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Effects of Gegen (Puerariae lobatae Radix) water extract on improving detrusor overactivity in spontaneously hypertensive rats.

ABSTRACT

Aim: Ex vivo experiments showed that the water extract of Puerariae lobatae Radix (named Gegen in Chinese) induced detrusor relaxation. The aim of this study was to prove the in vivo efficacy of Gegen on improving detrusor overactivity and its possible synergism with darifenacin (a first-line muscarinic receptor-3 inhibitor) in spontaneously hypertensive rats (SHR), a rat model exhibiting symptoms of detrusor overactivity.

Method: After daily oral administration of Gegen 30 (Gegen, 30mg/kg); Gegen 300 (Gegen, 300mg/kg); Low_Dar (darifenacin, 3mg/kg); High_Dar (darifenacin, 30mg/kg) Low_Dar+Gegen 30 or High_Dar+Gegen 30 for 3 weeks, bladder detrusor strips of the rats were isolated and assessed with different stimulators for the measurement of tonic and phasic contractile activities (including phasic amplitude and frequency). Modes of stimulation included the use of carbachol, isoprenaline and electrical field stimulation (EFS).

Results: All drug treatments significantly reduced carbachol-stimulated tonic contractile activities, but did not change the phasic amplitude. Meanwhile, the treatments with Gegen 300; Low_Dar; Low_Dar+Gegen 30; and High_Dar+Gegen 30 decreased carbachol-stimulated phasic frequency. Gegen 300 and Low_Dar+Gegen 30 showed stronger potency on lowering EFS-induced responses. Under isoprenaline-induced relaxation, only Gegen 300 significantly enhanced this relaxation by decreasing tonic contraction; Gegen 300; Low_Dar; Low_Dar+Gegen 30; and High_Dar+Gegen 30 increased the reduction of phasic frequency, but all treatment did not alter their phasic amplitude. Combination Index (Cl) showed that the combination with Low_Dar and Gegen 30 had very strong synergism (CI < 0.1) on inhibiting EFS-induced contractile response.

Conclusion: Gegen improved detrusor overactivity through neurogenic and anti-muscarinic mechanisms. Gegen and darifenacin together attained synergism for detrusor overactivity treatment via the neurogenic pathway.

Keywords:

Puerariae lobatae Radix

Gegen

Detrusor overactivity

Darifenacin

Synergism

Spontaneously hypertensive rat

Introduction

Overactive bladder, being one of the causes of urge incontinence, is common among the elderly women and men with benign prostatic hypertrophy. The prevalence of overactive bladder symptom was about 16.6% in adults over 40-year of age in six European countries (Milsom et al., 2001), and similar data were reported in the USA with approximately 34 million affected people (Asimakopoulos et al., 2012). This symptom is also prevalent globally as revealed in the recent systematic review (Milsom et al., 2014). Detrusor overactivity with high contractile frequency and urgency manifests as excessive and involuntary detrusor contraction in the bladder. It is hypothesized that detrusor overactivity is caused by neurogenic problem from central nervous system, while it can also be induced by myogenic and autonomic automation derived from the periphery (Drake et al., 2006).

Since the [M.sub.3] subtype of muscarinic receptors is mostly expressed in the bladder, [M.sub.3]-selective anti-muscarinic agents (e.g. darifenacin) provide the first-line treatment for detrusor overactivity, acting via the competition with acetylcholine at the [M.sub.3] receptors in the detrusor smooth muscle to inhibit detrusors overactivity (Andersson et al., 2009). Unfortunately, these [M.sub.3]-selective anti-muscarinic agents cause significant clinical adverse effects (e.g. constipation and dry mouth) in many patients; When the dosage was doubled to be effective, more than 30% of the patients had suffered from the side effects (FDA, 2004). It is therefore very much justified that new treatment strategies should be developed, which preserve the therapeutic efficacy against detrusor overactivity on one hand and minimize the adverse effects related to the effective dosages on the other.

