鹿皮胶的制备及功能研究
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摘要
目的：
制备鹿皮胶，在此基础上研究鹿皮胶对去势大鼠及正常大鼠血清激素、性器官及附性器官指数和内皮性一氧化氮合酶与5型磷酸二酯酶表达量的影响。方法：
按照已有文献的方法制备鹿皮胶。将雄性Wistar大鼠随机分为7组，其中两组制作去势模型。
去势7天后，进行动物处理，即分别灌以不同剂量浓度的鹿皮胶、丙酸睾丸酮、生理盐水，处理十天。处死大鼠，剪取其睾丸、阴茎、精液囊-前列腺称重并计算器官指数；采用全自动微粒子化学发光检测血清睾酮（T，testosterone)、促黄体生成素（LH，luteinizing hormone）和雌二醇( E2,estradiol )含量；分离阴茎海绵体，检测其中一氧化氮合酶（eNOS，endothelial nitric oxide synthase)和磷酸二酯酶( PDE-5，phosphodiesterase-5 )含量变化。实验所测数据以平均数±标准差的形式表示，运用SPSS18.0分析数据，以T检测比较实验组和对照组参数。结果：1.血清激素含量变化：
大鼠经去势后，血清睾酮、促黄体生成素含量显著降低，而雌二醇含量升高。去势组服用鹿皮胶后，血清睾酮(0.6±0.24mg/ml)、促黄体生成素(0.23±0.13mIU/ml)相对于去势对照组（0.3±0.22 mg/ml；0.14±0.13 mIU/ml）均有极显著提高（P>0.01）。正常大鼠各组中，低剂量组血清睾酮(2.26±1.08 mg/ml)、促黄体生成素含量(0.31±0.19 mIU/ml)与对照组(2.53±0.4 mg/ml; 0.14±0.01mIU/ml)差异不显著，而中剂量组（4.03±0.43 mg/ml；0.53±0.13 mIU/ml）、高剂量（4.97±1.87 mg/ml；0.53±0.13 mIU/ml）组相对于对照组则有极显著提高（P>0.01）。2.器官指数变化：
去势模型建立后大鼠的阴茎指数、精液囊-前列腺指数均显著下降。去势组服用鹿皮胶后，附性器官指数相对于去势对照组有所上升，但是不具有显著性差异。正常大鼠服用鹿皮胶后，与空白对照组相比，附性器官指数也有所提高，但是仅仅高剂量组精液囊-前列腺指数（6.99±0.86(mg/g)）相对于空白对照组（5.85±1.12(mg/g)）有显著差异( P < 0.01)，其他均无统计学差异性。3.一氧化氮合酶、磷酸二酯酶含量表达变化：
去势后，大鼠eNOS、PDE5含量降低，服用鹿皮胶后，eNOS表达量（0.29±0.09）、PDE5表达量（0.24±0.04）相对于去势对照组（0.21±0.04；0.2±0.03）含量有显著回升（P>0.05）。正常各组大鼠中，服用鹿皮胶后，低剂量组eNOS表达量（0.76±0.09）与空白对照组(0.58±0.16)相比有显著提高（P>0.05），而中剂量组（0.79±0.17）、高剂量组（0.86±0.18）则有极显著提高（P>0.01）。服用鹿皮胶后，各组大鼠的PDE5表达量（低剂量组0.63±0.11；中剂量组0.9±0.26；高剂量组1.04±0.05）与空白对照组(0.4±0.01)相比均有极显著提高（P>0.01）。结论：
鹿皮胶可以改善去势模型大鼠血清睾酮、促黄体生成素含量偏低的症状，同时可以提高正常大鼠上述激素含量水平；可以提高一氧化氮合酶和磷酸二酯酶的表达水平；对大鼠附性器官指数有提高趋势。
Aim:
Deer skin glue was prepared as described previously in the patent, and theninvestigated effects of deer skin glue on sexual hormones, sexual organ index andendothelial nitric oxide synthase and phosphodiesterase-5 expressions in castrated andnormal rats.Methods:
Deer skin glue was prepared. Forty-two male Wistar rats were randomized intoseven groups, two castrated groups (C2,L0) and five normal groups (C1,L1,L2,L3andA). The rats from castrated groups were underwent bilateral orchiectomy. One weekafter surgery, the rats were treated with normal water (groups C1and C2), oral deerskin glue (0.1g/[kg·day] for L1 group, 0.2 g/[kg·day] for L2 and L0 groups and 0.4g/[kg·day] for L3group) and intramuscular injection of Testosterone Propinas (5mg/[kg·day] for A group) for ten days. 24hours after the treatment, the weight of testis,penis, seminal vesicle-prostate, sexual hormones including serum testosterone(T) andluteinizing hormone (LH),the expressions of eNOS and PDE5 were evaluated.
