constituent ginsenoside Rh2 cause a high contractile
force of the urethra and a low contractile force of blood vessels. Our results provide a new insight into the pharmacologic treatment of SUI. This information may offer
clues to SUI pathogenesis and open additional avenues for potential therapeutic strategies.
Materials and Methods
Organ Bath Experiment
Organ bath experiment was used for functional evaluation of contractile urethra and blood vessel ex vivo for the contractility study (Ayajiki et al., 2008; Ramalingam et
al., 2010; Jang et al., 2012), female pig (5 – 6 months, 90 – 100 kg) urethra and blood vessel were prepared from the basal part of the bladder body and renal artery, respectively (Ramalingam et al., 2010). Urethra and renal artery were mounted between platinum-plate electrodes and secured by small clips in a double-jacketed organ bath containing 20 mL Krebs’ solution aerated with 95% O2 and 5% CO2 to
obtain a pH of 7.4 at 37C. The composition of the Krebs’ solution was (mM): 133 NaCl, 4.7 KCl, 2.5 CaCl2, 16.3 NaHCO3, 1.35 NaH2PO4, 0.6 MgSO4, and 7.8
dextrose. The tissues were left in the organ bath for equilibration period of 1 – 2 h. Resting tension (0.5 g) was applied to each strip and the change of tension was measured. After equilibration, regular phasic contractions were achieved. Isometric contraction was recorded with a computerized data acquisition program (Biobench, National Instruments Corporation, TX, USA) at a rate of 50 Hz and stored on a hard drive for later analyses. Testing with different stimuli was separated by at least 5 washes with drug-free Krebs’ solution over a 5-min period (equilibration period for 25 – 30 mins before PE and Rh2 treatment). At the end of the experiment, the size of each tissues were measured (~0.5 g and ~0.2 g for the urethra and vessel, respectively). The -adrenergic agonist PE was used as a positive control.
Cell Culture
Raw 264.7 cells were purchased as cryopreserved cultures from Bioresource
Collection and Research Center (Hsinchu, Taiwan). 1 106 cells were seeded into
100-mm petri dishes and incubated at 37C in 90% Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose + 10% fetal bovine serum. By which time the cells had reached 90% confluence, the cells were plated at 96-well or 10-cm plates for MTT assay or Western blot, respectively.
MTT Assay for Cell Viability
Cells were grown in 96-well plates at a concentration of 3 104 cells/well for 24 h,
and then serum-free medium with different drugs were added. The cells were collected at 24 h later. Mitochondrial dehydrogenase activity was measured as an index of cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Welder, 1992). In brief, MTT (0.5 mg/mL) was applied to the cells for 4 h to allow the conversion of MTT into formazan crystals, the cells were lysed with dimethyl sulfoxide (DMSO), and the absorbance read at 570 and 650 nm with a DIAS Microplate Reader (Molecular devices, CA, USA). The reduction in optical density caused by treatment was used as a measurement of cell viability, normalized to the cells incubated in control medium, which were considered 100% viable.
Nitric Oxide Assay
Cells were grown in 96-well plates at a concentration of 3 104 cells/well for 24 h,
were collected at 24 h later. Nitrite levels were measured by addition of 0.1 ml of the Griess reagent (1.5% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4) to 0.1 ml of culture supernatant in 96-well plates,
leaving in the dark for 10 min, and measuring the color intensity with an automated microtiter plate reader at 550 nm (Mur et al., 2011).
Experimental Animals and Study Design
Twenty virgin female C57BL/6 strain mice, aged approximately 6–8 weeks, were randomized into 5 groups: a non-instrumented control group; the other mice undergoing vaginal distension (VD) for 1 h with 8-mm dilators (compatible with the diameter of mouse newborn head), were randomized to receive PE (25 μg/kg, i.p.) (Sun et al., 2005), low- and high-dose ginsenoside Rh2 (4 and 40 mg/kg, i.p., respectively) (Hou et al., 2013), or the vehicle for 3 days. Four days after VD, mice underwent suprapubic bladder tubing (SPT) placement. Six days after VD, leak point pressure (LPP) and maximal urethral closure pressure (MUCP) measurements were assessed in these mice under urethane (1 g/kg, i.p.) anesthesia (Cannon et al., 2002; Chen et al., 2012), Blood pressure (BP) and heart rate (HR) were measured using a BP Monitor for Rats and Mice Model MK-2000 (Muromachi Kikai, Tokyo, Japan) according to the instructions of the manufacturer (Hikoso et al., 2009). After measuring LPP, MUCP, BP, and HR, the animals were sacrificed, and the urethras were removed for Western blot analysis. All experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University.
