Effect of td0014 on intracavernous pressure elicited with electrical stimulation of the cavernous nerve in male rats

Nội dung text: Effect of td0014 on intracavernous pressure elicited with electrical stimulation of the cavernous nerve in male rats

1. JOURNAL OF MEDICAL RESEARCH EFFECT OF TD0014 ON INTRACAVERNOUS PRESSURE ELICITED WITH ELECTRICAL STIMULATION OF THE CAVERNOUS NERVE IN MALE RATS Mai Phuong Thanh1, Pham Thi Van Anh1, Nguyen Trong Thong1, Nguyen Thi Huong Lien 2 1Department of Farmacology, Hanoi Medical University, 2Sunstar Joint Stock Company This study aimed to determine the effects of an herbal formula named TD0014 on the electrostimulation- induced erection in a rat model. Eighteen sixteen-week-old male Wistar rats were used. The intracavernous pressure and the arterial blood pressure were simultaneously monitored during cavernous nerve electrical stimulation before and after the administration of TD0014 (1.8 g/kg). Statistical analysis was performed on measurements of maximal intracavernous pressure (MIP), mean arterial blood pressure (MAP) and the MIP/MAP. The administration of TD0014 resulted in a significant increase in the baseline level of intracavernosal pressure (ICP) before stimulation in comparison to the control group (P < 0.05). The findings also showed that the maximal ICP and the area under the curve (AUC time × ICP curves) in TD0014 group were greater than distilled water group, however, this difference was not significant. TD0014 had no significant effect on the mean arterial pressure. These results indicated that the herbal formulation TD0014 may have positive and selective effects on improving erectile functions in male rats. Keywords: TD0014, cavernous nerve stimulation, intracavernous pressure, rat I. INTRODUCTION Erectile dysfunction (ED) is a term and negatively impact the quality of life of recommended by a panel of experts in afflicted men and their spouse [4]. Recently 1992 to replace the term “impotence” [1]. developed phosphodiesterase type-5 (PDE- ED is when a man is unable to get and/or 5) inhibitors have been widely used as first- keep an erection. It is not a disease, but line therapeutics to treat ED [5]. PDE5 is a symptom of some other problem, either an enzyme found primarily in the smooth physiological, psychological or a mixture muscle of the corpus cavernosum that of both [2]. Although physiological ED selectively cleaves and degrades cGMP itself is not life threatening, it is a strong to 5′-GMP. PDE5 inhibitors are similar in predictor of high-mortality diseases such as structure to cGMP; they competitively bind cardiovascular disease [3]. ED does directly to PDE5 and inhibit cGMP hydrolysis. The increased amounts of cGMP enhance the Corresponding author: Mai Phuong Thanh, Department effects of NO a potent vasodicator. This of Pharmacology, Hanoi Medical University increase in cGMP in the smooth muscle cells Email: maiphuongthanh@hmu.edu.vn is responsible for prolonging an erection [6]. Received: 05 June 2017 Although large, multicenter clinical trials Accepted: 16 November 2017 have shown the efficacy and tolerability of 36 JMR 111 E2 (2) - 2018
2. JOURNAL OF MEDICAL RESEARCH these drugs in patients with ED of various Herbal formula TD0014 preparation etiologies and a broad range of severity; TD0014 was manufactured as hard pills however, 30% to 35% of patients fail to according to the quality standard of Sunstar respond. The use of PDE - 5 inhibitors may Joint Stock Company, Vietnam. The major result in several side effects, including visual ingredients of the herbal formula are obtained disturbances, headache, facial flushing, from thirty-three plants (per 7.5g of serving): rhinitis, and indigestion. Other treatments Tribulus terrestris (4 g), Chrysanthemum for ED include penile injection therapy or sinense (1.83 g), Prunus persica (1.14 penile implants. However, such methods g), Vigna cylindrica (1.14 g), Eurycoma are invasive and irreversible, and are longifolia (0.69 g), Sophora japonica (0.57g), therefore not