Chinese medicinal herbs have been traditionally used for thousands of years in Asia. The safety of these herbs are well documented in ancient classics, but information about herbs for the treatment of detrusor overactivity is not available. We have made attempt to identify some Chinese herbs that might be useful for detrusor overactivity, and Puerariae lobatae Radix (named Gegen in Chinese) appeared to be a promising candidate (Liang et al., 2012). Gegen is the dried root of Pueraria lobata (Willd.) Ohwi, which is traditionally used for promoting circulation, increasing the blood flow, reducing dry mouth, and treating cardiovascular diseases such as hypertension, angina pectoris and type 2 diabetes mellitus (Zhang et al., 2013). In our previous ex vivo study, Gegen water extract induced detrusor relaxation in urothelium-independent manner on isolated bladder strips (Liang et al., 2012); Gegen also suppressed phasic amplitude but not phasic frequency. The ex vivo efficacy of Gegen on the bladder strips suggested its potential value as an adjunctive treatment for detrusor overactivity. Hence, the aim of this study was to prove the in vivo efficacy of Gegen on improving detrusor overactivity. A spontaneously hypertensive rat (SHR) model was used in this study, due to the fact that increased urinary frequency and urodynamic findings of filling-phase contractions have been well documented in the SHR (Steers et al., 1999). Meanwhile, Wistar Kyoto rats (WKY), a healthy animal model, were used as controls for comparison with SHR. However, in our preliminarily study using the current experimental protocol, significant difference between WKY and SHR detrusors from 12-week-old male rats was found, but not in female detrusors (Data not shown). Thus, male SHR model was used in this study. This study also investigated the possible synergistic effects of Gegen and darifenacin in the SHR model.

Methods and materials

Chemicals

Darifenacin hydrobromide, carbachol and other unspecified chemicals were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). The composition of Krebs' solution was as followed: NaCl, 119 mM; KC1, 4.6 mM; Mg[Cl.sub.2], 1.2 mM; Na[H.sub.2]P[O.sub.4], 1.2 mM; NaHC[O.sub.3], 15 mM; Ca[Cl.sub.2], 1.5 mM; and D-glucose, 11 mM.

Preparation of herbal extract

The dried root of Pueraria lobata (Willd.) Ohwi was purchased from Guangzhou Zhixin Pharmaceutical Co. (Guangzhou, China). It was chemically authenticated by thin layer chromatography (TLC) according to Chinese Pharmacopoeia 2010. Its voucher specimen (no. 2013-3409) was kept in the museum of the Institute of Chinese Medicine, The Chinese University of Hong Kong. The raw materials (500 g) of Gegen were extracted by boiling with water (5 L) for an hour and repeated once. After filtered, the supernatant was collected and subjected to freeze-drying for the collection of dried Gegen water extract. The dried extract was stored in the desiccator before use.

HPLC profile

Gegen powder was totally dissolved in water, filtered by 0.22 [micro]m filter, and then subjected to chemical analysis using Agilent 1100 Infinity HPLC system (Santa Clara, CA, USA) which was equipped with an online degasser, a binary-pump, an autosampler and a diode array detector. HPLC analysis was performed with an Alltima HPLC C18 column (250 mm x 4.6 mm, 5 [micro]m) guarded by a guard column with the same stationary phase. The column was maintained at room temperature, and the flow rate was set at 1 ml/min. The mobile phase consisted of (A) 0.1% acetate acid and (B) acetonitrile with the following gradient: 10-12.5% B from Omin to 20 min; 12.5-15% B from 20 to 30 min; 15-60% B from 30 min to 40 min; and 60-90 % B from 40 min to 45 min. The UV absorbance was detected at 254 nm, and injection volume was 10 [micro]l.

Animals

Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats (9-week old) were provided by the Laboratory Animal Services Centre, The Chinese University of Hong Kong (CUHK). Animals were kept with free access to food and water under a 12:12h light-dark cycle. All animal experiments have been approved by the CUHK Animal Experimentation Ethic Committee (AEEC No. 12/046/MIS-4) according to the guidance set by the Department of Health, Hong Kong SAR.