Vaginal Distension
Mice in the VD groups were anesthetized with 1.5% isoflurane. To avoid rupturing the vagina, vaginal accommodation of Hegar’s dilators was achieved by sequentially
inserting and removing different increasing sizes of Hegar’s dilators that were lubricated with Surgilube (Fougera, Melville, NY, USA). Finally, for the VD group, an 8 mm dilator was lubricated and inserted into the vagina (Lin et al., 2008). After 1 h, the 8 mm dilator was removed, and the animal was allowed to awaken from the anesthesia spontaneously. The non-instrumented control group did not undergo vaginal dilation.
Suprapubic Tube Implantation
The surgical procedure was carried out under 1.5% isoflurane anesthesia according to the methods (Cannon et al., 2001; Lin et al., 2008). Two days after VD, an SPT (PE-10 tubing, Clay Adams, Parsippany, NJ, USA) was implanted in the bladder. Key points of the operation were as follows: (1) a midline longitudinal abdominal incision was made, 0.5 cm above the urethral meatus; (2) a small incision was made in the bladder wall, and PE-10 tubing with a flared tip was implanted in the bladder dome; and (3) a purse-string suture with 8-0 silk was tightened around the catheter, which was tunneled subcutaneously to the neck, where it exited the skin.
LPP and MUCP Measurements
Two days after implanting the bladder catheter, the LPP was assessed in these mice under urethane anesthesia. The bladder catheter was connected to both a syringe pump and a pressure transducer. Pressure and force transducer signals were amplified and digitized for computer data collection at 10 samples per second (PowerLabs, AD Instruments, Bella Vista, Australia). The mice were placed supine at the level of zero pressure while bladders were filled with room temperature saline at 1 mL/h through the bladder catheter. If a mouse voided, the bladder was emptied manually using Crede’s maneuver. The average bladder capacity of each mouse was determined after
3–5 voiding cycles. Subsequently, the LPP and MUCP were measured in the following manner (Cannon et al., 2001; Lin et al., 2008). When half-bladder capacity was reached, gentle pressure with one finger was applied to the mouse’s abdomen. Pressure was gently increased until urine leaked, at which time the externally applied pressure was quickly removed. The peak bladder pressure was taken as the LPP. At least three data were obtained for each animal, and the mean LPP was calculated.
Urethral pressure profile (UPP) was assessed in these mice under urethane (1 g/kg, i.p.) anesthesia. The bladder catheter (PE-10 tubing, Clay Adams, Parsippany, NJ, USA) was connected to a syringe pump with room temperature saline at 1 mL/h. The urethral catheter was connected to a pressure transducer. A withdrawal speed of 10 μm per minute was used. Pressure and force transducer signals were amplified and digitized for computer data collection at 10 samples/second (PowerLabs, AD Instruments, Bella Vista, Australia). Three successive profiles were obtained in the supine position. The urethral closure pressure (Pclose) is the difference between the urethral pressure (Pure) and the bladder pressure (Pves): Pclose = Pure – Pves.(Hilton
et al., 1983) Maximum urethral pressure (MUP) and MUCP were determined from
the UPP measurements taken.
Western Blot Analysis
Cell extract or urethral tissue were prepared by homogenization of cells in a lysis buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, aprotinin (10 mg/mL), leupeptin (10 mg/mL), and phosphate-buffered
saline (PBS) (Abdelfadil et al., 2013). Cell lysates containing 100 g of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA, USA). The membrane was stained with Ponceau S to verify the integrity of the
transferred proteins and to monitor the unbiased transfer of all protein samples (Lin et
al., 2011a; Yin et al., 2011). Detection of myosin, inducible nitric oxide synthase
(iNOS), survival motor neuron (SMN) protein, -adrenergic receptor 1a (AdR1a), extracellular superoxide dismutase (SOD3), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the membranes was performed with an electrochemiluminescence kit (Amersham Life Sciences Inc, Arlington Heights, IL, USA) with the use of the antibody derived from rabbit (anti-myosin antibody, 1:200 dilution, Millipore, MA, USA; anti-iNOS antibody, 1:200 dilution, Abcam, Cambridge, UK; AdR1a antibody, 1:200 dilution, Abcam, Cambridge, UK; anti-GAPDH antibody, 1:3000 dilution, Abcam, Cambridge, UK). The intensity of each band was quantified using a densitometer (Molecular Dynamics, Sunnyvale, CA, USA) (Liu et al., 2010; Yang et al., 2011).