widely used [7]. Thus, there Dioscorea persimilis (0.43 g), Dioscorea is a continued need for the development of tokoro (0.4 g), Polygonum multiflorum (0.4 new noninvasive and effective therapies to g), Citrus deliciosa (0.34 g), Polyscias treat patients with ED. fruticosa (0.34 g), Tinospora sinensis Despite the remarkable developments (0.29 g), Chaenomeles lagenaria (0.29 g), of modern medicine, many people are Passiflora foetida (0.29 g), Zizyphus sativa still favorably disposed towards herbal (0.29 g), Rehmannia glutinosa (0.23 g), medicines owing to the aggressive Angelica sinensis (0.23 g), Alisma plantago- treatment protocols, toxicity, and drug aquatica L. var. orientalis Samuelsson tolerance associated with modern therapies. (0.23 g), Achyranthes bidentata (0.23 g), The widespread use of herbal medicines, Schizandra chinensis (0.23 g), Morinda however, requires scientific verification of offcinalis (0.23 g), Rosa laevigata (0.23 their effects. g), Allium sativum (0.2 g), Lycium sinense TD0014 is a preparation which (0.17 g), Glycyrrhiza uralensis (0.14 g), comprises of thirty-three medicinal plants. Panax ginseng (0.11 g), Ligusticum wallichii The composition of TD0014 has several (0.11 g), Cistanche tubulosa (0.11 g), medicinal herbs that have been studied Atractylodes macrocephala (0.11 g), Radix and used since ancient times in traditional Codonopsis (0.11 g), Cuscuta sinensis (0.11 folk medicine as an aphrodisiac [8 - 15]. g), Psoralea corylifolia (0.06 g), Cornu Cervi However, no studies have shown reliable parvum (7.2 mg). The experimental animals evidence of their effect on reproductive drank the test drug mixed with pure water. function, or toxicity when taken in TD0014. Animal groups Therefore, the purpose of this study was to Wistar male rats (250 - 300 g), 16 weeks explore the impact of TD0014 on erectile of age, were used in our study. They were capacity in adult male rats. housed in groups of six rats per cage in a 24 - hour air - conditioned room with access to II. MATERIALS AND METHODS standard certified rodent diet and water ad 1. Materials libitum. They were acclimated to housing for at least 1 week prior to investigation. JMR 111 E2 (2) - 2018 37
3. JOURNAL OF MEDICAL RESEARCH The rats were randomly separated into 3 groups of 6 animals each: 1) control, 2) sildenafil treatment, and 3) TD0014 treatment. In each group, animals were administered per os one- time with either distilled water (10 ml/kg b.w.), or sildenafil (6 mg/kg b.w.), or TD0014 (1.8 g/kg b.w.). 2 hours after treatment, erectile function was evaluated by measuring intracavernosal pressure (ICP) and maximal ICP/mean arterial pressure (MIP/MAP). 2. Methods Measurement of intracavernosal pressure Rats were anaesthetized with an intraperitoneal injection of ketamine at a dose of 25 mg/ kg (Rotexmedica, Germany). The penis was dissected and the corpus cavernosum and crus were exposed in a supine position. A low-midline incision was made to access the pelvis, and the pelvic ganglion lateral to the right prostate was exposed. The penile skin was degloved and the corpus cavernosum was identified. To measure ICP, a heparinized 23-gauge butterfly needle was inserted into the proximal portion of the corpus cavernosum. A bipolar electrical stimulator was placed on the ganglion to stimulate the cavernosal nerve for 60 seconds at 5 V and 20 Hz for 2 millisecond periods. The cavernosal nerve stimulation was conducted 3 times with a 10-minute interval between stimulations. Before and after each electrical stimulation, ICP was recorded on a computer by Powerlab system record software. ICP data was normalized by mean systemic arterial pressure (MAP) and the MAP was monitored simultaneously with ICP monitoring. The right carotid artery was dissected via a midline cervical incision under a microscope, and then PE-50 tubing was inserted into the carotid artery. The catheter was connected to both a pressure transducer and an amplifier unit which was connected to a data acquisition module. MAP was recorded on a computer by Powerlab system record software. Statistical analyses Data was analyzed employing with the Labchart pro software and Microsoft Excel 2010. All data is presented as mean ± standard deviation. Statistical significance was determined by Student's t-test and p < 0.05 were considered to be significant. III. RESULTS ** * p < 0.05; ** p < 0.01; *** p < 0.001; compared with control (Student’t-test) Figure 1. Intracavernous pressure (ICP) before and after electrical stimulation of the cavernous nerve in rats from each experimental group 38 JMR 111 E2 (2) - 2018