SPSS18.0 was used for the statistical assay. T-test was used to compare theexperimental and control groups. All the data were expressed as a mean±standarddeviation (SD) .Results:
1) Sexual Hormones Assay
The mean levels of testosterone and luteinizing hormone descreaced significantly incastrated rats，while fed with deer skin glue, the mean level of testosterone(0.6±0.24mg/ml) and the level of LH (0.23±0.13mIU/ml) increased significantly(P>0.01) coparing with the level of rats (0.3±0.22 mg/ml；0.14±0.13 mIU/ml) fromgroup C2.
In group L1, the levels of testosterone and luteinizing hormone were0.6±0.24mg/ml and 0.23±0.13mIU/ml. There was no significance with control group(0.3±0.22 mg/ml；0.14±0.13 mIU/ml). However, it was of statistical significances ingroup L2(4.03±0.43 mg/ml；0.53±0.13 mIU/ml) and L3 (4.97±1.87 mg/ml；0.53±0.13 mIU/ml)(P>0.01).
2) Sexual Organ Index Assay
The penis index, seminal vesicle and prostate index decreased after the bilateralorchiectomy, The index increased when the rat given deer skin glue, though there wasnot statistical significance. At the same time, the sexual organ index of the normal ratsgiven deer skin glue increased too, but it was of statistical significance only inseminal vesicle and prostate index (the mean value was 6.99±0.86(mg/g) in rats fromL3,comparing with the mean value was 5.85±1.12(mg/g) in rats from C1; P < 0.01).
3) Effects of Deer Skin Glue on eNOS and PDE5 Expressions
The expressions of eNOS and PDE5 in the rats uderwent bilateral orchiectomydecreased a lot , while fed with deer skin glue, the expression of eNOS (0.29±0.09)and PDE5 (0.24±0.04) recovered markedly (P>0.05) compared with that in C2 (0.21±0.04; 0.2±0.03).
In normal groups, the eNOS expression increased significantly compared withcontrol group ( 0.58±0.16) (0.76±0.09 in group L1, P>0.05; 0.79±0.17, in groupL2 and 0.86±0.18 in L3, P>0.01). While the PDE5 expressions increased remarkably(P>0.01) in all groups fed with deer skin glue(0.63±0.11 in group L1; 0.9±0.26 ingroup L2; 1.04±0.05 in group L3) compared with control group (0.4±0.01).Discussions:
The levels of sexual hormones could be increased, the expressions of eNOS andPDE5 could be enhanced, and the sexual organ index could be enhanced in bothnormal rats and castrated rats fed with deer skin glue. It implyed that deer skin gluemay have aphrodisiac function.