Statistical Analyses
The data are presented as mean standard deviation (S.D.) for each group. Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Fisher’s LSD as a post hoc test. All statistical tests were two-sided. A P-value less than 0.05 was considered statistically significant. All calculations were performed using the Statistical Package for Social Sciences (SPSS for Windows, release 8.0, SPSS Inc, Chicago, IL, USA).
Results
Organ Bath Experiment
The organ bath experiment is the gold standard for functional evaluation of contractile tissues ex vivo such as the urinary tract, including the ureters, urinary bladder, and urethra, and blood vessels (Malmgren et al., 1992; Fry, 2004). Fig. 1 shows that the
-adrenergic agonist PE induces high contractile forces in both the urethra and arteries. In contrast, ginsenoside Rh2 exerts a dose-dependent stimulating effect on urethral contraction but has less effect on renal artery contraction.
Effect of Ginsenoside Rh2 on Nitric Oxide Production and Protein Expression in the Cell Culture Model
The primary etiological factor of SUI is vaginal delivery (Thom et al., 1997), which may cause ischemic (Damaser et al., 2005) and inflammatory (Fry et al., 2010) damage of the urogenital tract (Lin et al., 2009). Ischemia and inflammation cause lower urinary tract dysfunction (Fry et al., 2010) and activate inducible nitric oxide synthase (iNOS) expression, thereby increasing NO synthesis and resulting in urethral relaxation (Canda et al., 2008; Chen et al., 2012). Thus, we evaluated the effect of ginsenoside Rh2 on NO production in vitro, and iNOS expression in the mouse leukemic monocyte macrophage cell line RAW 264.7. The effect of ginsenoside Rh2 on RAW 264.7 cell viability was first determined by an MTT assay. Cells cultured with ginsenoside Rh2 at concentrations of 0, 0.8, 1.6, 3.75, and 7.5 μM for 24 h did not show altered cell viability; however, ginsenoside Rh2 significantly reduced cell viability at high concentrations (15 μM) (Fig. 2A). Therefore, the effect of
ginsenoside Rh2 (7.5 μM) on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages was investigated. Nitrite accumulation in the culture
medium was estimated by the Griess reaction as an index for NO release from the cells. Nitrite concentration was measured 24 h after treatment with LPS (500 ng/mL). When RAW 264.7 macrophages were treated with different concentrations of ginsenoside Rh2 together with LPS for 24 h, high-dose ginsenoside Rh2 reduced nitrite production (Fig. 2B). In parallel, ginsenoside Rh2 dose-dependently inhibited LPS-induced iNOS expression (Fig. 2C).
SUI Treatment by Ginsenoside Rh2 in the Mouse Vaginal Distension Model
VD has been used to induce SUI in rats, as evidenced by LPP and MUCP on urodynamic testing (Cannon et al., 2002; Chen et al., 2012). Fig. 3A and 3B show LPP and MUCP, respectively; LPP and MUCP were significantly decreased in the VD group compared with those in the uninstrumented control group. Notably, treatment with either PE or ginsenoside Rh2 significantly reversed the physical signs of SUI. No significant change was found in the BP or HR in any of the experimental groups (Fig. 3C and 3D).
Protein Expression in the SUI Mice Model
Our preliminary proteomic analysis data related to SUI following VD revealed 68 differentially expressed proteins in the urethra (unpublished data). The majority of the VD-modulated proteins were involved in muscle contraction, metabolites and energy, oxidative stress, regulation of apoptosis, and glycolysis. We used western blotting to confirm several candidate proteins related to SUI following VD and attempted to explore the possible in vivo mechanisms of ginsenoside Rh2 on SUI. Fig. 4 shows that myosin expression in the urethra was significantly decreased in the VD group as compared with the control group; both PE and high-dose ginsenoside Rh2 treatments significantly reversed VD-induced myosin downregulation. No statistical differences
were found with regard to iNOS expression. PE treatment significantly increased the expression of superoxide dismutase 3 (SOD3), and ginsenoside Rh2 treatment
significantly increased the expressions of survival motor neuron (SMN) protein, -adrenergic receptor 1a (AdR1a), and SOD3.