Drug treatment for animals

According to the recommended dosage (10-15 g) of raw herb in the Chinese Pharmacopoeia 2010 and the yield (~47%) of Gegen water extract prepared in this study, the minimum human equivalent dose (HED) of Gegen water extract was about 4.7 g. As calculated by the body surface area normalization method with its HED and the translational factor (6.16) between human and rat, the rat dose of Gegen to HED was about 483 mg/kg (=HED/60 kg x 6.16). Then, a lower dose at 300 mg/kg was selected based on this calculation. For the study of detrusor overactivity in experimental SHR rats, Gegen 300 (Gegen, 300 mg/kg) and Gegen 30 (Gegen, 30 mg/kg) were given orally by gavage. According to pharmaceutical recommendations and basing on the acute treatment results described in the previous study, Low_Dar (darifenacin, 3 mg/kg) and High_Dar (darifenacin, 30 mg/kg), as the positive control, were administrated intragastrically to SHR rats, respectively (Patra and Thorneloe, 2011). To study the combination effect, Low_Dar+Gegen 30 and High_Dar+Gegen 30 were used. All rats received different treatments continuously for 3 weeks. SHR served as the disease model, while WKY rats served as the normal control. Gegen and darifenacin were dissolved in water, and control rats received water alone.

Ex vivo experiments

After 3-week treatments, rats were euthanized by C[O.sub.2] asphyxiation. The whole bladder was isolated and immediately put into carbogen-bubbled ice-cold Krebs' solution. The bladder base, one-third of the bladder, was discarded. Only tissues isolated from the bladder dome (detrusor) were used. Detrusor strips were prepared along the transverse and longitudinal orientations. Detrusor strips were mounted on a myograph system (Danish Myo Technology Model 800MS, Denmark) with carbogen-aerated Krebs' solution at 37[degrees]C. Isometric tension was recorded through the PowerLab data acquisition system with the LabChart software (ADInstruments Ltd., Chalgrove, Oxfordshire, UK). The detrusor strips were initially equilibrated for 30 min. Before agonists' stimulation, the resting tension was repeatedly adjusted to 1.5 [+ or -] 0.1 g. Since the response to carbachol in the longitudinal detrusor strips were stronger than in the transverse ones in the previous study (Lam et al., 2014), the longitudinal strips were stimulated by a muscarinic agonist carbachol ([10.sup.-8] - [10.sup.-4] M in Krebs' solution; with 5 min intervals between two concentrations) and subsequently subjected to electrical field stimulation (1-64 Hz; in 100 mA pulse trains of 1 ms duration with 3 min intervals between each two-fold frequency increment). Maximal tonic contractile force were measured. The transverse strips were stimulated by carbachol (3 x [10.sup.-5] M) for 30 min, and then treated with different concentrations ([10.sup.-7] - [10.sup.-5] M) of isoprenaline (a non-selective [beta]-adrenergic agonist) for 20 min each. The phasic contractile parameters (including amplitude and frequency) over 2 min before each addition of different concentrations of isoprenaline were recorded. During all experiments, the detrusor strips were aerated by carbogen. Between each two stimulations, the detrusor strips were washed out and balanced for 15 min. After the ex vivo experiments, the detrusor strips were dried in the oven overnight and then weighed.

Data analysis

Maximum values of tonic contractile force were obtained by the LabChart software. For the analysis of phasic contractile activity, phasic frequency and amplitude were computed from the Cyclic Measurements function of the LabChart software. Dry tissue weight was obtained for normalization of the tonic contractile and phasic data. Data represented as mean [+ or -] S.E.M. (standard error of mean). [EC.sub.50] values were calculated by Graphpad Prism v.4 (San Diego, CA, USA) using non-linear regression. Data were statistically analyzed by One-way ANOVA with Dunnet's post test or Two-way ANOVA with Bonferroni post test using Graphpad Prism v.4, where appropriate. A p value less than 0.05 was considered statistically significant.

For the combination of drug-herb synergism analysis, Combination Index (CI) was calculated by CompuSyn Software (ComboSyn, Inc. Paramus, NJ., USA). For comparison, CI < 1,=1, and > 1 indicate synergism, additivity and antagonism, respectively (Chou, 2008).