制备鹿皮胶，在此基础上研究鹿皮胶对去势大鼠及正常大鼠血清激素、性器官及附性器官指数和内皮性一氧化氮合酶与5型磷酸二酯酶表达量的影响。方法：
按照已有文献的方法制备鹿皮胶。将雄性Wistar大鼠随机分为7组，其中两组制作去势模型。
去势7天后，进行动物处理，即分别灌以不同剂量浓度的鹿皮胶、丙酸睾丸酮、生理盐水，处理十天。处死大鼠，剪取其睾丸、阴茎、精液囊-前列腺称重并计算器官指数；采用全自动微粒子化学发光检测血清睾酮（T，testosterone)、促黄体生成素（LH，luteinizing hormone）和雌二醇( E2,estradiol )含量；分离阴茎海绵体，检测其中一氧化氮合酶（eNOS，endothelial nitric oxide synthase)和磷酸二酯酶( PDE-5，phosphodiesterase-5 )含量变化。实验所测数据以平均数±标准差的形式表示，运用SPSS18.0分析数据，以T检测比较实验组和对照组参数。结果：1.血清激素含量变化：
大鼠经去势后，血清睾酮、促黄体生成素含量显著降低，而雌二醇含量升高。去势组服用鹿皮胶后，血清睾酮(0.6±0.24mg/ml)、促黄体生成素(0.23±0.13mIU/ml)相对于去势对照组（0.3±0.22 mg/ml；0.14±0.13 mIU/ml）均有极显著提高（P>0.01）。正常大鼠各组中，低剂量组血清睾酮(2.26±1.08 mg/ml)、促黄体生成素含量(0.31±0.19 mIU/ml)与对照组(2.53±0.4 mg/ml; 0.14±0.01mIU/ml)差异不显著，而中剂量组（4.03±0.43 mg/ml；0.53±0.13 mIU/ml）、高剂量（4.97±1.87 mg/ml；0.53±0.13 mIU/ml）组相对于对照组则有极显著提高（P>0.01）。2.器官指数变化：
去势模型建立后大鼠的阴茎指数、精液囊-前列腺指数均显著下降。去势组服用鹿皮胶后，附性器官指数相对于去势对照组有所上升，但是不具有显著性差异。正常大鼠服用鹿皮胶后，与空白对照组相比，附性器官指数也有所提高，但是仅仅高剂量组精液囊-前列腺指数（6.99±0.86(mg/g)）相对于空白对照组（5.85±1.12(mg/g)）有显著差异( P < 0.01)，其他均无统计学差异性。3.一氧化氮合酶、磷酸二酯酶含量表达变化：
去势后，大鼠eNOS、PDE5含量降低，服用鹿皮胶后，eNOS表达量（0.29±0.09）、PDE5表达量（0.24±0.04）相对于去势对照组（0.21±0.04；0.2±0.03）含量有显著回升（P>0.05）。正常各组大鼠中，服用鹿皮胶后，低剂量组eNOS表达量（0.76±0.09）与空白对照组(0.58±0.16)相比有显著提高（P>0.05），而中剂量组（0.79±0.17）、高剂量组（0.86±0.18）则有极显著提高（P>0.01）。服用鹿皮胶后，各组大鼠的PDE5表达量（低剂量组0.63±0.11；中剂量组0.9±0.26；高剂量组1.04±0.05）与空白对照组(0.4±0.01)相比均有极显著提高（P>0.01）。结论：
鹿皮胶可以改善去势模型大鼠血清睾酮、促黄体生成素含量偏低的症状，同时可以提高正常大鼠上述激素含量水平；可以提高一氧化氮合酶和磷酸二酯酶的表达水平；对大鼠附性器官指数有提高趋势。
Aim:
Deer skin glue was prepared as described previously in the patent, and theninvestigated effects of deer skin glue on sexual hormones, sexual organ index andendothelial nitric oxide synthase and phosphodiesterase-5 expressions in castrated andnormal rats.Methods:
Deer skin glue was prepared. Forty-two male Wistar rats were randomized intoseven groups, two castrated groups (C2,L0) and five normal groups (C1,L1,L2,L3andA). The rats from castrated groups were underwent bilateral orchiectomy. One weekafter surgery, the rats were treated with normal water (groups C1and C2), oral deerskin glue (0.1g/[kg·day] for L1 group, 0.2 g/[kg·day] for L2 and L0 groups and 0.4g/[kg·day] for L3group) and intramuscular injection of Testosterone Propinas (5mg/[kg·day] for A group) for ten days. 24hours after the treatment, the weight of testis,penis, seminal vesicle-prostate, sexual hormones including serum testosterone(T) andluteinizing hormone (LH),the expressions of eNOS and PDE5 were evaluated.