4. JOURNAL OF MEDICAL RESEARCH In sildenafil group, before and after the electrical stimulation of the cavernous nerve, the ICP significantly increased compared to the control group. In the flaccid state, TD0014 elevated the ICP level which was statistically different compared to the control animals (p < 0.05). In addition, the ICP after stimulation also increased in TD0014 group, however, it was not statistically significant. ** ΔΔΔ **p < 0.01 vs. control rats (Student’t-test); ∆∆∆p < 0.001 vs. sildenafil rats ((Student’t-test) Figure 2. Effect of TD0014 on time to the maximal ICP and response time to the electrical stimulation of the cavernous nerve Observing in Figure 2, the parameter of time to the maximal ICP was not different amongst treatment groups. The treatment with sildenafil increased the response time to electrical stimulation (p < 0.01). The animals treated with TD0014 were not able to extend the response time. Table 1. Total ICP (ICP vs stimulation time, area under curve) in treatment rats and control rats. Treatment Total ICP (mmHg*s) Group 1 (control group) 1375.06 ± 822.02 Group 2 (sildenafil) 2213.70 ± 1077.94* Group 3 (TD0014) 1722.96 ± 710.00 *p < 0.05, response significantly different from control rats In Table 1, statistical analysis indicated that sildenafil group showed significantly greater total ICP compared with the control group. TD0014 group exhibited an increase in total ICP, but it was not statistically significant. The effect of sildenafil and TD0014 on blood pressure is shown in Table 1. After sildenafil administration, MAP decreased by approximately 10 mmHg, but this was not a significant difference to the control (p > 0.05) MIP/MAP increased significantly compared with distilled water group and TD0014 group (p < 0.05 an p < 0.01 respectively). There was no statistical JMR 111 E2 (2) - 2018 39
5. JOURNAL OF MEDICAL RESEARCH differences in MAP and MIP/MAP values between the TD0014 group and the control group. Table 2. Effect of TD0014 on maximal ICP/MAP ratio Treatment MAP (mmHg) MIP/MAP Group 1 (control group) 106.34 ± 18.08 0.40 ± 0.11 Group 2 (sildenafil) 96.43 ± 13.76 0.56 ± 0.14* Group 3 (TD0014) 110.67 ± 5.05 0.44 ± 0.09∆∆ *p < 0.05 as compared with control; ∆∆p < 0.01 as compared with sildenafil IV. DISCUSSION Penile erection is a hemodynamic electrostimulation (53% and 25% increase, process which penile arteries, penis and respectively), prolong response time with penile venous system induced by neuro- stimulation, and increase total ICP (61% endocrine regulation [16]. Change in increase) compared to the control group. ICP induced by penile cavernous nerve In this study, the administration of stimulation is often used as an objective TD0014 at 1.8 g/kg/day showed an assessment index for the impact of drugs improvement of ICP level before the on erectile function in vivo [17]. start of electrostimulation, which means Stimulation of penile cavernous nerve the herbal preparation has a pro-erectile could activate nitric oxide synthase activity activity. Subsequently TD0014 can improve of non-adrenergic and non-cholinergic the ICP value during erection. After nerve endings, promote production and electrostimulating the cavernosal nerve, release of NO. No leads increased to cGMP compared to the control group, TD0014 concentration, which induces the relaxation tended to have an elevation in the maximal of smooth muscle and reults in erection [18]. ICP (12% increase), total