Results

HPLC profile

As shown in Fig. 1A, a typical HPLC-UV (254 nm) profile of Gegen showed that puerarin, daidzin and daidzein, as the typical markers of Gegen, were found in the Gegen water extract, when compared with the authentic standards (Fig. 1B).

Carbachol-induced tonic contractile activity in longitudinal detrusors

The responses to carbachol at different concentrations in the WKY control detrusors were all lower than those of the SHR control detrusors, showing that SHR was a suitable disease model for detrusor overactivity study (Supplemental Fig. SI). When compared with the SHR control, all treatments including Gegen, darifenacin, and their combination reduced the responses to carbachol stimulation. As shown in Table 1, the [EC.sub.50] values of all treatment groups except Gegen 30 were much lower than those of the SHR control. However, the maximal contractile response under carbachol stimulation in the SHR control detrusor was 2-3 folds higher than those of all treatments and the WKY control. In addition, there was no significant difference of the maximal contractile response among the groups of Low_Dar, Gegen 300, and Low_Dar+Gegen 30, showing that Gegen was an anti-muscarinic agent, and Gegen 30 did not decrease the therapeutic effect of darifenacin.

Electrical field stimulation (EFS)-induced contraction in longitudinal detrusors

The results of neurogenic tonic contractions stimulated by EFS at frequencies from 1 to 64 Hz were shown in Supplemental Fig. S2, showing that all treatments reduced the neurogenic tonic contractions. As summarized in Table 2, the maximal EFS-induced responses in the groups of Gegen 300; Low_Dar+Gegen 30; and High_Dar+Gegen 30 were significantly lower than those of SHR control by 50-60%.

Phasic frequency and phasic amplitude after carbachol stimulation in transverse detrusors

At the end of 30 min carbachol stimulation, the phasic frequency of WYK control was lower than SHR control by 37%, indicating that the SHR detrusor strips contracted more frequently than those of the WICY control. G300; Low_Dar; Low_Dar+Gegen 30 and High_Dar+Gegen 30 reduced carbachol-stimulated phasic frequency by 42%-58% (Fig. 2A). However, Gegen 30 and High_Dar did not significantly decrease this frequency.

As shown in Fig. 2B, the phasic amplitude of the WKY control detrusors was higher than that of the SHR control detrusors; that of High_Dar+Gegen 30 was also higher, but not significantly. Meanwhile, the phasic amplitudes of other treatment groups did not show significant differences against that of the SHR control detrusor.

Isoprenaline-induced relaxation in transverse detrusors

After 30 min carbachol stimulation, tonic contraction was reduced by isoprenaline more significantly on the detrusor strips isolated from Gegen 300-treated rats (Fig. 3A). However, there was no significant difference of tonic contraction on isoprenaline-induced relaxation among other groups. At the same time, phasic frequency in the treatment groups, such as Low_Dar; Gegen 300; Low_Dar+Gegen 30; and High_Dar+Gegen 30, were decreased more significantly by isoprenaline when compared to the SHR control (Fig. 3B). As shown in Fig. 3C, there was no significant difference of phasic amplitude between treatment groups and the SHR control group.

Synergistic effects of Gegen and darifenacin

When compared to controls, the maximal responses to EFS with treatments of Gegen, darifenacin and their combination were reduced by 35-64%, respectively (Table 2). According to these maximal responses, the Combination Index (CI) between Gegen 30 and Low_Dar or High_Dar was calculated. As shown in Fig. 4, the Combination Index plot indicated that Low_Dar and Gegen 30 attained very strong synergism (CI < 0.1) on the inhibition of the contractile response induced by EFS.

Discussion

Acetylcholine released from parasympathetic nerves reaches muscarinic receptors for the stimulation of the detrusor muscle, and anti-muscarinic drugs block this interaction to control detrusor overactivity. Currently, anti-muscarinic drugs have been the gold standard for detrusor overactivity treatment (Andersson et al., 2009). Muscarinic receptor-3 are rich in bladder, and its selective inhibitors have been highly recommended for detrusor overactivity treatment in urology clinics. In this study, darifenacin and Gegen 300 both effectively reduced the tonic contraction activity and phasic frequency induced by carbachol. It is therefore deduced that Gegen could be considered as an alternative anti-muscarinic agent. When used in combination with Gegen 30 and darifenacin, Gegen 30 did not alter the effects of darifenacin against carbachol's stimulation, indicating that Gegen would not be interfering with darifenacin's therapeutic effects through anti-muscarinic mechanism.