SPSS18.0 was used for the statistical assay. T-test was used to compare theexperimental and control groups. All the data were expressed as a mean±standarddeviation (SD) .Results:
1) Sexual Hormones Assay
The mean levels of testosterone and luteinizing hormone descreaced significantly incastrated rats，while fed with deer skin glue, the mean level of testosterone(0.6±0.24mg/ml) and the level of LH (0.23±0.13mIU/ml) increased significantly(P>0.01) coparing with the level of rats (0.3±0.22 mg/ml；0.14±0.13 mIU/ml) fromgroup C2.
In group L1, the levels of testosterone and luteinizing hormone were0.6±0.24mg/ml and 0.23±0.13mIU/ml. There was no significance with control group(0.3±0.22 mg/ml；0.14±0.13 mIU/ml). However, it was of statistical significances ingroup L2(4.03±0.43 mg/ml；0.53±0.13 mIU/ml) and L3 (4.97±1.87 mg/ml；0.53±0.13 mIU/ml)(P>0.01).
2) Sexual Organ Index Assay
The penis index, seminal vesicle and prostate index decreased after the bilateralorchiectomy, The index increased when the rat given deer skin glue, though there wasnot statistical significance. At the same time, the sexual organ index of the normal ratsgiven deer skin glue increased too, but it was of statistical significance only inseminal vesicle and prostate index (the mean value was 6.99±0.86(mg/g) in rats fromL3,comparing with the mean value was 5.85±1.12(mg/g) in rats from C1; P < 0.01).
3) Effects of Deer Skin Glue on eNOS and PDE5 Expressions
The expressions of eNOS and PDE5 in the rats uderwent bilateral orchiectomydecreased a lot , while fed with deer skin glue, the expression of eNOS (0.29±0.09)and PDE5 (0.24±0.04) recovered markedly (P>0.05) compared with that in C2 (0.21±0.04; 0.2±0.03).
In normal groups, the eNOS expression increased significantly compared withcontrol group ( 0.58±0.16) (0.76±0.09 in group L1, P>0.05; 0.79±0.17, in groupL2 and 0.86±0.18 in L3, P>0.01). While the PDE5 expressions increased remarkably(P>0.01) in all groups fed with deer skin glue(0.63±0.11 in group L1; 0.9±0.26 ingroup L2; 1.04±0.05 in group L3) compared with control group (0.4±0.01).Discussions:
The levels of sexual hormones could be increased, the expressions of eNOS andPDE5 could be enhanced, and the sexual organ index could be enhanced in bothnormal rats and castrated rats fed with deer skin glue. It implyed that deer skin gluemay have aphrodisiac function.
引文
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[63] Conti CR, Pepine CJ, Sweeney M. Efficacy and safety of sildenafil citrate inthe treatment of erectiledysfunction in patients with ischemic heart disease [J].The American Journal of Cardiology, 1999, 83(5): 29-34.
[64] Lugg JA, Rajfer J, Gonzalez-Cadavid NF. Dihydrotestosterone is the activeandrogen in the maintenance of nitric oxide-mediated penile erection in the rat[J]. Endocrinology, 1995, 136: 1495–1501.
[65] Muller SC, Hsieh JT, Lue TF, et al. Castration and erection.An animal study[J]. European Urology, 1988, 15: 118-124.
[66] Zvara P, Souifi R., Schipper HM, et al. Nitric oxide mediated erectile activityis a testosterone dependent event: a rat erection model [J]. InternationalJournal of Impotence Research, 1995, 7(4): 209-219.
[67] Park KH, Kim SW, Kim KD, et al. Effects of androgens on the expression ofnitric oxide synthase mRNAs in rat corpus cavernosum [J]. BJU International,1999. 83(3): 327–333.