Discussion
In this study, ex vivo organ bath assays revealed that the major constituent from
Panax ginseng, ginsenoside Rh2, induces a high contractile force of the urethra and a
low contractile force of blood vessels. In the VD-induced SUI mouse model, ginsenoside Rh2 treatment significantly reversed the physical signs of SUI without the side effect of hypertension. The modulation of several SUI-related proteins, including myosin, SMN, AdR1a, and SOD3, may be involved in SUI progression.
The primary etiological factor of SUI is VD (Thom et al., 1997), which may cause ischemic (Damaser et al., 2005) and inflammatory (Fry et al., 2010) amage to the urogenital tract (Lin et al., 2009). Ischemia and inflammation cause the lower urinary tract dysfunction (Fry et al., 2010), an activation of iNOS expression, and an increase in NO synthesis that ultimately results in urethral relaxation (Canda et al., 2008; Chen et al., 2012). In vitro models such as macrophage or other cell lines are useful because they have a steady high-level production of NO. Our results are consistent with previous studies reporting that ginsenoside inhibits LPS-induced iNOS expression and NO production (Park et al., 1996; Choi et al., 2013).
Moreover, these findings suggest the possible inhibitory effect of the ginsenoside Rh2 on macrophage-mediated inflammatory responses. NOS activation as a result of trauma (calcium influx) leads to NO production, which activates downstream receptors. This event may lead to one or more systemic effects, including dilatation in
the cardiovascular, respiratory, and urinary systems (Thippeswamy et al., 2006). However, NO has dual roles as a protective and toxic molecule. This paradoxical dichotomy challenges researchers to determine the net impact of NO (Mocellin et al., 2007). The mechanisms by which ginsenoside Rh2 inhibits urethral contraction needs to be further elucidated. A comprehensive and dynamic view of the cascade of molecular and cellular events underlying urinary biology will allow investigators to exploit the potential properties of NO.
Myosin molecules consist of two heavy chains. Myosin heavy chains play a crucial role in modulating smooth muscle contraction (Lin et al., 2011b). In an animal model, increasing load induces significant smooth muscle hypertrophy, which is associated with a downregulation of myosin heavy chain expression. This contributes to a decrease in smooth muscle contractility (Lin et al., 1994). We found that myosin expression in the urethra was significantly decreased in VD mice, and high-dose ginsenoside Rh2 treatment significantly reversed the myosin downregulation. It is possible that VD in the urethra induces smooth muscle cell dysfunction, which alters contractility, leading to altered urethral performance and decreased compliance. These data support the potential pharmacologic effect of ginsenoside Rh2 on smooth muscle contractile properties and myosin expression.
SMN protein is necessary for the assembly of Sm proteins onto small nuclear RNAs to form small nuclear ribonucleoproteins (snRNPs) (Liu et al., 2011). SMN mutations result in spinal muscular atrophy, a common neurodegenerative disease. However, the viability and sensitivity to stresses also need to be determined in other cell types. Liu et al. established a stable HeLa cell line with inducible SMN knockdown to study its viability and sensitivity to oxidative stress. Their results suggested that there was only a slight decrease in the proliferative rate of SMN knockdown cells. In contrast, the survival rate of these cells decreased significantly
after H2O2 reached certain concentrations. Their data indicate that SMN knockdown
alone is not critical for cell viability. However, the survival rate of SMN knockdown cells may decrease significantly under stress (Liu et al., 2011). In parallel, SOD3 (extracellular superoxide dismutase, EC-SOD) was shown to be the predominant form of SOD in extracellular fluids. Several studies have focused on the antioxidative effect of SOD3 in the lung and vascular wall, where it is highly expressed. The role of SOD3 as an antioxidative enzyme in ROS-mediated ischemia and inflammation has been extensively studied. However, Kwon et al. revealed that the role of SOD3 in inflammation is not simply because of radical scavenging; it also affects immune responses and signal initiation (Kwon et al., 2012), as VD may induce ischemic and oxidative damage in the urogenital tract (Lin et al., 2009). This data showed that ginsenoside Rh2 induced both SMN and SOD3 in the urethral tissue of mice, suggesting that SMN and SOD3 may play important roles in the pharmacological prevention of SUI.