Another kind of highly recommended drugs for detrusor overactivity are neurogenic toxins like botulinum toxin which blocks neurotransmitter release (Chung, 2015). In the present study, Gegen was found to reduce the response from EFS on isolated bladder strips, indicating its possible anti-neurogenic action. Few studies demonstrated the possible neurogenic mechanisms of Gegen. However, puerarin, one major component of Gegen, has been found to decrease the activation of toad's sciatic nerve trunk through blocking of the sodium channel (Dongji et al., 2014). The possible neurogenic mechanism of Gegen has to be further investigated. It could be summarized that through actions against the muscarinic receptor agonists and EFS, Gegen has been found to be a favorable agent for detrusor overactivity with possible mixed actions.

Adrenoceptor agonists have also been used against detrusor overactivity. In this study, isoprenaline, a non-selective beta-adrenergic agonist, had been shown to strengthen the reduction of the phasic frequency of carbachol-sustained stimulation in darifenacin-treated groups. However, it did not increase the reduction of tonic contraction and phasic amplitude, indicating that isoprenaline and darifenacin have possible synergism via the inhibition effects on different receptors. This is comparable to the clinical practice using the combination treatment with anti-muscarinic drugs and adrenoceptor agonists (Andersson et al., 2009; Ruggieri et al., 2005). In addition, Gegen 300 was shown to significantly reduce the tonic contraction and phasic frequency with isoprenaline, but not the phasic amplitude. According to the reduction level of the tonic contraction, Gegen 300 was more potent than darifenacin for the combination with isoprenaline. A major component of Gegen, genistein was speculated to be a candidate responsible for this reduction, since genistein had been found to potentiate the relaxation induced by beta1- and beta2-adrenoceptor activation in the rat aortic rings (Satake et al., 2000).

As an antimuscarinic agent, darifenacin alone effectively reduced carbachol's stimulation, but showed very weak inhibition (no more than 40%) on lowering EFS responses. After sub-acute treatments, Gegen 300 and Low_Dar+Gegen 30 showed stronger potency on lowering EFS responses. This indicated that Gegen and darifenacin possibly had synergistic effects on improving detrusor overactivity through inhibiting EFS response. As shown in Fig. 4, Combination Index demonstrated very strong synergism of Gegen and darifenacin on the inhibition of EFS responses. Thus, Gegen and darifenacin could be positively considered for concomitant application against detrusor overactivity. In order to enhance the effects in clinics, the dose of darifenacin could be doubled, basing on individual patients' response. However, the side effects (e.g. constipation, and dry mouth) could also increase dramatically (FDA, 2004). It is therefore speculated that under the clinical situation, when the individual response to the standard dose of darifenacin is not satisfactory, Gegen may be considered as an enhancer. Their observed synergism in our study platform could be truly utilized, while the side effects of darifenacin at high dosages could be avoided.

It is concluded that Gegen, as a potential therapeutic agent against detrusor overactivity, may function through neurogenic and anti-muscarinic mechanisms. Gegen may achieve synergistic effects with darifenacin via the neurogenic mechanism. It is suggested that the role of Gegen in the control of detrusor overactivity could be further studied in clinical trials, using it either as an individual item or in combination with darifenacin. The possible synergism between them and the reduction of adverse effects could be further worked out.

http://dx.doi.org/10.1016/j.phymed.2016.03.002

ARTICLE INFO

Article history:

Received 16 October 2015

Revised 13 January 2016

Accepted 4 March 2016

Abbreviations: CI, combination index; EFS, electrical field stimulation; High_Dar, darifenacin, 30 mg/kg; Low_Dar, darifenacin, 3 mg/kg; SHR, spontaneously hypertensive rat; WKY, Wistar Kyoto rat.