[68] Reilly CM, Zamorano P, Stopper VS, et al. Mills Androgenic regulation of NOavailability in rat penile erection [J]. Journal of Andrology, 1997. 18(2):110-115.
[69] Reilly CM, Lewis RW, Stopper VS, et al. Mills Androgenic maintenance of therat erectile response via a non-nitric-oxide-dependent pathway [J]. Journal ofAndrology, 1997, 18(6): 588-594.
[70] Garban H, Vernet D, Freedman A, et al. Effect of aging on nitricoxide-mediated penile erection in rats [J]. The American Journal ofPhysiology 1995, 268: 467-475.
[71] Penson DF, Ng C, Cai L, et al. Androgen and pituitary control of penile nitricoxide synthase and erectile function in the rat [J]. Biology of Reproduction1996, 5: 567–574.
[72] Shen Z, Chen Z, Lu Y, et al. Relationship between gene expression of nitricoxide synthase and androgens in rat corpus cavernosum [J]. Chinese MedicalJournal, 2000, 113(12): 1092-1095.
[73] Marin R, Escrig A, Abreu P, et al. Androgen-Dependent Nitric Oxide Releasein Rat Penis Correlates with Levels of Constitutive Nitric Oxide SynthaseIsoenzymes [J]. Biology of Reproduction, 1999, 61: 1012-1016
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[75] Seong S, Kim SW, Paick JS. The effects of androgen on penile reflex, erectileresponse to electrical stimulation and penile NOS activity in the rat [J]. Asian JAndrol, 1999, 1: 169-174.
[76] Baba K, Yajima M, Carrier S, et al. Effect of testosterone on the number ofNADPH diaphorase-stained nerve fibers in the rat corpus cavernosum anddorsal nerve [J]. Urology, 1999, 56(3): 533-538.
[77] Baba K, Yajima M, Carrier S, et al. Delayed testosterone replacement restoresnitric oxide synthase-containing nerve fibres and the erectile response in ratpenis [J]. BJU International, 2000, 85(7): 953–958.
[78] Armagan A, Kim NN, Goldstein I, et al. Dose–Response RelationshipBetween Testosterone and Erectile Function: Evidence for the Existence of aCritical Threshold [J]. Journal of Andrology, 2006, 27(4): 517–526.
[79] Traish AM, Park K, Dhir V, et al. Moreland and Irwin Goldstein, Effects ofCastration and Androgen Replacement on Erectile Function in a Rabbit Model[J]. Endocrinology, 1999, 140: 1861-1868
[80] Zhang XH, Morelli A, Luconi M, et al. Testosterone Regulates PDE5Expression and in vivo Responsiveness to Tadalafil in Rat CorpusCavernosum [J]. European Urology, 2005, 47(3): 409-416.
[81] Morelli A, Filippi S, Mancina R, et al. Androgens Regulate PhosphodiesteraseType 5 Expression and Functional Activity in Corpora Cavernosa [J].Endocrinology, 2004, 145: 2253-2263.
[82] Traish AM , Goldstein I, Kim NN. Testosterone and Erectile Function : FromBasic Research to a New Clinical Paradigm for Managing Men with AndrogenInsufficiency and Erectile Dysfunction [J]. European Urology, 2009, 52(1):54-70.
[83] Zhang XH, Filippi S, Morelli A. Testosterone Restores Diabetes- InducedErectile Dysfunction and Sildenafil Reponsiveness in Tow Distinct AnimalModels of Chemical Diabetes [J] . The Journal of Sexual Medcine ,2006, 3: 253–266.
[84] Mancina R, Filippi S, Marini M. Expression and functional activity ofphosphodiesterase type 5 in human and rabbit vas deferens [J]. MHR: BasicScience of Reproductive Medcine , 2004, 11(2): 107-115.
[85] Filippi S, Morelli A, Sandner P. Characterization and Functional Role ofAndrogen-Dependent PDE5 Activity in the Bladder [J]. GeneralEndocrinology, 2007, 148(3): 1019.