ICP (25% Sildenafil is a potent and selective inhibitor increase), and a prolonged response time of cyclic guanosine monophosphate (cGMP) to the stimulus, i.e prolong the duration of specific phosphodiesterase type 5 (PDE5) penile tumescence, however, these results which is responsible for the degradation showed no signitificant differences (p > 0.05). of cGMP in the corpus cavernosum of the Thus, in the current study, TD0014 given penis. Therefore, increased cGMP within once before nerve stimulation markedly the corpus cavernosum can lead to smooth increased baseline ICP, slightly increased muscle relaxation [19]. The results of Fig 1, both the MIP and the response time to Fig 2, and Tab 1 clearly demonstrated the the stimulus. These results suggested that beneficial effects of sildenafil on erectile TD0014 may be beneficial for patients with dysfunction, as shown by the ability to erectile dysfunction, and can improving the significantly increase ICP before and after patient's sexual life. 40 JMR 111 E2 (2) - 2018
6. JOURNAL OF MEDICAL RESEARCH TD0014 preparation contains several because of an associated contraction of medicinal plants that have been studied for corpus cavernosum and deep arteries in the effects on sexual functions, including the penis. On the contrary, drugs with an erectile capacity, which can partly explain effect of decreasing MAP will increase the the impacts of this herbal formula on the MIP/MAP. The optimal charge in MIP should enhancement of penile erection. Results be determined by a balanced change in the published by Do J et al. (2013) indicated MIP/MAP and MAP. The present study in that Tribulus terrestris extract caused rats showed that sildenafil administration relaxation of the cavernous smooth muscle slightly decreased MAP by approximately in a concentration-dependent manner, 10 mmHg, and subsequently increased and the mechanism included a reaction MIP/MAP (Table 2). Therefore, in clinical involving the NO/NOS pathway in the practice, sildenafil should be prescribed corpus cavernosum endothelium [8]. with caution in patients with cardiovascular Echinacoside, a phenylethanoid glycoside risk factors [19]. This current study results isolated from the medicinal herb Cistanche show that TD0014 could increase ICP tubulosa, had an effect on nitric oxide (NO)- without significantly impacting on MAP. The cGMP signaling pathway and resulted in data suggests that TD0014 could selectively an increased cGMP in corpus cavernosum enhance erectile function without effecting smooth muscle [9]. The neurotransmitter systemic blood pressure. Therefore it could inducing penile erection, NO release was limit the side effects on blood pressure and shown to be enhanced by ginsenosides, cardiovascular diseases, which are common saponins from Panax ginseng in rabbit’s undesirable effects of drugs currently used corpus cavernosum in vitro. Ginsenosides to treat ED. enhanced both acetylcholine-induced and V. CONCLUSION transmural nerve stimulation-activated relaxation associated with increased tissue On the basis of our results, we conclude cGMP [10]. Some other medicinal plants in that the herbal formulation TD0014 may TD0014 were proved to cause an increase have a positive and selective effect on in serum testosterone level [11 - 15], improving the erectile functions in male rats therefore ameliorated ED by reducing ROS without impacting systemic blood pressure. production and increasing the activity of the Treatment with TD0014 may significantly eNOS/cGMP [20]. increase the baseline ICP, improve maximal The MIP/MAP indicates the extent of ICP and the duration of penile tumescence. relaxation of corpus cavernosum and deep Acknowledgements arteries in the penis, while MAP represents This work was supported by the the capacity of pumping blood into the Department of Physiology, Military Medical penis. Generally, drugs with an effect of University. increasing MAP will decrease the MIP/MAP JMR 111 E2 (2) - 2018 41
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