Traditional -adrenergic stimulating drugs cause urethral contraction but have unwanted side effects, including vasomotor stimulation, restlessness, and insomnia (Alhasso et al., 2005; Canda et al., 2008). Sympathetically mediated urethral tone is essential for the maintenance of continence and involves the activation of
postjunctional 1-adrenoceptors. The contraction of urethral circular smooth muscle is
mediated via 1-adrenoceptors (Bagot et al., 2006). Yono et al. showed that
doxazosin (-blocker) treatment up-regulated mRNA levels of 1-adrenoceptors in
the rat urethra, indicating that chronic doxazosin treatment may alter the properties of
1-adrenoceptors (Yono et al., 2004). These results showed that ginsenoside Rh2
significantly increased AdR1a expression, suggesting that alterations in the properties
Integrative medicine is commonly used to treat health conditions in middle-aged women, and traditional Chinese herbal medicines have been used to ameliorate the symptoms of SUI. Panax ginseng is used as an aphrodisiac, stimulant, diabetes treatment, cure for sexual dysfunction, and incontinence therapy (Shergis et al., 2013). Modern studies have shown that Panax ginseng has several pharmacological properties, including antioxidant, anti-inflammatory, antitumor, antimicrobial, antiaging, and antidiabetic activities (Choi, 2008). Our study provides the first evidence that the active component of Panax ginseng, ginsenoside Rh2, has potential as an SUI treatment. Because one of the more obvious side effects of -agonists is the stimulation of vasomotor responses and BP elevation, we suggest the complementary value of Rh2 with conventional PE treatment. Moreover, ginsenoside Rh2 exhibited its antioxidant property by inducing expression of the antioxidative protein SMN (Liu
et al., 2011), suggesting a possible advantage of ginsenoside Rh2 over PE in SUI
treatment.
In conclusion, the major constituent of the Chinese medicinal herb Panax
ginseng, ginsenoside Rh2, significantly reversed VD-induced SUI physical symptoms
without the possible side effect of hypertension. The modulation of several SUI-related proteins, including myosin, SMN, AdR1a, and SOD3, may be involved in these effects. However, the exact mechanisms underlying Rh2’s effects remain unclear, and further experimental and clinical studies are required to elucidate the mechanism(s) responsible for Rh2’s pharmacological activities in SUI.
Conflict of Interests
Acknowledgments
This work was supported by Taiwan National Science Council (NSC101-2314-B-039-018 and NSC102-2320-B-039-025), China Medical University Hospital (DMR-102-059), and in part by the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004). The authors would like to thank Enago for the English language review.
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Figure Legends
Fig. 1. Urethra and artery contraction in organ bath experiment. Contractions induced by 10-5 M of PE in the control media were taken as 100%. Graphic representation of
concentration-response curves. *P < 0.05, compared to untreated group.
Fig. 2. (A) Raw 264.7 cell viability after culture with ginsenoside Rh2 for 24 h as
determined by MTT assay. (B) Effects of ginsenoside Rh2 on LPS-induced NO production of RAW264.7 macrophages. Cells were incubated for 24 h with 500 ng/mL of LPS in the absence or presence of ginsenoside Rh2. Nitrite concentration in the medium was determined using Griess reagent. (C) Inhibition of iNOS protein expression by ginsenoside Rh2 in LPS-stimulated RAW264.7 cells. GADPH was used as a loading control. The calculated data were presented as mean ± S.D. for at least three different experiments. *P < 0.05, compared to control group. #P < 0.05,
compared to LPS group.
Fig. 3. (A) LPP, (B) MUCP, (C) BP, and (D) HR values on the sixth day after VD in
the different groups. Each bar represents the mean S.D. of five individual mice. *P < 0.05, compared to control group. #P < 0.05, compared to VD group.
Fig. 4. Myosin, iNOS, SMN, AdR1a, and SOD3 expressions on the sixth day after
VD in the different groups. Each bar represents the mean S.D. of five individual mice. *P < 0.05, compared to control group. #P < 0.05, compared to VD group.