Conflict of interest

There are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

This project was financially supported by Health and Medical Research Fund (Ref. no. 10110411) of the Food and Health Bureau, Hong Kong SAR.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.03.002.

References

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Patra, P.B., Thomeloe, K.S., 2011. Enhanced sensitivity to afferent stimulation and impact of overactive bladder therapies in the conscious, spontaneously hypertensive rat. J. Pharmacol. Exp. Ther. 338 (1), 392-399.

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Xuelin Zhou (a,b), Wai Ping Lam (a,b), Hong Chai Tang (a,b), Chi-Man Koon (a,b), Ling Cheng (a,b), Clara Bik-San Lau (a,b), Willmann Liang (c), **, Ping-Chung Leung (a,b) *

(a) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

(b) State Key Laboratory of Phytochemistry and Plant Resources in West China. The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

(c) School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

* Corresponding author at: Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China. Tel.: +852 22528868; fax: +852 26325441.

** Corresponding author. Tel.: +852 39439427; fax: +852 26035022.

E-mail addresses: pingcleung@cuhk.edu.hk (P.-C. Leung), willmann@cuhk.edu.hk (W. Liang).

Table 1
Maximal contractile response and EC50 values of carbachol stimulation
on longitudinal detrusors isolated from Gegen and/or darifenacin-
treated male SHR rats (n = 7-10). Data was presented as Mean [+ or -]
SEM.

Rat                                               [EC.sub.50]
species  Treatment        Emax (g/mg tissue)      ([micro]M)

WKY      Control          3.73 [+ or -] 0.95 ***  0.31 [+ or -] 0.11 *
SHR      Control          9.97 [+ or -] 0.78      0.72 [+ or -] 0.08
         Gegen 30 mg/kg   4.64 [+ or -] 0.63 ***  0.87 [+ or -] 0.17
         Gegen 300 mg/kg  3.05 [+ or -] 0.58 ***  0.26 [+ or -] 0.02 *
         Low_Dar          3.01 [+ or -] 0.51 ***  0.48 [+ or -] 0.09
         High_Dar         3.31 [+ or -] 0.43 ***  0.36 [+ or -] 0.13
         Low_Dar+Gegen    2.95 [+ or -] 0.46 ***  0.10 [+ or -] 0.03 **
           30 mg/kg
         High_Dar+Gegen   4.83 [+ or -] 1.29 ***  0.26 [+ or -] 0.09 *
           30 mg/kg

* p < 0.05, ** p < 0.01 and *** p < 0.001 indicate significant
difference from SHR control. Low_Dar: Darifenacin, 3 mg/kg;
High_Dar: Darifenacin, 30 mg/kg.

Table 2
Maximum effects of electrical field stimulation on longitudinal
detrusors isolated from Gegen and-or darifenacin-treated male SHR
rats (n = 7-10). Data was presented as Mean [+ or -] SEM.

Rat                                                          % to SHR
species   Treatment                 Emax (g/mg tissue)       control

WKY       Control                   1.74 [+ or -] 0.24       63%
SHR       Control                   2.77 [+ or -] 0.44       100%
          Gegen 30 mg/kg            1.79 [+ or -] 0.31       65%
          Gegen 300 mg/kg           1.07 [+ or -] 0.31 ***   39%
          Low_Dar                   1.83 [+ or -] 0.22       66%
          High_Dar                  1.82 [+ or -] 0.57       65%
          Low_Dar+Gegen 30 mg/kg    1.18 [+ or -] 0.25 *     43%
          High_Dar+Gegen 30 mg/kg   1.43 [+ or -] 0.35 *     52%

* p < 0.05 and *** p < 0.001 indicate significant difference from
SHR control. Low_Dar: Darifenacin, 3 mg/kg; High_Dar: Darifenacin,
30 mg/kg.
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Author:Zhou, Xuelin; Lam, Wai Ping; Tang, Hong Chai; Koon, Chi-Man; Cheng, Ling; Lau, Clara Bik-San; Liang,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2016
Words:4357
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